Patent application title: Constitutively Translocating Cell Line
Inventors:
Robert H. Oakley (Durham, NC, US)
Christine C. Hudson (Durham, NC, US)
Assignees:
Molecular Devices Corporation
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid
Publication date: 2009-03-26
Patent application number: 20090081652
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Patent application title: Constitutively Translocating Cell Line
Inventors:
Robert H. Oakley
Christine C. Hudson
Agents:
MORGAN, LEWIS & BOCKIUS, LLP
Assignees:
Molecular Devices Corporation
Origin: SAN FRANCISCO, CA US
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Abstract:
The present invention relates to agonist-independent methods of screening
for compounds that alter GPCR desensitization. Included in the present
invention are cell lines containing GRKs, in which GPCRs are desensitized
in the absence of agonist; the GRKs may be modified. The present
invention relates to methods to determine if a GPCR is expressed at the
plasma membrane, and if the GPCR has an affinity for arrestin. Modified
GPCRs which have increased arrestin affinity are included in the present
invention. These modified GPCRs are useful in methods to screen for
compounds that alter desensitization, including both the
agonist-independent methods and agonist-dependent methods described
herein.Claims:
1-29. (canceled)
30. A host cell comprising a GPCR and a modified GRK.
31. The host cell of claim 30, wherein the GRK is inducible or over-expressed.
32. The host cell of claim 30, wherein said host cell further comprises arrestin, wherein said arrestin may be detectably labeled.
33. The host cell of claim 30, wherein at least one of the GPCR, GRK, another molecule involved in desensitization, or a molecule that interacts with a molecule involved in desensitization is detectably labeled.
34. A method of modifying a nucleic acid encoding a GRK in which a GPCR is constitutively internalized, comprising:(c) providing a nucleic acid encoding a GRK;(b) mutating the nucleic acid encoding a GCRK such that the encoded GRK comprises a CAAX motif, wherein said modified GRK phosphorylates a GPCR in the absence of agonist; and(c) expressing the modified GRK in a cell.
35. The method of claim 34, wherein the nucleic acid encoding a GRK comprises SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
36. A kit for identifying a compound that modulates the internalization of a GPCR, comprising the host cell of claim 30.
37-53. (canceled)
Description:
[0001]The present application is a Continuation Application of U.S. patent
application Ser. No. 10/788,197, filed on Feb. 26, 2004, which is a
Continuation-In-Part application of International Application No.
PCT/US03/14581, filed on May 12, 2003, which claims the benefit of U.S.
Provisional Application No. 60/379,986 filed on May 13, 2002; and U.S.
Provisional Application No. 60/401,698 filed on Aug. 7, 2002; which are
hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002]The present invention relates to methods of assaying GPCR desensitization in a agonist-independent manner, host cells useful in such methods, methods of the identification of compounds that alter GPCR desensitization, the compounds identified, and their use in disease treatment.
BACKGROUND
[0003]G protein-coupled receptors (GPCRs) are cell surface proteins that translate hormone or ligand binding into intracellular signals. GPCRs are found in all animals, insects, and plants. GPCR signaling plays a pivotal role in regulating various physiological functions including phototransduction, olfaction, neurotransmission, vascular tone, cardiac output, digestion, pain, and fluid and electrolyte balance. Although they are involved in numerous physiological functions, GPCRs share a number of common structural features. They contain seven membrane domains bridged by alternating intracellular and extracellular loops and an intracellular carboxyl-terminal tail of variable length.
[0004]GPCRs have been implicated in a number of disease states, including, but not limited to: cardiac indications such as angina pectoris, essential hypertension, myocardial infarction, supraventricular and ventricular arrhythmias, congestive heart failure, atherosclerosis, renal failure, diabetes, respiratory indications such as asthma, chronic bronchitis, bronchospasm, emphysema, airway obstruction, upper respiratory indications such as rhinitis, seasonal allergies, inflammatory disease, inflammation in response to injury, rheumatoid arthritis, chronic inflammatory bowel disease, glaucoma, hypergastrinemia, gastrointestinal indications such as acid/peptic disorder, erosive esophagitis, gastrointestinal hypersecretion, mastocytosis, gastrointestinal reflux, peptic ulcer, Zollinger-Ellison syndrome, pain, obesity, bulimia nervosa, depression, obsessive-compulsive disorder, organ malformations (for example, cardiac malformations), neurodegenerative diseases such as Parkinson's Disease and Alzheimer's Disease, multiple sclerosis, Epstein-Barr infection and cancer.
[0005]The magnitude of the physiological responses controlled by GPCRs is linked to the balance between GPCR signaling and signal termination. The signaling of GPCRs is controlled by a family of intracellular proteins called arrestins. Arrestins bind activated GPCRs, including those that have been agonist-activated and especially those that have been phosphorylated by G protein-coupled receptor kinases (GRKs).
[0006]Receptors, including GPCRs, have historically been targets for drug discovery and therapeutic agents because they bind ligands, hormones, and drugs with high specificity. Approximately fifty percent of the therapeutic drugs in use today target or interact directly with GPCRs. See e.g., Jurgen Drews, (2000) "Drug Discovery: A Historical Perspective," Science 287: 1960-1964.
[0007]There is a need for accurate, easy to interpret methods of detecting G protein-coupled receptor activity and methods of assaying GPCR activity. One method, as disclosed in Barak et al., U.S. Pat. Nos. 5,891,646 and 6,110,693, uses a cell expressing a GPCR and a conjugate of an arrestin and a detectable molecule, the contents of which are incorporated by reference in their entirety.
[0008]Although only several hundred human GPCRs are known, it is estimated that upwards of a thousand GPCRs exist in the human genome. Of these known GPCRs, many are orphan receptors that have yet to be associated with a ligand.
[0009]The majority of the existing methods for identifying GPCR antagonists are dependent on the presence of agonist. Assays for identifying compounds that prevent the activation of GPCRs typically require that the GPCR is first activated in order to identify interfering compounds. For receptors with known agonists, these agonists are currently used to activate these receptors. However, many GPCRs are orphan receptors with no known ligand or agonist.
[0010]The agonist-dependence of GPCR assays continues to be a problem because antagonist discovery for orphan receptors is typically dependent on the prior discovery of agonist or ligand. Agonist-independent methods to screen for compounds that alter GPCR desensitization will (1) eliminate the step of agonist-addition in screening methods, and (2) enable identification of compounds that alter the desensitization of orphan receptors. Agonist-independent methods will eliminate the step of identifying an agonist of an orphan receptor prior to screening for compounds that alter desensitization of the orphan receptor.
SUMMARY
[0011]The present invention relates to methods of identifying compounds which alter GPCR internalization.
[0012]A first aspect of the present invention is a method of identifying a compound which alters GPCR internalization, including: (a) providing a cell including a GPCR, an arrestin, and a modified GRK, wherein said GPCR is at least partially internalized in an agonist-independent manner upon expression of said GRK; (b) exposing said cell to the compound(s); (c) determining the cellular distribution of the GPCR, arrestin, or modified GRK; and (d) monitoring a difference between (1) the distribution of the GPCR, arrestin, or modified GRK in the cell in the presence of the compound(s) and (2) the distribution of the GPCR, arrestin, or modified GRK in the cell in the absence of the compound(s). An agonist may not be provided in the above method. In the method, a difference between (1) and (2) of step (d) may indicate modulation of GPCR internalization.
[0013]The GRK may be over-expressed, its expression may be inducible, and it may include a CAAX motif. The GRK may be GRK1, GRK2, GRK3, GRK4, GRK5, GRK6, or a biologically active fragment thereof.
[0014]The GPCR may be modified to have enhanced phosphorylation by a GRK. The GPCR may be β2AR(Y326A), a GPCR listed in FIG. 1A-1C, an orphan GPCR, a modified GPCR, a taste receptor, a Class A GPCR, a Class B GPCR, a mutant GPCR, or a biologically active fragment thereof.
[0015]The arrestin may be visual arrestin, cone arrestin, β-arrestin 1, β-arrestin-2, or a biologically active fragment thereof.
[0016]The GPCR, GRK, or arrestin may be detectably labeled. A molecule involved in desensitization may be detectably labeled, or a molecule that interacts with a molecule involved in desensitization may be detectably labeled.
[0017]In a further aspect, the present invention relates to a method of identifying a compound that alters GPCR phosphorylation, including: (a) providing a cell including a GPCR and a GRK; (b) exposing the cell to the compound(s); and (c) determining whether GRK phosphorylation of the GPCR is altered in the presence of the compound(s).
[0018]The cellular distribution of the GPCR or GRK may be determined. A difference may be monitored between (1) the distribution of the GPCR or GRK in the cell in the presence of the compound(s) and (2) the distribution of the GPCR or GRK in the cell in the absence of the compound(s). A difference may be correlated between (1) and (2) to the phosphorylation of the GPCR.
[0019]The GRK may not be located in the plasma membrane, indicating that GRK phosphorylation of the GPCR is altered. The phosphorylation state of the GPCR may be determined. The activity of the GRK may be determined. The ability of the GPCR to be internalized may be determined.
[0020]In an additional aspect, the present invention relates to a method of determining if a GPCR is expressed at the plasma membrane, including: (a) providing a cell including a GPCR, an arrestin, and a GRK, wherein the arrestin is detectably labeled; (b) determining the cellular distribution of the arrestin; and (c) correlating the cellular distribution of the arrestin to the ability of the GPCR to be expressed at the plasma membrane. The arrestin may be localized in vesicles, pits endosomes, or elsewhere in the desensitization pathway.
[0021]Additionally, the present invention relates to a further method of determining if a GPCR is expressed at the plasma membrane, including: (a) providing a cell including a GPCR and a GRK, wherein the GRK is detectably labeled; (b) determining the cellular distribution of the GRK, and (c) correlating the cellular distribution of the GRK to the ability of the GPCR to be expressed at the plasma membrane. The GRK may be localized at the plasma membrane.
[0022]In a further aspect, the present invention relates to a method of analyzing the ability of a GPCR to bind arrestin, including: (a) providing a cell including a GPCR, an arrestin, and a GRK, wherein the arrestin is detectably labeled; (b) determining the cellular distribution of the arrestin; and (c) correlating the cellular distribution of the arrestin to the ability of the GPCR to bind arrestin. The arrestin or the GPCR may be localized in vesicles, pits, or endosomes.
[0023]In an additional aspect, the present invention relates to a compound identified by a method of the present invention.
[0024]In a further aspect, the present invention is related to a method of treating a disease by modulating desensitization of a GPCR in a host cell, including: (a) providing a compound identified by a method of the present invention; and (b) administering the compound to a host.
[0025]Another aspect of the invention relates to a host cell including a GPCR and a modified GRK. The GRK may be inducible or over-expressed. The host cell may further include arrestin, wherein the arrestin may be detectably labeled. The GPCR, GRK or another molecule involved in desensitization, or a molecule that interacts with a molecule involved in desensitization may be detectably labeled.
[0026]A further aspect of the present invention relates to a method of modifying a nucleic acid encoding a GRK in which a GPCR is constitutively internalized, including: (a) providing a nucleic acid encoding a GRK; (b) mutating the nucleic acid encoding a GRK such that the encoded GRK includes a CAAX motif, wherein the modified GRK phosphorylates a GPCR in the absence of agonist; and (c) expressing the modified GRK in a cell. The nucleic acid encoding a GRK may include SEQ ID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or 34.
[0027]The present invention also relates to a kit for identifying a compound that modulates the internalization of a GPCR, including a host cell including a GPCR and a modified GRK.
[0028]In a further aspect, the present invention relates to a modified GPCR including a NPXXY motif, and a carboxyl terminal tail, wherein the carboxyl terminal tail includes a putative site of palmitoylation and one or more clusters of phosphorylation, wherein the carboxyl terminal tail includes a retained portion of a carboxyl-terminus region of a first GPCR portion fused to a portion of a carboxyl-terminus from a second GPCR, and wherein the second GPCR includes the one or more clusters of phosphorylation and further includes a second putative site of palmitoylation approximately 10 to 25 amino acid residues downstream of a second NPXXY motif. The first GPCR may be a Class A receptor. The first GPCR may be hGPR3, hGPR6, hGPR12, hSREB2, hSREB3, hGPR8, or hGPR22. The second GPCR may be a Class B receptor. The Class B receptor may be selected from the group consisting of a vasopressin V2 receptor, a neurotensin-1 receptor, a substance P receptor, and an oxytocin receptor.
[0029]The present invention relates to a nucleic acid encoding a modified GPCR. Included in the present invention are nucleic acids selected from the group consisting of SEQ ID Nos: 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90. Also included in the present invention are expression vectors including the nucleic acid. Host cells including the expression vector or the nucleic acid are also included.
[0030]In a further aspect, the present invention relates to a method of screening compounds for GPCR activity including the steps of: (a) providing a cell that expresses at least one modified GPCR, wherein the cell further includes arrestin conjugated to a detectable molecule; b) exposing the cell to the compound; (c) detecting location of the arrestin within the cell; (d) comparing the location of the arrestin within the cell in the presence of the compound to the location of the arrestin within the cell in the absence of the compound; and (e) correlating a difference between (1) the location of the arrestin within the cell in the presence of the compound and (2) the location of the arrestin within the cell in the absence of the compound. The arrestin may be detected in endosomes, endocytic vesicles, or pits.
[0031]A further aspect of the present invention is a kit for identifying a molecule that modulates the activity of a GPCR, including a cell that expresses at least one modified GPCR, wherein the cell further includes a molecule involved in desensitization conjugated to a detectable molecule.
BRIEF DESCRIPTION OF DRAWINGS
[0032]The objects and advantages of the invention will be understood by reading the following detailed description in conjunction with the drawings in which:
[0033]FIG. 1A-1C is a list of GPCRs that may be used with the present invention.
[0034]FIG. 2A-2Q is a list of GRKs that may be used with the present invention. Amino acid and nucleic acid sequences of certain GRKs are shown. The amino acid and nucleic acid sequences of GRK2-C20, a modified GRK, are shown.
[0035]FIG. 3A-3BB is a list of GPCRs that have been modified to have enhanced affinity for arrestin. The amino acid and nucleic acid sequences are shown.
[0036]FIG. 4 illustrates the agonist-independent translocation of arrestin-GFP to GPCRs in the presence of GRK2-C20.
[0037]FIG. 5 illustrates the agonist-independent translocation of arrestin-GFP to GPCRs in the presence of GRK2-C20.
[0038]FIG. 6 illustrates the agonist-independent translocation of arrestin-GFP to GPCRs in the presence of GRK2-C20.
[0039]FIG. 7 illustrates the agonist-independent translocation of arrestin-GFP to GPCRs in the presence of GRK2-C20.
[0040]FIG. 8 demonstrates that losartan, a nonpeptide antagonist/inverse agonist of the AT1AR, inhibits the ligand-independent translocation of arrestinGFP to the AT1AR induced by expression of GRK2-C20. Data plotted are the mean±SD for a representative experiment performed in triplicate.
[0041]FIG. 9 illustrates the percent inhibition of constitutive, GRK2-C20 induced, arrestinGFP translocation to the AT1AR by losartan treatment for 3 hours.
[0042]FIG. 10 illustrates the percent inhibition of constitutive, GRK2-C20 induced, arrestinGFP translocation to the AT1AR by losartan treatment for 18 hours.
DETAILED DESCRIPTION
[0043]In accordance with the present invention there may be employed conventional molecular biology, microbiology, immunology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (3rd edition, 2001); "Current Protocols in Molecular Biology" Volumes I-IV [Ausubel, R. M., ed. (2002 and updated bimonthly)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [J. E. Celis, ed. (1994)]; "Current Protocols in Immunology" Volumes I-IV [Coligan, J. E., ed. (2002 and updated bimonthly)]; "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B. D. Hames & S. J. Higgins eds. (1985)]; "Transcription And Translation" [B. D. Hames & S. J. Higgins, eds. (1984)]; "Culture of Animal Cells, 4th edition" [R. I. Freshney, ed. (2000)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1988); Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1998); Using Antibodies: A Laboratory Manual, Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1999); "G Protein-Coupled Receptors" [T. Haga, et al., eds. (1999)].
[0044]Unless otherwise stated, the following terms used in the specification and claims have the meanings given below:
[0045]A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
[0046]A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
[0047]A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
[0048]An "origin of replication" refers to those DNA sequences that participate in the initiation of DNA synthesis.
[0049]A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
[0050]Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
[0051]The expression of a coding sequence in a host cell may be inducible. By inducible, it is meant that the expression can be regulated. For example, the nucleic acid may be present in the cell, but it is not expressed until a necessary signal is provided. Typically, inducible expression of a protein is controlled by a promoter that requires a necessary signal to induce transcription of the protein. However, expression may also be induced by a process or sequence that increases the number of DNA sequences of interest in the cell. Such processes or sequences include origins of replication, as well as the physical addition of DNA to a cell.
[0052]A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
[0053]An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
[0054]A "signal sequence" can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
[0055]The term "oligonucleotide," as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
[0056]The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
[0057]The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
[0058]As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
[0059]A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.
[0060]Two DNA sequences are "substantially homologous" when at least about 65% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
[0061]It should be appreciated that also within the scope of the present invention are DNA sequences encoding the same amino acid sequence as SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, and 89, but also those which are degenerate to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90. By "degenerate to" is meant that a different three-letter codon is used to specify a particular amino acid.
[0062]"Arrestin" means all types of naturally occurring and engineered variants of arrestin, including, but not limited to, visual arrestin (sometimes referred to as Arrestin 1), cone arrestin (sometimes referred to as arrestin-4), β-arrestin 1 (sometimes referred to as Arrestin 2), and β-arrestin 2 (sometimes referred to as Arrestin 3).
[0063]"βARK1" is a GRK termed β-adrenergic receptor kinase 1, also called GRK2.
[0064]"βAR" is a GPCR termed a β-adrenergic receptor.
[0065]"Internalization" of a GPCR is the translocation of a GPCR from the cell surface membrane to an intracellular vesicular membrane, where it may be inaccessible to substances remaining outside the cell.
[0066]"Carboxyl-terminal tail" means the carboxyl-terminal tail of a GPCR following membrane span 7. The carboxyl-terminal tail of many GPCRs begins shortly after the conserved NPXXY motif that marks the end of the seventh transmembrane domain (i.e. what follows the NPXXY motif is the carboxyl-terminal tail of the GPCR). The carboxyl-terminal tail may be relatively long (approximately tens to hundreds of amino acids), relatively short (approximately tens of amino acids), or virtually non-existent (less than approximately ten amino acids). As used herein, "carboxyl-terminal tail" shall mean all three variants (whether relatively long, relatively short, or virtually non-existent), and may or may not contain palmitoylated cysteine residue(s).
[0067]"Class A receptors" preferably do not translocate together with arrestin proteins to endocytic vesicles or endosomes in association with arrestin-GFP in HEK-293 cells.
[0068]"Class B receptors" preferably do translocate together with arrestin proteins to endocytic vesicles or endosomes associated with arrestin-GFP in HEK-293 cells.
[0069]"DACs" mean any desensitization active compounds. Desensitization active compounds are any compounds that influence the GPCR desensitization mechanism by either stimulating or inhibiting the process. DACs may influence the GPCR desensitization pathway by acting on any cellular component of the process, as well as any cellular structure implicated in the process, including but not limited to: arresting, GRKs, GPCRs, phosphoinositide 3-kinase, AP-2 protein, clathrin, protein phosphatases, and the like. DACs may include, but are not limited to, compounds that inhibit arrestin translocating to a GPCR, compounds that inhibit arrestin binding to a GPCR, compounds that stimulate arrestin translocating to a GPCR, compounds that stimulate arrestin binding to a GPCR, compounds that inhibit GRK phosphorylation of a GPCR, compounds that stimulate GRK phosphorylation of a GPCR, compounds that stimulate or inhibit GRK binding to a GPCR, compounds that inhibit protein phosphatase dephosphorylation of a GPCR, compounds that stimulate protein phosphatase dephosphorylation of a GPCR, compounds that prevent GPCR internalization or recycling to the cell surface, compounds that regulate the release of arrestin from a GPCR, antagonists of a GPCR, inverse agonists and the like. DACs may inhibit or stimulate the GPCR desensitization process and may not bind to the same ligand binding site of the GPCR as traditional agonists and antagonists of the GPCR. DACs may act independently of the GPCR, i.e., they do not have high specificity for one particular GPCR or one particular type of GPCRs. DACs may bind the same site(s) as agonist or antagonist but do not desensitize the receptor (perhaps by not altering the receptor to be properly phosphorylated or bind to arrestin or any other protein). DACs may bind to allosteric sites on the receptor and inhibit or enhance desensitization.
[0070]"Detectable molecule" means any molecule capable of detection by spectroscopic, photochemical, biochemical, immunochemical, electrical, radioactive, and optical means, including but not limited to, fluorescence, phosphorescence, and bioluminescence and radioactive decay. Detectable molecules include, but are not limited to, GFP, luciferase, β-galactosidase, rhodamine-conjugated antibody, and the like. Detectable molecules include radioisotopes, epitope tags, affinity labels, enzymes, fluorescent groups, chemiluminescent groups, and the like. Detectable molecules include molecules which are directly or indirectly detected as a function of their interaction with other molecule(s).
[0071]"GFP" means Green Fluorescent Protein which refers to various naturally occurring forms of GFP which may be isolated from natural sources or genetically engineered, as well as artificially modified GFPs. GFPs are well known in the art. See, for example, U.S. Pat. Nos. 5,625,048; 5,777,079; and 6,066,476. It is well understood in the art that GFP is readily interchangeable with other fluorescent proteins, isolated from natural sources or genetically engineered, including but not limited to, yellow fluorescent proteins (YFP), red fluorescent proteins (RFP), cyan fluorescent proteins (CFP), blue fluorescent proteins, luciferin, UV excitable fluorescent proteins, or any wave-length in between. As used herein, "GFP" shall mean all fluorescent proteins known in the art.
[0072]"Unknown or Orphan Receptor" means a GPCR whose ligands are unknown.
[0073]"Downstream" means toward a carboxyl-terminus of an amino acid sequence, with respect to the amino-terminus.
[0074]"Upstream" means toward an amino-terminus of an amino acid sequence, with respect to the carboxyl-terminus.
[0075]Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site in order to allow formation of disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" residue (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces β-turns in the protein's structure.
[0076]Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.
[0077]A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
[0078]The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduce some feature of pathology such as for example, elevated blood pressure, respiratory output, etc.
[0079]A DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term "operatively linked" includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
[0080]"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine (A) and thymine (T) are complementary nucleobases that pair through the formation of hydrogen bonds.
[0081]The term "standard hybridization conditions" refers to salt and temperature conditions substantially equivalent to 5×SSC and 65° C. for both hybridization and wash. However, one skilled in the art will appreciate that such "standard hybridization conditions" are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of "standard hybridization conditions" is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20° C. below the predicted or determined Tm with washes of higher stringency, if desired.
[0082]By "animal" is meant any member of the animal kingdom including vertebrates (e.g., frogs, salamanders, chickens, or horses) and invertebrates (e.g., worms, etc.). "Animal" is also meant to include "mammals." Preferred mammals include livestock animals (e.g., ungulates, such as cattle, buffalo, horses, sheep, pigs and goats), as well as rodents (e.g., mice, hamsters, rats and guinea pigs), canines, felines, primates, lupine, camelid, cervidae, rodent, avian and ichthyes.
[0083]"Antagonist(s)" include all agents that interfere with wild-type and/or modified GPCR binding to an agonist, wild-type and/or modified GPCR desensitization, wild-type and/or modified GPCR binding arrestin, wild-type and/or modified GPCR endosomal localization, internalization, and the like, including agents that affect the wild-type and/or modified GPCRs as well as agents that affect other proteins involved in wild-type and/or modified GPCR signaling, desensitization, endosomal localization, resensitization, and the like.
[0084]"Modified GPCR" means a GPCR that has one or more modifications in the amino acid sequence of its carboxyl-terminal tail. As such, the carboxyl-terminal tail may be modified in whole or in part. These modifications in the amino acid sequence include mutations of one or more amino acids, insertion of one or more amino acids, deletion of one or more amino acids, and substitutions of one or more amino acids in which one or more amino acids are deleted and one or more amino acids are added in place of the deleted amino acids. Such modified GPCRs are described herein, as well as in U.S. Ser. No. 09/993,844, which is incorporated herein by reference in its entirety.
[0085]"GPCR" means G protein-coupled receptor and includes GPCRs naturally occurring in nature, as well as GPCRs which have been modified.
[0086]"Putative site of palmitoylation" means an expected site of palmitate addition, preferably a cysteine residue. In the GPCRs used in the present invention, the putative site of palmitoylation is preferably 10 to 25, preferably 15 to 20, amino acid residues downstream of the NPXXY motif.
[0087]"Clusters of phosphorylation sites" mean clusters of amino acid residues that may be efficiently phosphorylated and thus readily function as phosphorylation sites. The clusters of amino acids occupy two out of two, two out of three, three out of three positions, three out of four positions, four out of four, four out of five positions, five out of five, and the like consecutive amino acid positions in the carboxyl terminal tail of a GPCR. These clusters of phosphorylation sites are preferably clusters of serine (S) and/or threonine (T) residues. Clusters of phosphorylation sites may be substituted, inserted, or added on to a GPCR sequence so that the resulting modified GPCR binds arrestin with sufficient affinity to recruit arrestin into endosomes.
[0088]"NPXXY motif" means a conserved amino acid motif that marks the end of the seventh transmembrane domain. The conserved amino acid motif begins most frequently with asparagine and proline followed by two unspecified amino acids and then a tyrosine. The two unspecified amino acids may vary among GPCRs but the overall NPXXY motif is conserved.
[0089]"Abnormal GPCR desensitization" and "abnormal desensitization" mean that the GPCR desensitization pathway is disrupted such that the balance between active receptor and desensitized receptor is altered with respect to wild-type conditions. Either there is more active receptor than normal or there is more desensitized receptor than wild-type conditions. Abnormal GPCR desensitization may be the result of a GPCR that is constitutively active or constitutively desensitized, leading to an increase above normal in the signaling of that receptor or a decrease below normal in the signaling of that receptor.
[0090]"Biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject; wherein said sample can be blood, serum, a urine sample, a fecal sample, a tumor sample, a cellular wash, an oral sample, sputum, biological fluid, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture.
[0091]"Concurrent administration," "administration in combination," "simultaneous administration," or "administered simultaneously" mean that the compounds are administered at the same point in time or sufficiently close in time that the results observed are essentially the same as if the two or more compounds were administered at the same point in time.
[0092]"Conserved abnormality" means an abnormality in the GPCR pathway, including but not limited to, abnormalities in GPCRs, GRKs, arresting, AP-2 protein, clathrin, protein phosphatase and the like, that may cause abnormal GPCR signaling. This abnormal GPCR signaling may contribute to a GPCR-related disease.
[0093]"Desensitized GPCR" means a GPCR that presently does not have ability to respond to agonist and activate conventional G protein signaling.
[0094]"Desensitization pathway" means any cellular component of the desensitization process, as well as any cellular structure implicated in the desensitization process and subsequent processes, including but not limited to, arresting, GRKs, GPCRs, AP-2 protein, clathrin, protein phosphatases, and the like. In the methods of assaying of the present invention, the polypeptides may be detected, for example, in the cytoplasm, at a cell membrane, in clathrin-coated pits, in endocytic vesicles, endosomes, any stages in between, and the like.
[0095]"GPCR signaling" means GPCR induced activation of G proteins. This may result in, for example, cAMP production.
[0096]"G protein-coupled receptor kinase" (GRK) includes any kinase that has the ability to phosphorylate a GPCR. Certain GRKs which may be used in the present invention are listed in FIG. 2A-2Q. Splice variants, biologically active fragments, modified GRKs, and GRKs from animals and other organisms are included.
[0097]"Homo sapiens GPCR" means a naturally occurring GPCR in a Homo sapiens.
[0098]"Inverse agonist" means a compound that, upon binding to the GPCR, inhibits the basal intrinsic activity of the GPCR. An inverse agonist is a type of antagonist.
[0099]"Modified GRK" means a GRK modified such that it alters desensitization.
[0100]"Naturally occurring GPCR" means a GPCR that is present in nature.
[0101]"Odorant ligand" means a ligand compound that, upon binding to a receptor, leads to the perception of an odor including a synthetic compound and/or recombinantly produced compound including agonist and antagonist molecules.
[0102]"Odorant receptor" means a receptor protein normally found on the surface of olfactory neurons which, when activated (normally by binding an odorant ligand) leads to the perception of an odor.
[0103]The term "pharmaceutically acceptable carrier," as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.
[0104]"Sensitized GPCR" means a GPCR that presently has ability to respond to agonist and activate conventional G protein signaling.
[0105]"Modulation" includes at least an up-regulation or down-regulation of the expression, or an increase or decrease in activity of a protein. Modulation of a protein includes the up-regulation, down-regulation, increase or decrease in activity of a protein or compound that regulates a protein. Modulation also includes the regulation of the gene, the mRNA, or any other step in the synthesis of the protein of interest.
[0106]An "overexpressed" protein refers to a protein that is expressed at levels greater than wild-type expression levels.
[0107]"Modified GRK" means a GRK that has one or more modifications in the amino acid sequence at the C-terminus of the GRK. The modified GRK constitutively localizes to the plasma membrane. Preferably, the GRK is modified by the addition of a CAAX motif.
[0108]"CAAX" (SEQ ID NO: 95) motif means a four amino acid sequence, wherein C is cysteine; A is an aliphatic amino acid; and X is the C-terminal amino acid of the protein.
[0109]A "constitutive" activity means an activity that occurs in the absence of agonist. For example, the modified GRK constitutively localizes to the plasma membrane means that the modified GRK localizes to the plasma membrane in the absence of agonist.
[0110]"GRK-C20" refers to a modified GRK which has the ability to have a geranylgeranyl group added to it. GRK2-C20 is a GRK2 modified in this manner. Preferably, the GRK-C20 contains a CAAX motif.
[0111]The present inventors developed an agonist-independent method to screen for compounds that alter GPCR desensitization. They developed cell lines in which GPCRs are desensitized in the absence of agonist. These cell lines include GRKs, which may be modified. Using these cell lines, they developed methods to screen for compounds that alter GPCR desensitization in the absence of agonist. These methods eliminate the step of agonist addition from the screening method. The elimination of this step (1) creates more efficient screening methods for compounds that alter desensitization of GPCRs with known agonists, and (2) provides screening methods for compounds that alter desensitization of orphan GPCRs, which have no known agonist. They developed methods to determine if a GPCR is expressed at the plasma membrane, and determine if the GPCR has an affinity for arrestin; preferably these methods utilize an orphan GPCR and host cells containing a GRK, wherein the GPCR is at least partially internalized in an agonist-independent manner upon expression of the GRK, thus eliminating the need for agonist addition. They modified GPCRs to increase their affinities for arrestin. These modified GPCRs are useful in the agonist-independent methods to screen for compounds that alter desensitization.
[0112]GPCRs and Desensitization
[0113]The exposure of a GPCR to agonist produces rapid attenuation of its signaling ability that involves uncoupling of the receptor from its cognate heterotrimeric G-protein. The cellular mechanism mediating agonist-specific or homologous desensitization is a two-step process in which agonist-occupied receptors are phosphorylated by a G protein-coupled receptor kinases (GRKs) and then bind an arrestin protein.
[0114]It is known that after agonists bind GPCRs, G-protein coupled receptor kinases (GRKs) phosphorylate intracellular domains of GPCRs. After phosphorylation, an arrestin protein associates with the GRK-phosphorylated receptor and uncouples the receptor from its cognate G protein. The interaction of the arrestin with the phosphorylated GPCR terminates GPCR signaling and produces a non-signaling, desensitized receptor.
[0115]The arrestin bound to the desensitized GPCR targets the GPCR to clathrin-coated pits or other cellular machinery for endocytosis (i.e., internalization) by functioning as an adaptor protein, which links the GPCR to components of the endocytic machinery, such as adaptor protein-2 (AP-2) and clathrin. The internalized GPCRs are dephosphorylated and are recycled back to the cell surface resensitized, or are retained within the cell and degraded. The stability of the interaction of arrestin with the GPCR is one factor that dictates the rate of GPCR dephosphorylation, recycling, and resensitization. The involvement of GPCR phosphorylation and dephosphorylation in the desensitization process has been exemplified in U.S. Ser. No. 09/933,844, filed Nov. 5, 2001, the disclosure of which is hereby incorporated by reference in its entirety.
[0116]Using methods described herein, the present inventors identified certain GPCRs which do not have an affinity for arrestin. They modified these GPCRs to comprise one or more sites of phosphorylation, preferably clusters of phosphorylation sites, properly positioned in their carboxyl-terminal tail. This modification allows the modified GPCR to form a stable complex with an arrestin that will internalize as a unit into endosomes. These modified GPCRs may be useful in methods of assaying GPCR activity. These modified GPCRs may be useful to identify agonists of the GPCRs. These modified GPCRs may be useful in the agonist-independent screening methods described herein.
[0117]Agonist-independent screening methods using GPCRs altered to contain a DRY motif are described in U.S. Ser. No. 10/054,616, which is incorporated herein by reference in its entirety. The alteration of the GPCR is included in that screening process; each GPCR to be utilized must be altered in that manner.
[0118]The present inventors developed agonist-independent screening methods using GRKs, which may be modified. These GRKs phosphorylate GPCRs in the absence of agonist. These phosphorylated GPCRs internalize in the absence of agonist. The present inventors developed agonist-independent methods of screening for antagonists of GPCR internalization utilizing these modified GRKs. These methods do not require the GPCR alterations described in U.S. Ser. No. 10/054,616.
[0119]Previously, certain GRKs were shown to constitutively localize in the plasma membrane. Inglese et al constructed GRK2-C20 which was constitutively isoprenylated and localized to the membranes.
[0120]The present inventors determined that cellular expression of GRKs that constitutively localize in the plasma membrane results in constitutive desensitization of GPCRs. These GRKs may be over-expressed, their expression may be inducible, the nucleic acids encoding them may be located in a vector or integrated into the genome. The present inventors constructed host cell expressing a GRK that constitutively localizes in the plasma membrane. These host cells may also express arrestin. To these host cells, they introduced a GPCR of interest. Using the GRK-containing cells, they developed methods to determine if a GPCR of interest is expressed at the plasma membrane, analyze the ability of a GPCR to bind arrestin, and detect constitutively desensitized GPCRs. They built upon these desensitization methods and developed agonist-independent methods of identifying compounds that alter GPCR desensitization. These methods are useful for the identification of compounds that alter the internalization of GPCRs, whether the GPCR agonist is known or unknown.
[0121]The present inventors also determined that increased expression of wild-type or modified GRKs increased desensitization, irrespective of whether the GRK constitutively localized in the plasma membrane.
[0122]The present invention is related to modified GPCRs, polypeptides of modified GPCRs, nucleic acid molecules that encode the modified GPCRs, vectors containing the nucleic acid molecules which encode the modified GPCRs, vectors enabling the nucleic acid construction of the modified GPCRs, and cells containing modified GPCRs. The invention further relates to assay systems using the modified GPCRs, assay systems using the cells containing modified GPCRs, compounds identified using the assay systems, methods of treatment using the compounds identified, methods of disease diagnosis using the assay systems, and kits containing assay reagents of the present invention and cells of the present invention.
[0123]Mutations can be made in the GPCR or modified GPCR such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.
[0124]In a particular embodiment, the modified GPCRs of the present invention include GPCRs that have been modified to have one or more sites of phosphorylation, preferably clusters of phosphorylation sites, properly positioned in its carboxyl-terminal tail. These modified GPCRs recruit arrestin to endosomes within approximately 30 minutes of agonist stimulation. These modified GPCRs recruit arrestin to endosomes in the cells described herein, in which the GPCR is phosphorylated in an agonist-independent manner.
[0125]The modified GPCRs of the present invention comprise one or more sites of phosphorylation, preferably one or more clusters of phosphorylation sites, properly positioned in its carboxyl-terminal tail. The present inventors have discovered that GPCRs containing one or more sites of phosphorylation, preferably clusters of phosphorylation sites, properly positioned in its carboxyl-terminal tail have an increased affinity for arrestin and colocalize with arrestin in endosomes upon GPCR phosphorylation, either after stimulation with agonist or in an agonist-independent manner as described herein. The present inventors have also discovered that the one or more sites of phosphorylation, preferably clusters of phosphorylation sites, must be optimally positioned within the GPCR tail for the GPCR to have an increased affinity for arrestin. Therefore, the modified GPCRs may be constructed such that the one or more sites of phosphorylation, preferably clusters of phosphorylation sites, are optimally positioned within the carboxyl-terminal tail. The portions of polypeptides, which are to be fused together to form the modified GPCR, are chosen such that the one or more sites of phosphorylation, preferably clusters of phosphorylation sites, are reliably positioned properly within the carboxyl-terminal tail. In the alternative, the location of discrete point mutations to create the modified GPCR may be chosen so that the one or more sites of phosphorylation, preferably clusters of phosphorylation sites, are properly positioned within the carboxyl-terminal tail.
[0126]The present inventors have discovered that the modified GPCRs of the present invention are useful in assays for screening compounds that may alter G protein-coupled receptor (GPCR) activity. Examples of assays in which the present invention may be used include, but are not limited to, those as described in U.S. Pat. Nos. 5,891,646 and 6,110,693, the disclosures of which are hereby incorporated by reference in their entireties. Additional examples of assays in which the present invention may be used include, but are not limited to, assays using Fluorescent Resonance Energy Transfer (FRET) and assays using Bioluminescence Resonance Energy Transfer (BRET) technology as described in Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, "β2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET)," Proc. Natl, Acad. Sci. USA 97, 7: 3684-3689.
[0127]The present inventors have determined that these modified GPCRs are useful in agonist-independent assays for screening compounds that may alter GPCR internalization. Examples of assays in which the present invention may be used include, but are not limited to, assays described herein.
Methods of Enhancing GPCR Desensitization
[0128]Provided in the present invention are methods of enhancing GPCR desensitization. One embodiment is related to the expression of GRKs, which may be modified. The GRKs may be over-expressed or their expression may be inducible. These methods may be used to analyze the desensitization of a GPCR, including a modified GPCR, an orphan GPCR, a taste receptor, a mutant GPCR, the β2AR Y326A GPCR mutant, or another GPCR. Certain GPCRs useful in the present invention are listed in FIG. 1A-1C.
[0129]In a preferred embodiment, a cell is provided that contains an expression system and a nucleic acid encoding a GRK. The GRK may be modified such that the expression of the GRK results in constitutive desensitization of the GPCR. The GRK may be over-expressed and its expression may be inducible.
[0130]Preferably, host cells are provided which include a GRK, which may be modified, and arrestin. A GPCR is then added to these cells. The agonist-independent desensitization of the GPCR is detected. FIGS. 4, 5, 6, and 7 are examples of this method. Detection methods are described below.
[0131]The present invention provides methods of determining if the GPCR of interest is expressed at the plasma membrane. GPCRs expressed at the plasma membrane are useful in the previously mentioned methods of compound identification.
[0132]A preferred method of determining if a GPCR of interest is expressed at the plasma membrane includes: (a) providing a cell including a GPCR, an arrestin, and a GRK, wherein the arrestin is detectably labeled; (b) determining the cellular distribution of the arrestin; and (c) correlating the cellular distribution of the arrestin to the ability of the GPCR to be expressed at the plasma membrane.
[0133]Preferred embodiments of this aspect of the invention are described in Examples 2, 3, 4, 5, 6, and 7 and illustrated in FIGS. 4, 5, 6, and 7.
[0134]Another method of determining if a GPCR of interest is expressed at the plasma membrane includes: (a) providing a cell comprising a GPCR and a GRK, wherein the GRK is detectably labeled; (b) determining the cellular distribution of the GRK; and (c) correlating the cellular distribution of the GRK to the ability of the GPCR to be expressed at the plasma membrane.
[0135]The present invention provides methods of analyzing the ability of a GPCR to bind arrestin. GPCRs which bind arrestin are useful in the previously mentioned methods of compound identification.
[0136]A preferred method of analyzing the ability of a GPCR to bind arrestin includes: (a) providing a cell including a GPCR, an arrestin, and a GRK, wherein the arrestin is detectably labeled; (b) determining the cellular distribution of the arrestin; and (c) correlating the cellular distribution of the arrestin to the ability of the GPCR to bind arrestin.
[0137]Preferred embodiments of this aspect of the invention are described in Examples 2, 3, 4, 5, 6, and 7, and illustrated in FIGS. 4, 5, 6, and 7.
[0138]Using this method, certain GPCRs will bind arrestin and desensitize. However, certain GPCRs will not desensitize without modification of the GPCR, as described in U.S. Ser. No. 09/993,844. The present inventors modified several GPCRs, including known and orphan GPCRs, listed in FIG. 3A-3BB. Upon modification, these modified GPCRs constitutively desensitized in the above system.
Modified GPCRs
[0139]The present invention is related to modified GPCRs. Modified GPCRs of the present invention may comprise one or more modifications in their carboxyl-terminal tail. These modifications may comprise inserting one or more sites of phosphorylation, preferably clusters of phosphorylation sites, within certain regions of the carboxyl-terminal tail. As such, the carboxyl-terminal tail may be modified in whole or in part. The carboxyl-terminal tail of many GPCRs begins shortly after a conserved NPXXY motif that marks the end of the seventh transmembrane domain (i.e. what follows the NPXXY motif is the carboxyl-terminal tail of the GPCR). The carboxyl-terminal tail of many GPCRs comprises a putative site of palmitoylation approximately 10 to 25 amino acid residues, preferably 15 to 20 amino acid residues, downstream of the NPXXY motif. This site is typically one or more cysteine residues. The carboxyl-terminal tail of a GPCR may be relatively long, relatively short, or virtually non-existent. The present inventors have determined that the carboxyl-terminal tail of a GPCR determines the affinity of arrestin binding.
[0140]The present inventors have discovered that specific amino acid motifs in the carboxyl-terminal tail promote formation of a stable GPCR/arrestin complex and thus ultimately may promote recruitment of arrestin to endosomes. These amino acid motifs comprise one or more amino acids, preferably clusters of amino acid residues, that may be efficiently phosphorylated and thus readily function as phosphorylation sites. The clusters of amino acids may occupy two out of two, two out of three, three out of three, three out of four positions, four out of four, four out of five positions, five out of five, and the like consecutive amino acid positions. Accordingly, the clusters of amino acids that promote formation of a stable GPCR/arrestin complex are "clusters of phosphorylation sites." These clusters of phosphorylation sites are preferably clusters of serine and threonine residues.
[0141]GPCRs that form stable complexes with arrestin comprise one or more sites of phosphorylation, preferably clusters of phosphorylation sites. In addition to the presence of the one or more sites of phosphorylation, preferably clusters of phosphorylation sites, it has been discovered that the sites must be properly positioned within the carboxyl-terminal tail to promote formation of a stable GPCR/arrestin complex. To promote formation of a stable GPCR/arrestin complex, the one or more sites of phosphorylation, preferably one or more clusters of phosphorylation, may be approximately 15 to 35 (preferably 15 to 25) amino acid residues downstream of a putative site of palmitoylation of the GPCR. In addition, the one or more sites of phosphorylation, preferably one or more clusters of phosphorylation, may be approximately 20 to 55 (preferably 30 to 45) amino acid residues downstream of the NPXXY motif of the GPCR. GPCRs containing one or more sites of phosphorylation, preferably clusters of phosphorylation sites, properly positioned are typically Class B receptors.
[0142]By way of example, it has been discovered that the V2R receptor comprises a cluster of phosphorylation sites (SSS) that promotes formation of a stable GPCR/arrestin complex at 19 amino acid residues downstream of the putative site of palmitoylation and 36 amino acid residues downstream of the NPXXY motif. The NTR-2 receptor comprises a cluster of phosphorylation sites (STS) that promotes formation of a stable GPCR/arrestin complex at 26 amino acid residues downstream of the putative site of palmitoylation and 45 amino acid residues downstream of the NPXXY motif. The oxytocin receptor (OTR) receptor comprises two clusters of phosphorylation sites (SSLST and STLS) that promote formation of a stable GPCR/arrestin complex, one at 20 amino acid residues downstream of the putative site of palmitoylation and the other at 29 amino acid residues downstream of the putative site of palmitoylation, and one at 38 amino acid residues downstream of the NPXXY motif and the other at 47 amino acid residues downstream of the NPXXY motif, respectively. The substance P receptor (SPR, also known as the neurokinin-1 receptor) comprises a cluster of phosphorylation sites (TTIST) that promotes formation of a stable GPCR/arrestin complex at 32 amino acid residues downstream of the putative site of palmitoylation and 50 amino acid residues downstream of the NPXXY motif.
[0143]The present inventors have determined that GPCRs that lack one or more sites of phosphorylation, preferably clusters of phosphorylation, properly positioned within the carboxyl terminal tail form GPCR/arrestin complexes that are less stable and dissociate at or near the plasma membrane. These GPCRs are typically Class A receptors, olfactory receptors, taste receptors, and the like. However, the present inventors have discovered that stable GPCR/arrestin complexes may be achieved with GPCRs naturally lacking one or more sites of phosphorylation and having a lower affinity for arrestin by modifying the carboxyl-terminal tails of these receptors. Preferably, the carboxyl-terminal tails are modified to include one or more sites of phosphorylation, preferably one or more clusters of phosphorylation sites, properly positioned within the carboxyl terminal tail.
[0144]The present invention includes the polypeptide sequences of these modified GPCRs. The modified GPCRs of the present invention include GPCRs that have been modified to have one or more sites of phosphorylation, preferably one or more clusters of phosphorylation, properly positioned in their carboxyl terminal tails. The polypeptide sequences of the modified GPCRs of the present invention also include sequences having one or more additions, deletions, substitutions, or mutations. These mutations are preferably substitution mutations made in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. The present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.
[0145]The modified GPCRs of the present invention include GPCRs containing a NPXXY motif, a putative site of palmitoylation approximately 10 to 25 amino acid residues (preferably 15 to 20 amino acids) downstream of the NPXXY motif, and a modified carboxyl-terminal tail. The modified carboxyl-terminal tail has one or more sites of phosphorylation, preferably one or more clusters of phosphorylation sites, such that the phosphorylation sites are approximately 15 to 35, preferably 15 to 25, amino acid residues downstream of the putative site of palmitoylation of the modified GPCR. The modified carboxyl-terminal tail may have one or more sites of phosphorylation, preferably one or more clusters of phosphorylation sites, such that the phosphorylation sites are approximately 20 to 55, preferably 30 to 45, amino acid residues downstream of the NPXXY of the modified GPCR.
[0146]The present invention further includes isolated nucleic acid molecules that encode modified GPCRs. It should be appreciated that also within the scope of the present invention are DNA sequences encoding modified GPCRs which code for a modified GPCR having the same amino acid sequence as the modified GPCRs, but which are degenerate. By "degenerate to" it is meant that a different three-letter codon is used to specify a particular amino acid.
[0147]As one of skill in the art would readily understand, the carboxyl-tail of many GPCRs may be identified by the conserved NPXXY motif that marks the end of the seventh transmembrane domain.
[0148]To create a modified GPCR containing a modified carboxyl-terminus region according to the present invention, a GPCR lacking phosphorylation sites or clusters of phosphorylation sites or with a lower or unknown affinity for arrestin may have one or more additions, substitutions, deletions, or mutations of amino acid residues in its carboxyl-terminal tail. These additions, substitutions, deletions, or mutations are performed such that the carboxyl-terminal tail is modified to comprise one or more sites of phosphorylation, preferably clusters of phosphorylation sites. By way of example, discrete point mutations of the amino acid residues may be made to provide a modified GPCR. By way of example three consecutive amino acids may be mutated to serine residues to provide a modified GPCR. These mutations are made such that the one or more sites of phosphorylation, preferably clusters of phosphorylation sites, are properly positioned within the carboxyl terminal tail.
[0149]In addition, to create a modified GPCR containing a modified carboxyl-terminal tail region, mutations may be made in a nucleic acid sequence of a GPCR lacking sites of phosphorylation or clusters of phosphorylation sites or with a lower or unknown affinity for arrestin such that a particular codon is changed to a codon which codes for a different amino acid, preferably a serine or threonine. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein to create one or more sites of phosphorylation, preferably clusters of phosphorylation sites. Also by way of example, discrete point mutations of the nucleic acid sequence may be made. The phosphorylation sites are positioned such that they are located approximately 15 to 35 amino acid residues downstream of the putative site of palmitoylation of the modified GPCR.
[0150]Furthermore, to provide modified GPCRs of the present invention, a GPCR lacking properly positioned phosphorylation sites or with a lower or unknown affinity for arrestin may also have its carboxyl-terminal tail, in whole or in part, exchanged with that of a GPCR having properly positioned clusters of phosphorylation sites. The site of exchange may be after or including the conserved NPXXY motif. As an alternative, a putative site of palmitoylation of a GPCR may be identified at approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of the conserved NPXXY motif, and the site of exchange may be after or including the palmitoylated cysteine(s). Preferably, the carboxyl-terminal tail of a GPCR lacking properly positioned clusters of phosphorylation sites or with a lower or unknown affinity for arrestin is exchanged at an amino acid residue in close proximity to a putative site a palmitoylation. More preferably, the carboxyl-terminal tail of a GPCR lacking properly positioned clusters of phosphorylation sites or with a lower or unknown affinity for arrestin is exchanged at a putative site of palmitoylation approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of the NPXXY motif, such that the palmitoylated cysteine residue is maintained. Exchanging in the preferred manner allows the clusters of phosphorylation sites to be reliably positioned properly within the carboxyl-terminal tail of the modified GPCR. The tails may be exchanged and the modified GPCRs may be constructed accordingly by manipulation of the nucleic acid sequence or the corresponding amino acid sequence.
[0151]In a further alternative, the carboxyl-tail of a GPCR, for example a GPCR not containing the NPXXY motif, may be predicted from a hydrophobicity plot and the site of exchange may be selected accordingly. Based on a hydrophobicity plot, one of skill in the art may predict a site where it is expected that the GPCR may anchor in the membrane and then predict where to introduce a putative site of palmitoylation accordingly. Using this technique GPCRs having neither a NPXXY motif nor a putative site of palmitoylation may be modified to create a point of reference (e.g. a putative site of palmitoylation). The introduced putative site of palmitoylation may then be used to position a tail exchange.
[0152]The carboxyl-terminal tail used for the exchange may be from a second GPCR having one or more properly positioned clusters of phosphorylation sites and having a putative site of palmitoylation approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of a NPXXY motif. The tail as identified may be exchanged, after or including the conserved NPXXY motif. As an alternative, a putative site of palmitoylation of a GPCR may be identified at approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of the conserved NPXXY motif, and the tail may be exchanged after or including the palmitoylated cysteine(s). Preferably, the carboxyl-terminal tail of a GPCR having clusters of phosphorylation sites is exchanged at an amino acid residue in close proximity to a putative site of palmitoylation. More preferably, the carboxyl-terminal tail of a GPCR having clusters of phosphorylation sites is exchanged at a putative site of palmitoylation approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of the NPXXY motif, such that the portion of the carboxyl-terminal tail containing the clusters of phosphorylation sites begins at the amino acid residue immediately downstream of the palmitoylated cysteine residue. Exchanging in the preferred manner allows the clusters of phosphorylation sites to be reliably positioned properly within the carboxyl-terminal tail of the modified GPCR. The carboxyl-terminal tail having clusters of phosphorylation sites used for the exchange may have a detectable molecule conjugated to the carboxyl-terminus. The tails may be exchanged and the modified GPCRs may be constructed accordingly by manipulation of the nucleic acid sequence or the corresponding amino acid sequence.
[0153]In addition, the carboxyl-terminal tail portion used for the exchange may originate from a polypeptide synthesized to have an amino acid sequence corresponding to an amino acid sequence from a GPCR having one or more sites of phosphorylation, preferably one or more clusters of phosphorylation sites. The synthesized polypeptide may have a putative site of palmitoylation approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of a NPXXY motif. The synthesized polypeptide may have one or more additions, substitutions, mutations, or deletions of amino acid residues that does not affect or alter the overall structure and function of the polypeptide.
[0154]Furthermore, the carboxyl-terminal tail portion used for the exchange may originate from a naturally occurring polypeptide recognized to have an amino acid sequence corresponding to an amino acid sequence from a GPCR having one or more clusters of phosphorylation sites. The polypeptide may have a putative site of palmitoylation approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of a NPXXY motif. The polypeptide may have one or more additions, substitutions, mutations, or deletions of amino acid residues that does not affect or alter the overall structure and function of the polypeptide.
[0155]A modified GPCR containing a modified carboxyl-terminus region may be created by fusing a first carboxyl-terminal tail portion of a GPCR lacking properly positioned clusters of phosphorylation sites or with a lower or unknown affinity for arrestin with a second carboxyl-terminal tail portion of a GPCR or polypeptide having one or more clusters of phosphorylation sites. The second GPCR or polypeptide used for the exchange may have a putative site of palmitoylation approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of a NPXXY motif. Accordingly, the modified carboxyl-terminus region of the modified GPCR comprises a portion of a carboxyl-terminal tail from a GPCR lacking properly positioned clusters of phosphorylation sites or with a lower or unknown affinity for arrestin fused to a portion of a carboxyl-terminal tail of a GPCR or polypeptide having clusters of phosphorylation sites. The tail of a GPCR lacking properly positioned clusters of phosphorylation sites may be exchanged after or including the conserved NPXXY motif, and fused to a carboxyl-terminal tail containing clusters of phosphorylation sites, after or including the conserved NPXXY motif. As an alternative, the tail of a GPCR lacking properly positioned clusters of phosphorylation sites may be exchanged after or including the palmitoylated cysteine(s), and fused to a tail containing clusters of phosphorylation sites, after or including the palmitoylated cysteine(s). The tails may be exchanged and the modified GPCRs may be constructed accordingly by manipulation of the nucleic acid sequence or the corresponding amino acid sequence.
[0156]In a further alternative, the carboxyl-tail of a GPCR, for example a GPCR not containing the NPXXY motif, may be predicted from a hydrophobicity plot and exchanged accordingly. The site of exchange may be selected according to the hydrophobicity plot. Based on a hydrophobicity plot, one of skill in the art may predict a site where it is expected that the GPCR may anchor in the membrane and then predict where to introduce a putative site of palmitoylation accordingly. Using this technique GPCRs having neither a NPXXY motif nor a putative site of palmitoylation may be modified to create a point of reference (e.g. a putative site of palmitoylation). The introduced putative site of palmitoylation may be then used to position a tail exchange. After introduction of a putative site of palmitoylation, the resulting tail may be fused with a second carboxyl-terminal tail portion of a GPCR or polypeptide having one or more clusters of phosphorylation sites and having a putative site of palmitoylation approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of a NPXXY motif.
[0157]Preferably, the modified carboxyl-terminus region of the modified GPCR is fused at amino acid residues in close proximity to a putative site of palmitoylation. More preferably, the modified carboxyl-terminus region of the modified GPCR is fused such that the portion from the first GPCR with a lower affinity for arrestin comprises amino acid residues from the NPXXY motif through a putative site of palmitoylation approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of the NPXXY motif and the portion from the second GPCR having clusters of phosphorylation sites and a putative site of palmitoylation approximately 10 to 25 (preferably 15 to 20) amino acid residues downstream of a NPXXY motif comprises amino acid residues beginning with an amino acid residue immediately downstream of the putative site of palmitoylation of the second GPCR extending to the end of the carboxyl-terminus. This fusion is preferred because the clusters of phosphorylation sites are reliably positioned properly within the carboxyl-terminal tail and the modified GPCR maintains its structure and ability to function.
[0158]By way of example, a Class A receptor or an orphan receptor may have a portion of its carboxyl-terminal tail exchanged with a portion of a carboxyl-terminal tail from a known Class B receptor. Further, receptors having virtually non-existent carboxyl-terminal tails, for example, olfactory receptors and taste receptors, may have a portion of their carboxyl-terminal tails exchanged with a portion of a carboxyl-terminal tail from a known Class B receptor. The Class B receptor tail used for these exchanges may have a detectable molecule fused to the carboxyl-terminus.
[0159]Modified GPCRs may be generated by molecular biological techniques standard in the genetic engineering art, including but not limited to, polymerase chain reaction (PCR), restriction enzymes, expression vectors, plasmids, and the like. By way of example, vectors, such as a pEArrB (enhanced arrestin binding), may be designed to enhance the affinity of a GPCR lacking clusters of phosphorylation sites for arrestin. To form a vector, such as a pEArrB vector, PCR amplified DNA fragments of a GPCR carboxyl-terminus, which forms stable complexes with arrestin, may be digested by appropriate restriction enzymes and cloned into a plasmid. A schematic of one such plasmid is illustrated in FIG. 4A. The DNA of a GPCR, which is to be modified, may also be PCR amplified, digested by restriction enzymes at an appropriate location, and subcloned into the vector, such as pEArrB, as illustrated in FIG. 4B. When expressed, the modified GPCR will contain a polypeptide fused to the carboxyl-terminus. The polypeptide will comprise clusters of phosphorylation sites. Preferably, the polypeptide originates from the GPCR carboxyl-terminus of a receptor that forms stable complexes with arrestin.
[0160]Such modified GPCRs may also occur naturally as the result of aberrant gene splicing or single nucleotide polymorphisms. Such naturally occurring modified GPCRs would be predicted to have modified endocytic targeting. These naturally occurring modified GPCRs may be implicated in a number of GPCR-related disease states.
[0161]As shown in FIG. 3A-3BB, the present inventors modified several GPCRs. The β2-adrenergic receptor, dopamine D1A receptor, mu opiod receptor, orphan GPR3, orphan GPR6, orphan GPR12, orphan GPR7, orphan GPR8, orphan GPR55, orphan SREB2, and orphan SREB3 were modified as described herein. These modified GPCRs contain a properly positioned V2R cluster of phosphorylation sites (SSS) within the modified GPCR's tail.
[0162]As may be shown by standard receptor binding assays, the modified receptors are essentially indistinguishable from their wild-type counterparts except for an increased affinity for arrestin and thus an increased stability of their complex with arrestin and in their ability to traffic with arrestin and in their ability to recycle and resensitize. For example, the modified receptors are appropriately expressed at the membrane and possess similar affinity for agonists or ligands. However, the modified GPCRs have an increased affinity for arrestin and thus form a more stable complex with arrestin than their wild-type counterparts and may remain bound to arrestin when trafficking to endosomes.
[0163]These modified GPCRs are useful in assays to screen for an agonist of the GPCR, as well as in agonist-independent assays to identify compounds that alter GPCR desensitization.
Methods of Assaying GPCR Activity Using the Modified GPCRs
[0164]The modified GPCRs of the present invention are useful in methods of assaying GPCR activity. The modified GPCRs of the present invention may be used in assays to study GPCRs that have weaker than desired interactions or associations with arrestins and GPCRs that have unknown interactions or associations with arresting. Methods of the present invention that use the modified GPCRs provide a sensitive assay and may provide for enhanced detection, for example, of arrestin/GPCRs in endosomes. The assays using the modified GPCRs of the present invention may be useful for screening compounds and sample solutions for ligands, agonists, antagonists, inverse agonists, desensitization active compounds, and the like. Once identified, these compounds may be useful as drugs capable of modulating GPCR activity and useful in the treatment of one or more of the disease states in which GPCRs have been implicated.
[0165]In a preferred assay according to the present invention, cells are provided that express modified GPCRs of the present invention and these cells may further contain a conjugate of an arrestin and a detectable molecule.
[0166]Arrestin coupled to a detectable molecule may be detected and monitored as it functions in the GPCR pathway. The location of the arrestin may be detected, for example, evenly distributed in the cell cytoplasm, concentrated at a cell membrane, concentrated in clathrin-coated pits, localized on endosomes, and the like. In response to agonist stimulation, the proximity of arrestin to a GPCR may be monitored, as well as the proximity to any other cell structure. For example, in response to agonist stimulation arrestin may be detected in proximity to GPCRs at a cell membrane, concentrated with GPCRs in clathrin-coated pits, colocalized with a GPCR on endosomes, and the like.
[0167]The modified GPCRs of the present invention have an increased affinity for arrestin and provide a stable complex of the GPCR with arrestin, and thereby promote colocalization of the GPCR with arrestin into endosomes. In the methods of assaying of the present invention, arrestin may be detected, for example, in the cytoplasm, concentrated in proximity to GPCRs at a cell membrane, concentrated in proximity to GPCRs in clathrin-coated pits, colocalized with a GPCR on endosomes, and the like. Preferably the arrestin may be detected colocalized with a GPCR on endosomes.
[0168]The association of arrestin with a GPCR at a cell membrane may be rapidly detected after agonist addition, for example, approximately 1 second to 2 minutes. The colocalization of arrestin with GPCR on endosomes may be detected within several minutes of agonist addition, for example, approximately 3 to 15 minutes, and may persist for extended periods of time, for example, after 1 hour. The association of arrestin with GPCR on endosomes may give a strong, readily recognizable signal. Under magnification of 40× objective lens, the signal may be doughnut-like in appearance. The signal resulting from the compartmentalization of arrestin and GPCR colocalized in endosomes vesicles is typically easy to detect and may persist for extended periods of time.
[0169]A preferred method of assessing GPCR pathway activity of the present invention comprises (a) providing a cell that expresses at least one modified GPCR of the present invention and that further comprises a conjugate of an arrestin and a detectable molecule; (b) inducing translocation of the arrestin; and (c) detecting interaction of the arrestin with the modified GPCR along the translocation pathway.
[0170]Interaction of the arrestin with the modified GPCR may be detected, for example, in endosomes, in clathrin-coated pits, concentrated in proximity to a cell membrane, and the like. Preferably, interaction of the arrestin with the modified GPCR is detected in endosomes. Interaction of arrestin with a GPCR in endosomes may be detected within several minutes of agonist addition, for example, approximately 3 to 15 minutes, and may persist for extended periods of time, for example, after 1 hour. The association of arrestin with a GPCR in endosomes may give a strong, readily recognizable signal that persists for extended periods of time.
[0171]In a method of screening compounds for GPCR activity of the present invention a cell that expresses at least one modified GPCR is provided. The cell further contains arrestin conjugated to a detectable molecule. The cell is exposed to the compounds to be tested. The location of the arrestin within the cell is detected. The location of the arrestin within the cell in the presence of the compound is compared to the location of the arrestin within the cell in the absence of the compound, and a difference is correlated between (1) the location of the arrestin within the cell in the presence of the compound and (2) the location of the arrestin within the cell in the absence of the compound.
[0172]By way of example, compounds and sample solutions may be screened for GPCR agonist activity using the modified GPCRs of the present invention. In this method, cells that express at least one modified GPCR of the present invention and that further comprise a conjugate of an arrestin and a detectable molecule are provided. The cells are exposed to compounds or sample solutions to be tested. It is detected whether interaction of the arrestin with the modified GPCR is increased after exposure to the test compound or solution, an increase in interaction being an indication that the compound or solution has GPCR agonist activity. Interaction of the arrestin with the GPCR may be detected in endosomes, in clathrin-coated pits, in proximity to a cell membrane, and the like. The modified GPCR may also be conjugated to a detectable molecule, preferably at the carboxyl-terminus. As explained above modifications to GPCRs as in the present invention should not affect the GPCRs' natural affinity for agonists or ligands.
[0173]Also by way of example, compounds and sample solutions may be screened for GPCR antagonist or inverse agonist activity using the modified GPCRs of the present invention. Cells that express at least one modified GPCR of the present invention and that further comprise a conjugate of an arrestin and a detectable molecule are provided. The cells are exposed to compounds or sample solutions to be tested and to a known agonist for the GPCR. It is detected whether interaction of the arrestin with the modified GPCR is decreased after exposure to the test compound or solution, a decrease in interaction being an indication that the compound or solution has GPCR antagonist or inverse agonist activity. Interaction of the arrestin with the GPCR may be detected in endosomes, in clathrin-coated pits, in proximity to a cell membrane, and the like. The modified GPCR may also be conjugated to a detectable molecule, preferably at the carboxyl-terminus. As explained above modifications to GPCRs as in the present invention should not affect the GPCRs' natural affinity for antagonists or inverse agonists.
[0174]Further by way of example, compounds and sample solutions may be screened for GPCR desensitization activity using the modified GPCRs of the present invention. First cells that express at least one first modified GPCR of the present invention and that further comprise a conjugate of an arrestin and a detectable molecule are provided. The first cells are exposed to compounds or sample solutions to be tested and to a known agonist for the first GPCR. It is detected whether interaction of the arrestin with the first modified GPCR is decreased or not increased after exposure to the test compound or solution, a decrease or lack of increase in interaction being an indication that the compound or solution has GPCR desensitization activity. Interaction of the arrestin with the GPCR may be detected in endosomes, in clathrin-coated pits, in proximity to a cell membrane, and the like. Then second cells that express at least one second modified GPCR of the present invention and that further comprise a conjugate of an arrestin and a detectable molecule are provided. The second modified GPCR is not related to the first modified GPCR. The second cells are exposed to the compounds or sample solutions to be tested and to a known agonist for the second GPCR. It is detected whether interaction of the arrestin with the second modified GPCR is decreased or not increased after exposure to the test compound or solution, a decrease or lack of increase in interaction being an indication that the compound or solution has GPCR desensitization activity independent of the GPCR expressed. Interaction of the arrestin with the GPCR may be detected in endosomes, in clathrin-coated pits, in proximity to a cell membrane, and the like.
[0175]The methods of assessing GPCR pathway activity of the present invention also include cell-free assays. In cell-free assays of the present invention, a substrate having deposited thereon a modified GPCR of the present invention is provided. A fluid containing a conjugate of an arrestin and a detectable molecule is also provided. Translocation of the arrestin is induced and interaction of the arrestin with the GPCR is detected. The GPCR and arrestin may be obtained from whole cells and used in the cell-free assay after purification. The modified GPCR has arrestin binding sites and agonist binding sites and may be supported in a multilayer or bilayer lipid vesicle. The vesicle supporting the modified GPCR may be deposited on the substrate, and the modified GPCR may be supported in the lipid vesicle and deposited on the substrate such that the arrestin binding sites are exposed to arrestin and the receptor binding sites are accessible to agonists. The substrate may be any artificial substrate on which the GPCR may be deposited, including but not limited to, glass, plastic, diamond, ceramic, semiconductor, silica, fiber optic, diamond, biocompatible monomer, biocompatible polymer, polymer beads (including organic and inorganic polymers), and the like.
[0176]The present invention relates to the compounds identified as ligands, agonists, antagonists, inverse agonists, or DACs by the methods of assaying of the present invention. These compounds may be used to treat any one of the disease states in which GPCRs have been implicated. The compounds identified may be administered to a human or a non-human in therapeutically effective doses to treat or ameliorate a condition, disorder, or disease in which GPCRs have been implicated. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of such a condition, disorder or disease.
Methods to Identify Compounds that Modulate GPCR Desensitization
[0177]The present invention relates to methods of screening for compounds that modulate GPCR desensitization. The methods utilize modified GRKs which constitutively phosphorylate GPCRs, resulting in constitutive desensitization. These may be used to identify compounds that alter the desensitization of GPCRs, even if the GPCR agonist is unknown. Once identified, these compounds may be useful as drugs capable of modulating GPCR activity and useful in the treatment of one or more of the disease states in which GPCRs have been implicated.
[0178]In a preferred method according to the present invention, cells are provided that contain an expression system and a nucleic acid encoding a modified GRK, resulting in constitutive desensitization of GPCRs expressed in the cell. These cells may further contain an arrestin conjugated to a GFP.
[0179]A preferred method of identifying a compound which inhibits GPCR internalization includes: (a) providing a cell including a GPCR, an arrestin, and a modified GRK; (b) exposing the cell to the compound(s); (c) determining the cellular distribution of the GPCR or arrestin; and (d) correlating a difference between (1) the location of the labeled molecule in the cell in the presence of the compound(s) and (2) the location of the labeled molecule in the cell in the absence of the compound(s) to modulation of GPCR internalization. Non-limiting embodiments of this method are described in FIGS. 4, 5, 6, and 7 and Examples 2, 3, 4, 5, 6, and 7.
[0180]The GRK of step (a), as described above, may be GRK 1, 2, 3, 4, 5, 6, or any other GRK, including splice variants, biologically active fragments, or modified GRKs. The GRK may be overexpressed and/or its expression may be inducible. The GRK may include a CAAX motif.
[0181]In the above method, agonist may or may not be provided.
[0182]Methods of detecting the labeled molecules and determining the cellular distribution of the GPCR or arrestin are described below.
[0183]GPCRs useful in the present invention include, but are not limited to GPCRs which have known agonists, GPCRs which do not have known agonists, GPCRs listed in FIG. 1A-1C, GPCRs illustrated in FIGS. 3, 4, 5, 6, and 7, AT1AR, Class A GPCRs, Class B GPCRs, taste receptors, odorant receptors, orphan receptors, modified GPCRs, GPCRs as described in U.S. patent application Ser. Nos. 10/054,616, 09/993,844, 10/095,620, 10/101,235, 09/631,468, 10/141,725, 10/161,916, 09/469,554, 09/772,644, 60/393,789, and 60/379,986, which are herein incorporated by reference, or biologically active fragments of the above GPCRs.
Vectors and Nucleic Acids, Host Cells for Protein Expression
[0184]The present invention relates to modified GRKs, including GRKs which are over-expressed, or their expression is inducible.
[0185]Nucleic acids encoding modified GRKs are provided. The present invention relates to the expression, over-expression, and the inducible expression of these proteins. The expression may be carried out by a suitable expression system contained in a vector, as described below.
[0186]One aspect of the present invention relates to the combination of (1) nucleic acids encoding a modified GRK with (2) a system for expression of modified GRKs resulting in constitutive desensitization of GPCRs. This system for expression of modified GRKs may include a promoter or origin of replication.
[0187]Another aspect of the present invention relates to modified GPCRs, nucleic acids encoding modified GPCRs, and host cell for modified GPCR expression.
[0188]Nucleic acids encoding modified GPCRs are provided. The present invention relates to the expression, over-expression, and the inducible expression of these proteins. The expression may be carried out by a suitable expression system contained in a vector, as described below.
[0189]A feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
[0190]Such operative linking of a DNA sequence of this invention to an expression control sequence, of course, includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
[0191]A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmids col El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof, vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
[0192]Any of a wide variety of expression control sequences--sequences that control the expression of a DNA sequence operatively linked to it--may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast α-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
[0193]A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, plant cells, nematode cells, and animal cells, such as HEK-293, U2OS, CHO, R1.1, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture. In one aspect of the present invention, the host cells include a GRK-C20 and an arrestin. In a further aspect of the present invention, the host cells include a GRK-C20, an arrestin, and a GPCR.
[0194]It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered.
[0195]In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.
[0196]Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.
[0197]It is further intended that modified GRK analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention. Analogs, such as fragments, may be produced, for example, by pepsin digestion of GRK material. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of GRK coding sequences. Analogs exhibiting "GRK activity" such as small molecules, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays.
[0198]As mentioned above, a DNA sequence encoding a modified GRK6 can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the GRK amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem., 259:6311 (1984).
[0199]Synthetic DNA sequences allow convenient construction of genes which will express GRK analogs or "muteins". Alternatively, DNA encoding muteins can be made by site-directed mutagenesis of native or modified GRK genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
[0200]A general method for site-specific incorporation of unnatural amino acids into proteins is described in Christopher J. Noren, Spencer J. Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science, 244:182-188 (April 1989). This method may be used to create analogs with unnatural amino acids.
[0201]Additional motifs, such as epitope tags or sequences to aid in purification, may be incorporated into the nucleic acids encoding the modified GRKs or modified GPCRs. Preferably, the nucleic acids encoding the motifs may be at the 5' or 3' end of the nucleic acid, resulting in the presence of the motif at the N or C terminus of the protein.
The Conjugates
[0202]The cells used in the methods of assaying of the present invention may comprise a conjugate of an arrestin protein and a detectable molecule. In the cells and methods of the present invention, the cells may also comprise a conjugate of a modified GPCR of the present invention and a detectable molecule.
[0203]All forms of arrestin, naturally occurring and engineered variants, including but not limited to, visual arrestin, cone arrestin, Parrestin 1 and Parrestin 2, may be used in the present invention. The modified GPCRs of the present invention may interact to a detectable level with all forms of arrestin.
[0204]Detectable molecules that may be used to conjugate with the arrestin include, but are not limited to, molecules that are detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, radioactive, and optical means, including but not limited to bioluminescence, phosphorescence, and fluorescence. Detectable molecules include, but are not limited to, GFP, luciferase, β-galactosidase, rhodamine-conjugated antibody, and the like. Detectable molecules include radioisotopes, epitope tags, affinity labels, enzymes, fluorescent groups, chemiluminescent groups, and the like. Detectable molecules include molecules which are directly or indirectly detected as a function of their interaction with other molecule(s). These detectable molecules should be a biologically compatible molecule and should not compromise the ability of the arrestin to interact with the GPCR system and the interaction of the arrestin with the GPCR system must not compromise the ability of the detectable molecule to be detected. Preferred detectable molecules are optically detectable molecules, including optically detectable proteins, such that they may be excited chemically, mechanically, electrically, or radioactively to emit fluorescence, phosphorescence, or bioluminescence. More preferred detectable molecules are inherently fluorescent molecules, such as fluorescent proteins, including, for example, Green Fluorescent Protein (GFP). The detectable molecule may be conjugated to the arrestin protein by methods as described in Barak et al. (U.S. Pat. Nos. 5,891,646 and 6,110,693). The detectable molecule may be conjugated to the arrestin at the front-end, at the back-end, or in the middle.
[0205]The GPCR or biologically active fragments thereof may also be conjugated with a detectable molecule. Preferably, the carboxyl-terminus of the GPCR is conjugated with a detectable molecule. A carboxyl-terminal tail conjugated or attached to a detectable molecule can be used in a carboxyl-terminal tail exchange to provide the detectably labeled GPCR.
[0206]If the GPCR is conjugated with a detectable molecule, proximity of the GPCR with the arrestin may be readily detected. In addition, if the GPCR is conjugated with a detectable molecule, compartmentalization of the GPCR with the arrestin may be readily confirmed. The detectable molecule used to conjugate with the GPCRs may include those as described above, including, for example, optically detectable molecules, such that they may be excited chemically, mechanically, electrically, or radioactively to emit fluorescence, phosphorescence, or bioluminescence. Preferred optically detectable molecules may be detected by immunofluorescence, luminescence, fluorescence, and phosphorescence.
[0207]For example, the GPCRs may be antibody labeled with an antibody conjugated to an immunofluorescence molecule or the GPCRs may be conjugated with a luminescent donor. In particular, the GPCRs may be conjugated with, for example, luciferase, for example, Renilla luciferase, or a rhodamine-conjugated antibody, for example, rhodamine-conjugated anti-HA mouse monoclonal antibody. Preferably, the carboxyl-terminal tail of the GPCR may be conjugated with a luminescent donor, for example, luciferase. The GPCR, preferably the carboxyl-terminal tail, also may be conjugated with GFP as described in L. S. Barak et al. "Internal Trafficking and Surface Mobility of a Functionally Intact β2-Adrenergic Receptor-Green Fluorescent Protein Conjugate", Mol. Pharm. (1997) 51, 177-184.
Cell Types and Substrates
[0208]The cells of the present invention may express at least one modified GPCR of the present invention. The cells may further comprise a conjugate of an arrestin protein and a detectable molecule. Useful cells include eukaryotic and prokaryotic cells, including, but not limited to, bacterial cells, yeast cells, fungal cells, insect cells, nematode cells, plant cells, and animal cells. Suitable animal cells include, but are not limited to, HEK-293 cells, U2OS cells, HeLa cells, COS cells, and various primary mammalian cells. An animal model expressing a conjugate of an arrestin and a detectable molecule throughout its tissues or within a particular organ or tissue type, may also be used.
[0209]The cells of the present invention may express one modified protein that results in agonist-independent localization of GPCRs to endocytic vesicles or endosomes.
[0210]A substrate may have deposited thereon a plurality of cells of the present invention. The substrate may be any suitable biologically substrate, including but not limited to, glass, plastic, ceramic, semiconductor, silica, fiber optic, diamond, biocompatible monomer, or biocompatible polymer materials.
Methods of Detection
[0211]Methods of detecting the intracellular location of the detectably labeled arrestin, the intracellular location of a detectably labeled GPCR, or interaction of the detectably labeled arrestin, or other member of GPCR/arrestin complex with a GPCR or any other cell structure, including for example, the concentration of arrestin or GPCR at a cell membrane, colocalization of arrestin with GPCR in endosomes, and concentration of arrestin or GPCR in clathrin-coated pits, and the like, will vary dependent upon the detectable molecule(s) used.
[0212]One skilled in the art readily will be able to devise detection methods suitable for the detectable molecule(s) used. For optically detectable molecules, any optical method may be used where a change in the fluorescence, bioluminescence, or phosphorescence may be measured due to a redistribution or reorientation of emitted light. Such methods include, for example, polarization microscopy, BRET, FRET, evanescent wave excitation microscopy, and standard or confocal microscopy.
[0213]In a preferred embodiment arrestin may be conjugated to GFP and the arrestin-GFP conjugate may be detected by confocal microscopy. In another preferred embodiment, arrestin may conjugated to a GFP and the GPCR may be conjugated to an immunofluorescent molecule, and the conjugates may be detected by confocal microscopy. In an additional preferred embodiment, arrestin may conjugated to a GFP and the carboxy-terminus of the GPCR may be conjugated to a luciferase and the conjugates may be detected by bioluminescence resonance emission technology. In a further preferred embodiment arrestin may be conjugated to a luciferase and GPCR may be conjugated to a GFP, and the conjugates may be detected by bioluminescence resonance emission technology. The methods of the present invention are directed to detecting GPCR activity. The methods of the present invention allow enhanced monitoring of the GPCR pathway in real time.
[0214]In a preferred embodiment, the localization pattern of the detectable molecule is determined. In a further preferred embodiment, alterations of the localization pattern of the detectable molecule may be determined. The localization pattern may indicate cellular distribution of the detectable molecule. Certain methods of detection are described in U.S. Ser. No. 10/095,620, filed Mar. 12, 2002, which claims priority to U.S. Provisional Patent Application No. 60/275,339, filed Mar. 13, 2001, the contents of which are incorporated by reference in their entirety.
[0215]Molecules may also be detected by their interaction with another detectably labeled molecule, such as an antibody.
Disease Treatment
[0216]Another aspect of the invention relates to methods of treating a human or non-human subject suffering from a GPCR-related disease, such as cardiovascular disease, heart failure, asthma, nephrogenic diabetes insipidus, or hypertension. For example, compounds which alter AT1AR internalization may be useful to treat diseases and conditions related to AT1AR. Such diseases and conditions related to AT1AR include, but are not limited to: renal disease, diabetes and nephropathy, diabetes mellitus, type 2 diabetes, nephropathy, hypertension, congesive heart failure, endothelial dysfunction, vascular inflammation, and various heart diseases. Such treatment can be performed either by administering to a subject in need of such treatment, an amount of the compounds identified by the present method sufficient to treat the GPCR-related disease, or at least to lessen the symptoms thereof.
[0217]Treatment may also be effected by administering to the subject the naked modified nucleic acid sequences of the invention, such as by direct injection, microprojectile bombardment, delivery via liposomes or other vesicles, or by means of a vector which can be administered by one of the foregoing methods. Gene delivery in this manner may be considered gene therapy.
Pharmaceutical Compositions
[0218]The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
[0219]Losartan is a known angiotensin receptor antagonist, of the following formula:
##STR00001##
The present invention describes a new use of losartan: losartan is useful in methods of altering arrestin translocation to the AT1AR receptor (FIGS. 8-9). This method my be used to treat a patient by administering an effective amount of losartan to a patient in need thereof.
[0220]A GPCR agonist, antagonist, or DAC obtained by the methods disclosed herein can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
[0221]The therapeutic compositions are conventionally administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent (i.e., carrier, or vehicle).
[0222]The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compositions lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range which includes the IC50 (i.e., the concentration of the test composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
[0223]The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of modulation of GPCR activity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.001 to 30, preferably about 0.01 to about 25, and more preferably about 0.1 to 20 milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.
[0224]The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to, the severity of the disease or condition, disorder, or disease, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the composition(s) can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated with the composition in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of the composition used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
[0225]The therapeutic compositions may further include an effective amount of the GPCR agonist, antagonist, or DAC and one or more of the following active ingredients: an antibiotic, a steroid, and the like.
[0226]The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention can be prepared as SATE ((S-acetyl-2-thioethyl) phosphate) derivatives according to the methods disclosed for example in WO 93/24510 and in WO 94/26764.
[0227]The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. The compounds for modulating any of the disclosed genes, gene transcripts or proteins encoded thereby include antisense compounds as well as other modulatory compounds.
[0228]Pharmaceutically acceptable base addition salts for use with antisense as well as other modulatory compounds are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge et al., "Pharmaceutical Salts," J. Pharma. Sci., 1977, 66: 1-19). The base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a pharmaceutical addition salt includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are known in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid); with organic carboxylic, sulfonic, sulfo- or phospho-acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid.
[0229]Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.
[0230]For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.
[0231]The antisense compounds and other modulatory compounds described herein can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound or other modulatory compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.
[0232]The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding a gene identified using the systematic discovery technique or a mRNA transcript thereof. Such hybridization allows the use of sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding a gene or gene transcript identified by a systematic discovery method can be detected by means known in the art. Such means may include, for example, conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of a transcript of a gene in a sample may also be prepared.
[0233]The present invention also includes pharmaceutical antisense compositions and formulations which include the antisense compounds and other modulatory compounds and compositions of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
[0234]In certain embodiments, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment. This may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
[0235]For topical application, the compositions may be combined with a carrier so that an effective dosage is delivered, based on the desired activity.
[0236]Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
[0237]For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer, salts, flavoring, coloring and sweetening agents as appropriate.
[0238]Preparations for oral administration may be suitably formulated to give controlled release of the active composition.
[0239]The compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0240]For administration by inhalation, the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray, presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the composition and a suitable powder base such as lactose or starch.
[0241]The compositions may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
[0242]The compositions may, if desired, be presented in a pack or dispenser device that may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
[0243]Pharmaceutical compositions (e.g., gene, gene transcript or protein product modulatory agents as described herein) of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
[0244]The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
[0245]In one embodiment of the present invention, the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature, these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
[0246]The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 m in diameter. See, e.g., Idson, in Pharmaceutical Dosage Forms v. 1, p. 199 (Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York); Rosoff, in Pharmaceutical Dosage Forms, v. 1, p. 245; Block in Pharmaceutical Dosage Forms, v. 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences 301 (Mack Publishing Co., Easton, Pa., 1985). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.
[0247]Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms v. 1, p. 199 (Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York).
[0248]Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, v. 1, p. 285; Idson, in Pharmaceutical Dosage Forms, v. 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms).
[0249]Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers, especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, non-swelling clays (e.g., bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate), pigments and nonpolar solids (e.g., carbon or glyceryl tristearate).
[0250]A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, v. 1 p. 385 (Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York)).
[0251]Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers, such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
[0252]Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers (e.g., tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene) or reducing agents (e.g., ascorbic acid and sodium metabisulfite), and antioxidant synergists (e.g., citric acid, tartaric acid, and lecithin).
[0253]The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, v. 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms, v. 1, p. 245 (Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York); Idson, in Pharmaceutical Dosage Forms). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
[0254]In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, v. 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in Controlled Release of Drugs: Polymers and Aggregate Systems, 185-215 (Rosoff, M., Ed., 1989, VCH Publishers, New York). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, 271 (Mack Publishing Co., Easton, Pa., 1985).
[0255]Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with co-surfactants. The co-surfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
[0256]Microemulsions may, however, be prepared without the use of co-surfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
[0257]Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharm. Res., 1994, 11:1385-90; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13: 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., 1994; Ho et al., J. Pharm. Sci., 1996, 85: 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids and other active agents from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids and other active agents within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.
[0258]Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories-surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Crit. Rev. Therap. Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
[0259]There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, are useful because of their specificity and the duration of action. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
[0260]Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo. Selection of the appropriate liposome depending on the agent to be encapsulated would be evident given what is known in the art.
[0261]In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
[0262]Further advantages of liposomes include: (a) liposomes obtained from natural phospholipids are biocompatible and biodegradable; (b) liposomes can incorporate a wide range of water and lipid soluble drugs; (c) liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
[0263]Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
[0264]Another embodiment also contemplates the use of liposomes for topical administration. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin. Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.
[0265]Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Comm., 1987, 147:980-985).
[0266]Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., J. Controlled Release, 1992, 19: 269-74).
[0267]Another contemplated liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
[0268]"Sterically stabilized" liposomes, which refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids are also contemplated. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM 1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Lett., 1987, 223: 42; Wu et al., Can. Res., 1993, 53: 3765).
[0269]Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. See, e.g., Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53: 2778) described liposomes comprising a nonionic detergent, 2C12 15G, that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167: 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268: 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029: 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by, e.g., Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.). Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
[0270]Methods of encapsulating nucleic acids in liposomes is also known in the art. See, WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes.
[0271]Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, p. 285 (Marcel Dekker, Inc., New York, N.Y., 1988, p. 285)).
[0272]If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
[0273]If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
[0274]If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
[0275]If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
[0276]The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, 285 (Marcel Dekker, Inc., New York, N.Y., 1988).
[0277]In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids and other agents, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
[0278]Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
[0279]Another embodiment of the invention contemplates pharmaceutical compositions comprising surfactants. Surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Crit. Rev. Therap. Drug Carrier Systems, 1991, 92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40: 252).
[0280]Another embodiment contemplates the use of various fatty acids and their derivatives to act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, and the like) (Lee et al., 1991; Muranishi, Crit. Rev. Therap. Drug Carrier Systems, 1990, 7: 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44: 651-4).
[0281]The compositions comprising the active agents of the invention may further comprise bile salts. The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, N.Y., 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., 1991; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, 1990; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263: 25; Yamashita et al., J. Pharm. Sci., 1990, 79: 579-83).
[0282]The invention further contemplates compositions comprising chelating agents. Chelating agents can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers for use when the active agent is an antisense agent, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618: 315-39). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., 1991; Muranishi, 1990; Buur et al., J. Control Rel., 1990, 14: 43-51).
[0283]The invention also contemplates pharmaceutical compositions comprising active agents and non-chelating non-surfactants. Non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants, but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, 1990). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., 1991); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39: 621-6).
[0284]For pharmaceutical compositions comprising oligonucleotides, agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al., U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.
[0285]Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes (e.g., limonene and menthone).
[0286]Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5: 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6: 177-183).
[0287]The pharmaceutical compositions disclosed herein may also comprise an excipients. In contrast to carrier compounds described above, these excipients include a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids or other active agents to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid or other active agent and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).
[0288]Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
[0289]Formulations for topical administration of nucleic acids and other contemplated active agents may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids or other contemplated active agents can be used.
[0290]The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
[0291]Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Test Kits
[0292]In a further embodiment of this invention, commercial test kits including an assay system for screening potential drugs effective to modulate the activity of the GPCR may be prepared. The test kits may include cells, nucleic acids, or proteins described herein. The test kits may be used to carry out any of the methods described herein. A GPCR of interest may be introduced into host cells of the test kit. The test kit may be useful for determining if the GPCR is expressed at the plasma membrane, if the phosphorylated or unphosphorylated GPCR binds arrestin, or if the phosphorylated or unphosphorylated GPCR is internalized. The test kit may be useful for the identification of compounds that alter the desensitization of the GPCR of interest.
[0293]The GPCR may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the GPCR activity (e.g., signaling, recycling, affinity for arrestin, and the like) in the cells.
[0294]The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
EXAMPLES
Example 1
Experimental Procedures
[0295]The present inventors subcloned the Bovine GRK2-C20 cDNA (Inglese et al., Nature 1992) into the expression vector pcDNA3.1zeo+ (Invitrogen). Expression of this cDNA produces GRK2 with a CAAX motif (where C is cysteine, A is a small aliphatic residue, and X is an uncharged amino acid) added to the carboxyl terminus. The specific CAAX motif added to the end of GRK2, CVLL, directs the geranylgeranylation (C20 isoprenylation) of this protein. The enzyme-directed covalent attachment of the 20 carbon geranylgeranyl lipid group to the carboxyl terminus of GRK2 allows it to be localized at the plasma membrane (Inglese et al., Nature 1992).
[0296]Cell Culture
[0297]Human embryonic kidney (HEK-293) cells were purchased from the American Type Culture Collection (ATCC) and grown in Eagle's minimum essential medium (EMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum and gentamicin (100 μg/ml). HEK-293 cells stably expressing arrestin-GFP (HEK293-ArrGFP) were generated by standard procedures using G418 selection (0.4 mg/ml). HEK-293 cells were transiently transfected with arrestin-GFP, the GPCR of interest, and GRK2-C20. For control experiments performed in parallel, HEK-293 cells were transiently transfected with arrestin-GFP, the GPCR of interest, and no GRK2-C20. HEK293 cells were transiently transfected with arrestin-GFP, the GPCR of interest and GRK2-C20. For control experiments performed in parallel, the HEK293 cells were transiently transfected with arrestin-GFP were transiently transfected with the GPCR of interest and no GRK2-C20. All transfections were performed by the calcium phosphate coprecipitation method as previously described (Oakley et al., 1999). Following the transfection, cells were maintained in the culture medium (EMEM supplemented with 10% FCS and 10 μg/ml gentamycin) for approximately 24 hours. The cells were then plated on 35 mm glass bottom dishes (MatTek) and incubated for an additional 16-24 hours. Transfected GPCRs included both known GPCRs (receptors for which the natural ligand is know) and orphan GPCRs (receptors for which the natural ligand has not yet been identified).
[0298]Confocal Microscopy
[0299]Transfected HEK-293 cells were plated on 35 mm glass bottom dishes (MatTek) and cultured overnight. The next day, the medium was removed and replaced with serum-free medium supplemented with 10 mM HEPES for an additional 1 hour incubation at 37° C. The distribution of arrestin-GFP was then assessed using a Zeiss laser scanning confocal microscope (LSM 5 Pascal). Images were acquired with a 63× oil objective from live cells using single line excitation (488 nm) and a LP505 emission filter.
Example 2
Agonist-Independent Desensitization of Known GPCRs Upon Expression of a Modified GRK
[0300]The present inventors determined that overexpression of the GRK2-C20, which is expressed at the plasma membrane (Inglese et al., Nature 1992), in a cell line expressing arrestin-GFP promoted the binding of arrestin-GFP to GPCRs in the absence of added ligand.
[0301]The HEK293 cells transiently transfected with arrestin-GFP were transiently transfected with the GPCR of interest and with or without GRK2-C20. Using confocal microscopy, the distribution of the arrestin-GFP was determined. The localization of the arrestin-GFP at clathrin coated pits, endocytic vesicles, endosomes, or other stages in the desensitization pathway indicated arrestin-GFP binding to the GPCR. Thus, GPCR desensitization, visualized by the binding of arrestin-GFP to the GPCRs, was analyzed.
[0302]In the absence of added agonist, arrestin-GFP localized in small puncta (presumably clathrin coated pits) at the plasma membrane in cells expressing GRK2-C20 and either the cannabinoid type 2 receptor (CB2R) (FIG. 4). Moreover, in the absence of added agonist, arrestin-GFP localized in endocytic vesicles in cells expressing GRK2-C20 and either the angiotensin II type IA receptor (AT1AR), vasopressin V2 receptor (V2R), (FIG. 4) or neurokinin-1/substance P receptor (NK-1 or SPR). In control cells expressing each of the receptors (CB2R, AT1AR, V2R, or SPR) but lacking GRK2-C20, arrestin-GFP was diffusely expressed in the cytoplasm and did not localize to any significant extent in pits at the plasma membrane or vesicles inside the cell (FIG. 4).
Example 3
Agonist-Independent Desensitization of Orphan GPCRs Upon Expression of a Modified GRK
[0303]The present inventors determined that overexpression of the GRK2-C20, which is expressed at the plasma membrane (Inglese et al., Nature 1992), in a cell line expressing arrestin-GFP promoted the binding of arrestin-GFP to GPCRs in the absence of added ligand.
[0304]As above, the HEK293 cells transiently transfected with arrestin-GFP were transiently transfected with the GPCR of interest and with or without GRK2-C20. Using confocal microscopy, the distribution of the arrestin-GFP was determined. The localization of the arrestin-GFP at clathrin coated pits, endocytic vesicles, endosomes, or other stages in the desensitization pathway indicated arrestin-GFP binding to the GPCR. Thus, GPCR desensitization, visualized by the binding of arrestin-GFP to the GPCRs, was analyzed.
[0305]In the absence of added agonist, arrestin-GFP localized in small puncta (presumably clathrin coated pits) at the plasma membrane in cells expressing the orphan receptor GPR55 (FIG. 7). In control cells expressing GPR55 but lacking GRK2-C20, arrestin-GFP was diffusely expressed in the cytoplasm and did not localize to any significant extent in pits at the plasma membrane or vesicles inside the cell (FIG. 7). Other orphan GPCRs are described below.
Example 4
Method of Analyzing the Ability of a GPCR to Bind Arrestin
[0306]The present inventors developed a method to determine if a GPCR of interest is expressed at the plasma membrane. Preferably, the expression of orphan GPCRs may be analyzed in host cells in which GPCRs desensitize in an agonist-independent manner, as described herein.
[0307]As above, the HEK293 cells transiently transfected with arrestin-GFP were transiently transfected with the GPCR of interest and with or without GRK2-C20. Using confocal microscopy, the distribution of the arrestin-GFP was determined. The localization of the arrestin-GFP at clathrin coated pits, endocytic vesicles, endosomes, or other stages in the desensitization pathway indicated arrestin-GFP binding to the GPCR. Thus, GPCR desensitization, visualized by the binding of arrestin-GFP to the GPCRs, was analyzed.
[0308]Certain GPCRs, as described above, localized in clathrin coated pits, endocytic vesicles, endosomes, or other stages in the desensitization pathway. This localization indicated that the GPCRs had the ability to bind arrestin, because arrestin binding is requisite for subsequent localization in the desensitization pathway. A GPCR that does not bind arrestin would not enter or localize in the desensitization pathway. GPCRs that do not bind arrestin may be altered such that they do bind arrestin. The present inventors modified certain GPCRs to enhance arrestin affinity, as described below.
Example 5
Method of Increasing the Ability of a GPCR to Bind Arrestin
[0309]The present inventors modified GPCRs to enhance their binding to arrestin. These modifications are described in U.S. Ser. No. 09/993,844. GPCRs were modified at their C-terminal tails to be better phosphorylated by GRKs. These modified and phosphorylated GPCRs then had enhanced binding to arrestin. They demonstrated increased internalization. The letter E (for enhanced phosphorylation) is added to the end of the name of the GPCR which has been modified in this manner.
[0310]Modified GPCR constructs were generated by polymerase chain reaction following standard protocols and contain the HA epitope. Chimeric receptors were constructed in which the carboxyl-terminal tails of the GPCR and V2R were exchanged (FIG. 3A-3BB), one for the other, after the putative sites of palmitoylation. Sequences of the DNA constructs were confirmed by DNA sequencing.
[0311]The nucleic acids of the GPCR of interest were PCR-amplified with primers that introduced a Not I restriction enzyme site (gcggccgc) immediately after the codon for a cysteine residue (a putative site of palmitoylation) 10 to 25 amino acids (preferably 15 to 20) downstream of the NPXXY that is to be fused to the V2R carboxyl terminus. The amplified receptor DNA fragment was then subcloned into the pEArrB-1 vector (described in U.S. patent application Ser. No. 09/993,844) using the Not I restriction enzyme site and an additional restriction enzyme site upstream of the receptor atg start codon. When expressed, the modified GPCR will contain a 31 amino acid peptide fused to the receptor carboxyl terminus. The first two amino acids will be Ala residues contributed by the Not I site, and the last 29 amino acids will be from the V2R carboxyl terminus.
[0312]The present inventors modified the carboxyl-terminal tails of the following receptors as described above and in the U.S. patent application Ser. No. 09/993,844: the β2-adrenergic receptor (β2ARE), dopamine D1A receptor (D1ARE), mu opiod receptor (MORE), orphan GPR3 (GPR3E), orphan GPR6 (GPR6E), orphan GPR12 (GPR12E), orphan GPR7 (GPR7E), orphan GPR8 (GPR8E), orphan GPR55 (GPR55E), orphan SREB2 (SREB2E), and orphan SREB3 (SREB3E). The "E" stands for "enhanced arrestin binding". In the absence of added agonist, arrestin-GFP localized in endocytic vesicles for each of the modified GPCRs listed above when co-expressed with GRK2-C20 (FIGS. 4, 5, 6, and 7). For some of these modified receptors (such as orphan GPR6E), a small but significant amount of arrestin-GFP was observed to localize in intracellular vesicles in the control cells lacking the GRK2-C20 (FIG. 5). However, overexpression of GRK2-C20 with these receptors promoted a marked increase in this response (FIG. 5). The amino acid and nucleic acid sequences of these modified GPCRs, the wild-type sequences, and sequences of HA-tagged modified GPCRs are shown in FIG. 3A-3BB and in SEQ ID Nos: 35-90.
Example 6
Method of Determining if a GPCR of Interest is Expressed at the Plasma Membrane
[0313]The present inventors developed a method to determine if a GPCR of interest is expressed at the plasma membrane.
[0314]The HEK293 cells transiently transfected with arrestin-GFP were transiently transfected with the GPCR of interest and with or without GRK2-C20. Using confocal microscopy, the distribution of the arrestin-GFP was determined. The localization of the arrestin-GFP at clathrin coated pits, endocytic vesicles, endosomes, or other stages in the desensitization pathway indicated arrestin-GFP binding to the GPCR. Thus, GPCR desensitization, visualized by the binding of arrestin-GFP to the GPCRs, was analyzed.
[0315]Certain GPCRs, as described above, localized in clathrin coated pits, endocytic vesicles, endosomes, or other stages in the desensitization pathway. This localization indicated that the GPCRs were expressed at the plasma membrane, because plasma membrane expression is requisite for subsequent localization in the desensitization pathway. A GPCR that was not expressed at the plasma membrane would not localize in the desensitization pathway. GPCRs that do not express at the plasma membrane may be altered such that they do express at the plasma membrane. For example, the expression of the GPCR may be altered, the amino acid sequence of the GPCR may be altered, or the GPCR may be introduced into another host cell.
Example 7
Monitoring Desensitization of GPCR Mutants
[0316]Desensitization may be monitored in cells including GPCR mutants. The desensitization of the GPCR mutant may be dependent on GRK overexpression.
[0317]A vector including the human β2AR-E-Y326A containing a point mutation, the Tyrosine residue 326 converted to Alanine, will be transfected into cells expressing arrestin-GFP and a GRK, which may be modified. The "E" indicates that the GPCR has been modified, as described above. The Y326A mutation causes the GPCR to be dependent on overexpressed GRK for phosphorylation and subsequent desensitization. The β2AR-Y326A will desensitize in the absence of agonist upon expression of GRK-C20. The expression of the GRK may be altered, including methods of altering the amount of GRK nucleic acids in the cell using an inducible promoter, replication controlling machinery such as the origin of replication, or manually altering the amount of vector in the cells.
[0318]The cells will be seeded in 96 well or higher density plates and incubated overnight. The next morning the activator of the inducible system or vehicle only will be added to the wells to induce overexpression of the GRK or modified GRK. Agonist will be added to cells expressing the GRK (not the modified GRK).
[0319]Compounds of interest will then be added to the wells to see if they alter the internalization of arrestin-GFP. The cells will then be fixed with 2% paraformaldehyde and the amount of arrestin-GFP translocation will be measured using image analysis systems.
Example 8
Non Peptide Antagonist/Inverse GPCR Agonist Inhibits Constitutive Translocation of arrestinGFP Induced by Expression of GRK2-C20
[0320]U2OS cells stably expressing arrestinGFP and the angiotensin II type 1A receptor (AT1AR) were transiently transduced with human GRK2-C20 using a baculovirus expression system. A range of GRK2-C20 expression levels was obtained by using varying amounts (serial dilution) of the GRK2-C20 baculovirus. As shown by the black bars in FIG. 8, addition of GRK2-C20 promotes ligand-independent translocation of arrestinGFP to the AT1AR. A maximum arrestinGFP translocation response of 305±25 Fgrains and a minimum of 77±13 Fgrains was achieved with the 1:4 and 1:256 dilutions, respectively, of GRK2-C20 baculovirus. In the absence of added GRK2-C20 (none), no translocation was observed (18±3 Fgrains).
[0321]To test whether the ligand-independent arrestinGFP translocation could be blocked by an antagonist/inverse agonist of the AT1AR, the cells were treated with losartan. Losartan is a nonpeptide molecule that functions as an antagonist/inverse agonist of the AT1AR. Losartan (1 μM final concentration) was added to the cells either immediately after transduction for an 18 hour incubation or 15 hours after transduction for a 3 hour incubation. At the end of the incubation, the cells were analyzed for arrestinGFP translocation using the INCell Analyzer system.
[0322]As shown in FIGS. 8, 9, and 10, losartan treatment blocks the ligand-independent translocation of arrestinGFP to the AT1AR induced by expression of GRK2-C20. The most dramatic effect was observed after an 18 hour incubation with losartan (FIGS. 8 and 10). At the lower levels of GRK2-C20 expression (1:64, 1:128, and 1:256 dilutions), this treatment paradigm results in the inhibition of greater than 90% of the ligand-independent translocation of arrestinGFP to the AT1AR.
[0323]While the invention has been described and illustrated herein by references to various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art.
[0324]The following is a list of documents related to the above disclosure and particularly to the experimental procedures and discussions. The following documents, as well as any documents referenced in the foregoing text, should be considered as incorporated by reference in their entirety. [0325]Attramadal, H., Arriza, J. L., Aoki, C., Dawson, T. M., Codina, J., Kwatra, M. M., Snyder, S. H., Caron, M. G. & Lefkowitz, R. J. (1992) J. Biol. Chem. 267, 17882-17890 [0326]Barak, L. S., Oakley, R. H., Laporte, S. A. and Caron, M. G. (2001) Proc. Natl. Acad. Sci. USA 98, 93-98 [0327]Barak, L. S., Warabi, K., Feng, X., Caron, M. G. & Kwatra, M. M. (1999) J. Biol. Chem. 274, 7565-7569 [0328]Barak, L. S., Ferguson, S. S., Zhang, J. & Caron, M. G. (1997) J. Biol. Chem. 272, 27497-27500 [0329]Barak, L. S., Ferguson, S. S., Zhang, J., Martenson, C., Meyer, T. & Caron, M. G. (1997) Mol. Pharmacol. 51, 177-184 [0330]Barak, L. S., Menard, L., Ferguson, S. S., Colapietro, A. M. & Caron, M. G. (1995) Biochemistry 34, 15407-15414 [0331]Ferguson, S. S., Barak, L. S., Zhang, J. & Caron, M. G. (1996) Can. J. Physiol. Pharmacol. 74, 1095-1110 [0332]Ferguson, S. S., Menard, L., Barak, L. S., Koch, W. J., Colapietro, A. M. & Caron, M. G. (1995) J. Biol. Chem. 270, 24782-24789 [0333]Inglese, J., Koch, W. J., Caron, M. G., Lefkowitz, R. J. (1992) Nature 359:147-150 [0334]Kim, K.-M., Valenzano, K. J., Robinson, S. R., Yao, W. D., Barak, L. S., Caron, M. G. (2001) J. Biol. Chem. 276: 37409-37414 [0335]Laporte, S. A., Oakley, R. H., Holt, J. A., Barak, L. S. & Caron, M. G. (2000) J. Biol. Chem. 275, 23120-23126 [0336]Laporte, S. A., Oakley, R. H., Zhang, J., Holt, J. A., Ferguson, S. S., Caron, M. G. & Barak, L. S. (1999) Proc. Natl. Acad. Sci. USA 96, 3712-3717 [0337]Menard, L., Ferguson, S. S., Zhang, J., Lin, F. T., Lefkowitz, R. J., Caron, M. G. & Barak, L. S. (1997) Mol. Pharmacol. 51, 800-808 [0338]Mhaouty-Kodja, S., Barak, L. S., Scheer, A., Abuin, L., Diviani, D., Caron, M. G. & Cotecchia, S. (1999) Mol. Pharmacol. 55, 339-347 [0339]Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., Caron, M. G. (2001). J. Biol. Chem. 276: 19452-19460 [0340]Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G. & Barak, L. S. (2000) J. Biol. Chem. 275, 17201-17210 [0341]Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S. & Caron, M. G. (1999) J. Biol. Chem. 274, 32248-32257 [0342]Zhang, J., Barak, L. S., Anborgh, P. H., Laporte, S. A., Caron, M. G. & Ferguson, S. S. (1999) J. Biol. Chem. 274, 10999-11006 [0343]Zhang, J., Barak, L. S., Winkler, K. E., Caron, M. G. & Ferguson, S. S. (1997) J. Biol. Chem. 272, 27005-27014
Sequence CWU
1
951689PRTBos taurus 1Met Ala Asp Leu Glu Ala Val Leu Ala Asp Val Ser Tyr
Leu Met Ala1 5 10 15Met
Glu Lys Ser Lys Ala Thr Pro Ala Ala Arg Ala Ser Lys Lys Ile20
25 30Leu Leu Pro Glu Pro Ser Ile Arg Ser Val Met
Gln Lys Tyr Leu Glu35 40 45Asp Arg Gly
Glu Val Thr Phe Glu Lys Ile Phe Ser Gln Lys Leu Gly50 55
60Tyr Leu Leu Phe Arg Asp Phe Cys Leu Lys His Leu Glu
Glu Ala Lys65 70 75
80Pro Leu Val Glu Phe Tyr Glu Glu Ile Lys Lys Tyr Glu Lys Leu Glu85
90 95Thr Glu Glu Glu Arg Leu Val Cys Ser Arg
Glu Ile Phe Asp Thr Tyr100 105 110Ile Met
Lys Glu Leu Leu Ala Cys Ser His Pro Phe Ser Lys Ser Ala115
120 125Ile Glu His Val Gln Gly His Leu Val Lys Lys Gln
Val Pro Pro Asp130 135 140Leu Phe Gln Pro
Tyr Ile Glu Glu Ile Cys Gln Asn Leu Arg Gly Asp145 150
155 160Val Phe Gln Lys Phe Ile Glu Ser Asp
Lys Phe Thr Arg Phe Cys Gln165 170 175Trp
Lys Asn Val Glu Leu Asn Ile His Leu Thr Met Asn Asp Phe Ser180
185 190Val His Arg Ile Ile Gly Arg Gly Gly Phe Gly
Glu Val Tyr Gly Cys195 200 205Arg Lys Ala
Asp Thr Gly Lys Met Tyr Ala Met Lys Cys Leu Asp Lys210
215 220Lys Arg Ile Lys Met Lys Gln Gly Glu Thr Leu Ala
Leu Asn Glu Arg225 230 235
240Ile Met Leu Ser Leu Val Ser Thr Gly Asp Cys Pro Phe Ile Val Cys245
250 255Met Ser Tyr Ala Phe His Thr Pro Asp
Lys Leu Ser Phe Ile Leu Asp260 265 270Leu
Met Asn Gly Gly Asp Leu His Tyr His Leu Ser Gln His Gly Val275
280 285Phe Ser Glu Ala Asp Met Arg Phe Tyr Ala Ala
Glu Ile Ile Leu Gly290 295 300Leu Glu His
Met His Asn Arg Phe Val Val Tyr Arg Asp Leu Lys Pro305
310 315 320Ala Asn Ile Leu Leu Asp Glu
His Gly His Val Arg Ile Ser Asp Leu325 330
335Gly Leu Ala Cys Asp Phe Ser Lys Lys Lys Pro His Ala Ser Val Gly340
345 350Thr His Gly Tyr Met Ala Pro Glu Val
Leu Gln Lys Gly Val Ala Tyr355 360 365Asp
Ser Ser Ala Asp Trp Phe Ser Leu Gly Cys Met Leu Phe Lys Leu370
375 380Leu Arg Gly His Ser Pro Phe Arg Gln His Lys
Thr Lys Asp Lys His385 390 395
400Glu Ile Asp Arg Met Thr Leu Thr Met Ala Val Glu Leu Pro Asp
Ser405 410 415Phe Ser Pro Glu Leu Arg Ser
Leu Leu Glu Gly Leu Leu Gln Arg Asp420 425
430Val Asn Arg Arg Leu Gly Cys Leu Gly Arg Gly Ala Gln Glu Val Lys435
440 445Glu Ser Pro Phe Phe Arg Ser Leu Asp
Trp Gln Met Val Phe Leu Gln450 455 460Lys
Tyr Pro Pro Pro Leu Ile Pro Pro Arg Gly Glu Val Asn Ala Ala465
470 475 480Asp Ala Phe Asp Ile Gly
Ser Phe Asp Glu Glu Asp Thr Lys Gly Ile485 490
495Lys Leu Leu Asp Ser Asp Gln Glu Leu Tyr Arg Asn Phe Pro Leu
Thr500 505 510Ile Ser Glu Arg Trp Gln Gln
Glu Val Ala Glu Thr Val Phe Asp Thr515 520
525Ile Asn Ala Glu Thr Asp Arg Leu Glu Ala Arg Lys Lys Thr Lys Asn530
535 540Lys Gln Leu Gly His Glu Glu Asp Tyr
Ala Leu Gly Lys Asp Cys Ile545 550 555
560Met His Gly Tyr Met Ser Lys Met Gly Asn Pro Phe Leu Thr
Gln Trp565 570 575Gln Arg Arg Tyr Phe Tyr
Leu Phe Pro Asn Arg Leu Glu Trp Arg Gly580 585
590Glu Gly Glu Ala Pro Gln Ser Leu Leu Thr Met Glu Glu Ile Gln
Ser595 600 605Val Glu Glu Thr Gln Ile Lys
Glu Arg Lys Cys Leu Leu Leu Lys Ile610 615
620Arg Gly Gly Lys Gln Phe Val Leu Gln Cys Asp Ser Asp Pro Glu Leu625
630 635 640Val Gln Trp Lys
Lys Glu Leu Arg Asp Ala Tyr Arg Glu Ala Gln Gln645 650
655Leu Val Gln Arg Val Pro Lys Met Lys Asn Lys Pro Arg Ser
Pro Val660 665 670Val Glu Leu Ser Lys Val
Pro Leu Ile Gln Arg Gly Ser Cys Val Leu675 680
685Leu22070DNABos taurus 2atggcggacc tggaggcggt gctggccgac
gtgagctacc tgatggccat ggagaagagc 60aaggccacgc cggcggcgcg cgccagcaag
aagatcctgc tgcccgagcc cagcatccgc 120agcgtcatgc agaagtacct ggaggaccgg
ggcgaggtga cttttgagaa gatcttctcc 180cagaagctgg ggtacctgct tttccgagac
ttctgcctga agcacctgga ggaggccaag 240cccttggtag agttctacga ggagatcaag
aaatacgaga agctggagac agaggaggag 300cgcctggtct gcagccgaga gatcttcgac
acgtacatca tgaaggagct gctggcctgc 360tcacatcctt tctcgaagag cgccattgag
cacgtccagg gccatctggt gaagaagcag 420gtgcctccgg atctcttcca gccatatatt
gaagaaattt gccagaacct ccgaggagac 480gtgttccaga aattcatcga gagcgataaa
ttcacacgat tttgccagtg gaagaatgta 540gagctcaaca tccacctgac catgaacgac
ttcagtgtgc accgcatcat cgggcgaggc 600ggcttcggtg aggtctacgg ctgccggaag
gccgacacgg gcaagatgta cgccatgaag 660tgtctggaca agaagcgcat caagatgaag
caaggggaga ctctggccct gaatgagcgc 720atcatgctgt cgctcgtcag caccggggac
tgcccgttca tcgtctgcat gtcatacgcc 780ttccacacac cggacaagct cagcttcatc
ctggatctca tgaacggcgg ggacctgcac 840taccacctgt cccagcacgg ggtcttctcc
gaggccgaca tgcgtttcta cgccgccgag 900atcatcctgg gcctggagca catgcacaac
cgcttcgtgg tctaccggga cctgaagccg 960gccaacatcc tgctggacga gcacggccac
gtgcgcatct cagacctggg cctggcctgt 1020gacttctcca agaagaagcc tcacgccagt
gtgggcaccc acgggtacat ggctcccgag 1080gttctacaga agggtgtggc ctacgacagc
agcgccgact ggttctccct gggctgcatg 1140ctcttcaagc tgctgcgagg gcatagccct
ttccggcagc acaagaccaa agacaagcat 1200gagatcgaca gaatgacatt gacaatggct
gtggagctgc ctgactcctt ctcccctgag 1260ctccgctcct tgctggaggg gctgctgcag
agggatgtca accggaggct aggctgcctg 1320ggccgagggg cccaggaggt gaaggagagc
cccttcttcc gttccctgga ctggcagatg 1380gtctttttac aaaagtaccc tcccccgttg
atccccccac gaggggaggt gaatgcagcc 1440gacgcctttg acattggctc cttcgatgag
gaggacacaa aaggaatcaa gctactggac 1500agtgaccagg agctctaccg caacttcccc
ctgaccatct cggagcggtg gcagcaggag 1560gtagcagaga ctgtctttga caccatcaat
gctgagacgg accggctgga ggcccgcaag 1620aaaaccaaaa acaagcagtt gggccacgag
gaagactacg ccctgggcaa ggactgcatc 1680atgcatggct acatgtccaa gatgggcaac
cccttcctga cccagtggca gcggcggtac 1740ttctacctgt tccctaaccg gctcgagtgg
cggggcgagg gcgaggcccc gcagagcctg 1800ctgaccatgg aggagatcca gtcggtggag
gagacgcaga tcaaggagcg aaagtgcctc 1860ctcctcaaga tccgaggtgg caagcagttt
gtcctgcagt gcgatagtga cccagagctg 1920gtgcagtgga agaaggagct tcgagacgcc
taccgcgagg cccagcagct agtgcagcgg 1980gtgcccaaga tgaagaacaa gccgcgctcg
cccgtcgtgg agctgagcaa ggtgccactg 2040atccagcgcg gcagttgtgt gcttctttag
20703689PRTHomo sapiens 3Met Ala Asp Leu
Glu Ala Val Leu Ala Asp Val Ser Tyr Leu Met Ala1 5
10 15Met Glu Lys Ser Lys Ala Thr Pro Ala Ala Arg
Ala Ser Lys Lys Ile20 25 30Leu Leu Pro
Glu Pro Ser Ile Arg Ser Val Met Gln Lys Tyr Leu Glu35 40
45Asp Arg Gly Glu Val Thr Phe Glu Lys Ile Phe Ser Gln
Lys Leu Gly50 55 60Tyr Leu Leu Phe Arg
Asp Phe Cys Leu Asn His Leu Glu Glu Ala Arg65 70
75 80Pro Leu Val Glu Phe Tyr Glu Glu Ile Lys
Lys Tyr Glu Lys Leu Glu85 90 95Thr Glu
Glu Glu Arg Val Ala Arg Ser Arg Glu Ile Phe Asp Ser Tyr100
105 110Ile Met Lys Glu Leu Leu Ala Cys Ser His Pro Phe
Ser Lys Ser Ala115 120 125Thr Glu His Val
Gln Gly His Leu Gly Lys Lys Gln Val Pro Pro Asp130 135
140Leu Phe Gln Pro Tyr Ile Glu Glu Ile Cys Gln Asn Leu Arg
Gly Asp145 150 155 160Val
Phe Gln Lys Phe Ile Glu Ser Asp Lys Phe Thr Arg Phe Cys Gln165
170 175Trp Lys Asn Val Glu Leu Asn Ile His Leu Thr
Met Asn Asp Phe Ser180 185 190Val His Arg
Ile Ile Gly Arg Gly Gly Phe Gly Glu Val Tyr Gly Cys195
200 205Arg Lys Ala Asp Thr Gly Lys Met Tyr Ala Met Lys
Cys Leu Asp Lys210 215 220Lys Arg Ile Lys
Met Lys Gln Gly Glu Thr Leu Ala Leu Asn Glu Arg225 230
235 240Ile Met Leu Ser Leu Val Ser Thr Gly
Asp Cys Pro Phe Ile Val Cys245 250 255Met
Ser Tyr Ala Phe His Thr Pro Asp Lys Leu Ser Phe Ile Leu Asp260
265 270Leu Met Asn Gly Gly Asp Leu His Tyr His Leu
Ser Gln His Gly Val275 280 285Phe Ser Glu
Ala Asp Met Arg Phe Tyr Ala Ala Glu Ile Ile Leu Gly290
295 300Leu Glu His Met His Asn Arg Phe Val Val Tyr Arg
Asp Leu Lys Pro305 310 315
320Ala Asn Ile Leu Leu Asp Glu His Gly His Val Arg Ile Ser Asp Leu325
330 335Gly Leu Ala Cys Asp Phe Ser Lys Lys
Lys Pro His Ala Ser Val Gly340 345 350Thr
His Gly Tyr Met Ala Pro Glu Val Leu Gln Lys Gly Val Ala Tyr355
360 365Asp Ser Ser Ala Asp Trp Phe Ser Leu Gly Cys
Met Leu Phe Lys Leu370 375 380Leu Arg Gly
His Ser Pro Phe Arg Gln His Lys Thr Lys Asp Lys His385
390 395 400Glu Ile Asp Arg Met Thr Leu
Thr Met Ala Val Glu Leu Pro Asp Ser405 410
415Phe Ser Pro Glu Leu Arg Ser Leu Leu Glu Gly Leu Leu Gln Arg Asp420
425 430Val Asn Arg Arg Leu Gly Cys Leu Gly
Arg Gly Ala Gln Glu Val Lys435 440 445Glu
Ser Pro Phe Phe Arg Ser Leu Asp Trp Gln Met Val Phe Leu Gln450
455 460Lys Tyr Pro Pro Pro Leu Ile Pro Pro Arg Gly
Glu Val Asn Ala Ala465 470 475
480Asp Ala Phe Asp Ile Gly Ser Phe Asp Glu Glu Asp Thr Lys Gly
Ile485 490 495Lys Leu Leu Asp Ser Asp Gln
Glu Leu Tyr Arg Asn Phe Pro Leu Thr500 505
510Ile Ser Glu Arg Trp Gln Gln Glu Val Ala Glu Thr Val Phe Asp Thr515
520 525Ile Asn Ala Glu Thr Asp Arg Leu Glu
Ala Arg Lys Lys Ala Lys Asn530 535 540Lys
Gln Leu Gly His Glu Glu Asp Tyr Ala Leu Gly Lys Asp Cys Ile545
550 555 560Met His Gly Tyr Met Ser
Lys Met Gly Asn Pro Phe Leu Thr Gln Trp565 570
575Gln Arg Arg Tyr Phe Tyr Leu Phe Pro Asn Arg Leu Glu Trp Arg
Gly580 585 590Glu Gly Glu Ala Pro Gln Ser
Leu Leu Thr Met Glu Glu Ile Gln Ser595 600
605Val Glu Glu Thr Gln Ile Lys Glu Arg Lys Cys Leu Leu Leu Lys Ile610
615 620Arg Gly Gly Lys Gln Phe Ile Leu Gln
Cys Asp Ser Asp Pro Glu Leu625 630 635
640Val Gln Trp Lys Lys Glu Leu Arg Asp Ala Tyr Arg Glu Ala
Gln Gln645 650 655Leu Val Gln Arg Val Pro
Lys Met Lys Asn Lys Pro Arg Ser Pro Val660 665
670Val Glu Leu Ser Lys Val Pro Leu Val Gln Arg Gly Ser Ala Asn
Gly675 680 685Leu42070DNAHomo sapiens
4atggcggacc tggaggcggt gctggccgac gtgagctacc tgatggccat ggagaagagc
60aaggccacgc cggccgcgcg cgccagcaag aagatactgc tgcccgagcc cagcatccgc
120agtgtcatgc agaagtacct ggaggaccgg ggcgaggtga cctttgagaa gatcttttcc
180cagaagctgg ggtacctgct cttccgagac ttctgcctga accacctgga ggaggccagg
240cccttggtgg aattctatga ggagatcaag aagtacgaga agctggagac ggaggaggag
300cgtgtggccc gcagccggga gatcttcgac tcatacatca tgaaggagct gctggcctgc
360tcgcatccct tctcgaagag tgccactgag catgtccaag gccacctggg gaagaagcag
420gtgcctccgg atctcttcca gccatacatc gaagagattt gtcaaaacct ccgaggggac
480gtgttccaga aattcattga gagcgataag ttcacacggt tttgccagtg gaagaatgtg
540gagctcaaca tccacctgac catgaatgac ttcagcgtgc atcgcatcat tgggcgcggg
600ggctttggcg aggtctatgg gtgccggaag gctgacacag gcaagatgta cgccatgaag
660tgcctggaca aaaagcgcat caagatgaag cagggggaga ccctggccct gaacgagcgc
720atcatgctct cgctcgtcag cactggggac tgcccattca ttgtctgcat gtcatacgcg
780ttccacacgc cagacaagct cagcttcatc ctggacctca tgaacggtgg ggacctgcac
840taccacctct cccagcacgg ggtcttctca gaggctgaca tgcgcttcta tgcggccgag
900atcatcctgg gcctggagca catgcacaac cgcttcgtgg tctaccggga cctgaagcca
960gccaacatcc ttctggacga gcatggccac gtgcggatct cggacctggg cctggcctgt
1020gacttctcca agaagaagcc ccatgccagc gtgggcaccc acgggtacat ggctccggag
1080gtcctgcaga agggcgtggc ctacgacagc agtgccgact ggttctctct ggggtgcatg
1140ctcttcaagt tgctgcgggg gcacagcccc ttccggcagc acaagaccaa agacaagcat
1200gagatcgacc gcatgacgct gacgatggcc gtggagctgc ccgactcctt ctcccctgaa
1260ctacgctccc tgctggaggg gttgctgcag agggatgtca accggagatt gggctgcctg
1320ggccgagggg ctcaggaggt gaaagagagc ccctttttcc gctccctgga ctggcagatg
1380gtcttcttgc agaagtaccc tcccccgctg atccccccac gaggggaggt gaacgcggcc
1440gacgccttcg acattggctc cttcgatgag gaggacacaa aaggaatcaa gttactggac
1500agtgatcagg agctctaccg caacttcccc ctcaccatct cggagcggtg gcagcaggag
1560gtggcagaga ctgtcttcga caccatcaac gctgagacag accggctgga ggctcgcaag
1620aaagccaaga acaagcagct gggccatgag gaagactacg ccctgggcaa ggactgcatc
1680atgcatggct acatgtccaa gatgggcaac cccttcctga cccagtggca gcggcggtac
1740ttctacctgt tccccaaccg cctcgagtgg cggggcgagg gcgaggcccc gcagagcctg
1800ctgaccatgg aggagatcca gtcggtggag gagacgcaga tcaaggagcg caagtgcctg
1860ctcctcaaga tccgcggtgg gaaacagttc attttgcagt gcgatagcga ccctgagctg
1920gtgcagtgga agaaggagct gcgcgacgcc taccgcgagg cccagcagct ggtgcagcgg
1980gtgcccaaga tgaagaacaa gccgcgctcg cccgtggtgg agctgagcaa ggtgccgctg
2040gtccagcgcg gcagtgccaa cggcctctga
20705589PRTHomo sapiens 5Met Glu Leu Glu Asn Ile Val Ala Asn Thr Val Leu
Leu Lys Ala Arg1 5 10
15Glu Gly Gly Gly Gly Asn Arg Lys Gly Lys Ser Lys Lys Trp Arg Gln20
25 30Met Leu Gln Phe Pro His Ile Ser Gln Cys
Glu Glu Leu Arg Leu Ser35 40 45Leu Glu
Arg Asp Tyr His Ser Leu Cys Glu Arg Gln Pro Ile Gly Arg50
55 60Leu Leu Phe Arg Glu Phe Cys Ala Thr Arg Pro Glu
Leu Ser Arg Cys65 70 75
80Val Ala Phe Leu Asp Gly Val Ala Glu Tyr Glu Val Thr Pro Asp Asp85
90 95Lys Arg Lys Ala Cys Gly Arg Gln Leu Thr
Gln Asn Phe Leu Ser His100 105 110Thr Gly
Pro Asp Leu Ile Pro Glu Val Pro Arg Gln Leu Val Thr Asn115
120 125Cys Thr Gln Arg Leu Glu Gln Gly Pro Cys Lys Asp
Leu Phe Gln Glu130 135 140Leu Thr Arg Leu
Thr His Glu Tyr Leu Ser Val Ala Pro Phe Ala Asp145 150
155 160Tyr Leu Asp Ser Ile Tyr Phe Asn Arg
Phe Leu Gln Trp Lys Trp Leu165 170 175Glu
Arg Gln Pro Val Thr Lys Asn Thr Phe Arg Gln Tyr Arg Val Leu180
185 190Gly Lys Gly Gly Phe Gly Glu Val Cys Ala Cys
Gln Val Arg Ala Thr195 200 205Gly Lys Met
Tyr Ala Cys Lys Lys Leu Glu Lys Lys Arg Ile Lys Lys210
215 220Arg Lys Gly Glu Ala Met Ala Leu Asn Glu Lys Gln
Ile Leu Glu Lys225 230 235
240Val Asn Ser Arg Phe Val Val Ser Leu Ala Tyr Ala Tyr Glu Thr Lys245
250 255Asp Ala Leu Cys Leu Val Leu Thr Leu
Met Asn Gly Gly Asp Leu Lys260 265 270Phe
His Ile Tyr His Met Gly Gln Ala Gly Phe Pro Glu Ala Arg Ala275
280 285Val Phe Tyr Ala Ala Glu Ile Cys Cys Gly Leu
Glu Asp Leu His Arg290 295 300Glu Arg Ile
Val Tyr Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu Asp305
310 315 320Asp His Gly His Ile Arg Ile
Ser Asp Leu Gly Leu Ala Val His Val325 330
335Pro Glu Gly Gln Thr Ile Lys Gly Arg Val Gly Thr Val Gly Tyr Met340
345 350Ala Pro Glu Val Val Lys Asn Glu Arg
Tyr Thr Phe Ser Pro Asp Trp355 360 365Trp
Ala Leu Gly Cys Leu Leu Tyr Glu Met Ile Ala Gly Gln Ser Pro370
375 380Phe Gln Gln Arg Lys Lys Lys Ile Lys Arg Glu
Glu Val Glu Arg Leu385 390 395
400Val Lys Glu Val Pro Glu Glu Tyr Ser Glu Arg Phe Ser Pro Gln
Ala405 410 415Arg Ser Leu Cys Ser Gln Leu
Leu Cys Lys Asp Pro Ala Glu Arg Leu420 425
430Gly Cys Arg Gly Gly Ser Ala Arg Glu Val Lys Glu His Pro Leu Phe435
440 445Lys Lys Leu Asn Phe Lys Arg Leu Gly
Ala Gly Met Leu Glu Pro Pro450 455 460Phe
Lys Pro Asp Pro Gln Ala Ile Tyr Cys Lys Asp Val Leu Asp Ile465
470 475 480Glu Gln Phe Ser Thr Val
Lys Gly Val Glu Leu Glu Pro Thr Asp Gln485 490
495Asp Phe Tyr Gln Lys Phe Ala Thr Gly Ser Val Pro Ile Pro Trp
Gln500 505 510Asn Glu Met Val Glu Thr Glu
Cys Phe Gln Glu Leu Asn Val Phe Gly515 520
525Leu Asp Gly Ser Val Pro Pro Asp Leu Asp Trp Lys Gly Gln Pro Pro530
535 540Ala Pro Pro Lys Lys Gly Leu Leu Gln
Arg Leu Phe Ser Arg Gln Arg545 550 555
560Ile Ala Val Glu Thr Ala Ala Thr Ala Arg Lys Ser Ser Pro
Pro Ala565 570 575Ser Ser Pro Gln Pro Glu
Ala Pro Thr Ser Ser Trp Arg580 58561770DNAHomo sapiens
6atggagctcg agaacatcgt agcgaacacg gtgctactca aggcccggga aggtggcggt
60ggaaatcgca aaggcaaaag caagaaatgg cggcagatgc tccagttccc tcacatcagc
120cagtgcgaag agctgcggct cagcctcgag cgtgactatc acagcctgtg cgagcggcag
180cccattgggc gcctgctgtt ccgagagttc tgtgccacga ggccggagct gagccgctgc
240gtcgccttcc tggatggggt ggccgagtat gaagtgaccc cggatgacaa gcggaaggca
300tgtgggcggc agctaacgca gaattttctg agccacacgg gtcctgacct catccctgag
360gtcccccggc agctggtgac gaactgcacc cagcggctgg agcagggtcc ctgcaaagac
420cttttccagg aactcacccg gctgacccac gagtacctga gcgtggcccc ttttgccgac
480tacctcgaca gcatctactt caaccgtttc ctgcagtgga agtggctgga aaggcagcca
540gtgaccaaaa acaccttcag gcaataccga gtcctgggca aaggtggctt tggggaggtg
600tgcgcctgcc aggtgcgggc cacaggtaag atgtatgcct gcaagaagct agagaaaaag
660cggatcaaga agcggaaagg ggaggccatg gcgctgaacg agaagcagat cctggagaaa
720gtgaacagta ggtttgtagt gagcttggcc tacgcctatg agaccaagga cgcgctgtgc
780ctggtgctga cactgatgaa cgggggcgac ctcaagttcc acatctacca catgggccag
840gctggcttcc ccgaagcgcg ggccgtcttc tacgccgccg agatctgctg tggcctggag
900gacctgcacc gggagcgcat cgtgtacagg gacctgaagc ccgagaacat cttgctggat
960gaccacggcc acatccgcat ctctgacctg ggactagctg tgcatgtgcc cgagggccag
1020accatcaaag ggcgtgtggg caccgtgggt tacatggctc cggaggtggt gaagaatgaa
1080cggtacacgt tcagccctga ctggtgggcg ctcggctgcc tcctgtacga gatgatcgca
1140ggccagtcgc ccttccagca gaggaagaag aagatcaagc gggaggaggt ggagcggctg
1200gtgaaggagg tccccgagga gtattccgag cgcttttccc cgcaggcccg ctcactttgc
1260tcacagctcc tctgcaagga ccctgccgaa cgcctggggt gtcgtggggg cagtgcccgc
1320gaggtgaagg agcaccccct ctttaagaag ctgaacttca agcggctggg agctggcatg
1380ctggagccgc cgttcaagcc tgacccccag gccatttact gcaaggatgt tctggacatt
1440gaacagttct ctacggtcaa gggcgtggag ctggagccta ccgaccagga cttctaccag
1500aagtttgcca caggcagtgt gcccatcccc tggcagaacg agatggtgga gaccgagtgc
1560ttccaagagc tgaatgtctt tgggctggat ggctcagttc ccccagacct ggactggaag
1620ggccagccac ctgcacctcc taaaaaggga ctgctgcaga gactcttcag tcgccaaagg
1680attgctgtgg aaactgcagc gacagcgagg aagagctccc cacccgcctc tagcccccag
1740cccgaggccc ccaccagcag ttggcggtag
17707560PRTHomo sapiens 7Met Glu Leu Glu Asn Ile Val Ala Asn Thr Val Leu
Leu Lys Ala Arg1 5 10
15Glu Gly Gly Gly Gly Asn Arg Lys Gly Lys Ser Lys Lys Trp Arg Gln20
25 30Met Leu Gln Phe Pro His Ile Ser Gln Cys
Glu Glu Leu Arg Leu Ser35 40 45Leu Glu
Arg Asp Tyr His Ser Leu Cys Glu Arg Gln Pro Ile Gly Arg50
55 60Leu Leu Phe Arg Glu Phe Cys Ala Thr Arg Pro Glu
Leu Ser Arg Cys65 70 75
80Val Ala Phe Leu Asp Gly Val Ala Glu Tyr Glu Val Thr Pro Asp Asp85
90 95Lys Arg Lys Ala Cys Gly Arg Gln Leu Thr
Gln Asn Phe Leu Ser His100 105 110Thr Gly
Pro Asp Leu Ile Pro Glu Val Pro Arg Gln Leu Val Thr Asn115
120 125Cys Thr Gln Arg Leu Glu Gln Gly Pro Cys Lys Asp
Leu Phe Gln Glu130 135 140Leu Thr Arg Leu
Thr His Glu Tyr Leu Ser Val Ala Pro Phe Ala Asp145 150
155 160Tyr Leu Asp Ser Ile Tyr Phe Asn Arg
Phe Leu Gln Trp Lys Trp Leu165 170 175Glu
Arg Gln Pro Val Thr Lys Asn Thr Phe Arg Gln Tyr Arg Val Leu180
185 190Gly Lys Gly Gly Phe Gly Glu Val Cys Ala Cys
Gln Val Arg Ala Thr195 200 205Gly Lys Met
Tyr Ala Cys Lys Lys Leu Glu Lys Lys Arg Ile Lys Lys210
215 220Arg Lys Gly Glu Ala Met Ala Leu Asn Glu Lys Gln
Ile Leu Glu Lys225 230 235
240Val Asn Ser Arg Phe Val Val Ser Leu Ala Tyr Ala Tyr Glu Thr Lys245
250 255Asp Ala Leu Cys Leu Val Leu Thr Leu
Met Asn Gly Gly Asp Leu Lys260 265 270Phe
His Ile Tyr His Met Gly Gln Ala Gly Phe Pro Glu Ala Arg Ala275
280 285Val Phe Tyr Ala Ala Glu Ile Cys Cys Gly Leu
Glu Asp Leu His Arg290 295 300Glu Arg Ile
Val Tyr Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu Asp305
310 315 320Asp His Gly His Ile Arg Ile
Ser Asp Leu Gly Leu Ala Val His Val325 330
335Pro Glu Gly Gln Thr Ile Lys Gly Arg Val Gly Thr Val Gly Tyr Met340
345 350Ala Pro Glu Val Val Lys Asn Glu Arg
Tyr Thr Phe Ser Pro Asp Trp355 360 365Trp
Ala Leu Gly Cys Leu Leu Tyr Glu Met Ile Ala Gly Gln Ser Pro370
375 380Phe Gln Gln Arg Lys Lys Lys Ile Lys Arg Glu
Glu Val Glu Arg Leu385 390 395
400Val Lys Glu Val Pro Glu Glu Tyr Ser Glu Arg Phe Ser Pro Gln
Ala405 410 415Arg Ser Leu Cys Ser Gln Leu
Leu Cys Lys Asp Pro Ala Glu Arg Leu420 425
430Gly Cys Arg Gly Gly Ser Ala Arg Glu Val Lys Glu His Pro Leu Phe435
440 445Lys Lys Leu Asn Phe Lys Arg Leu Gly
Ala Gly Met Leu Glu Pro Pro450 455 460Phe
Lys Pro Asp Pro Gln Ala Ile Tyr Cys Lys Asp Val Leu Asp Ile465
470 475 480Glu Gln Phe Ser Thr Val
Lys Gly Val Glu Leu Glu Pro Thr Asp Gln485 490
495Asp Phe Tyr Gln Lys Phe Ala Thr Gly Ser Val Pro Ile Pro Trp
Gln500 505 510Asn Glu Met Val Glu Thr Glu
Cys Phe Gln Glu Leu Asn Val Phe Gly515 520
525Leu Asp Gly Ser Val Pro Pro Asp Leu Asp Trp Lys Gly Gln Pro Pro530
535 540Ala Pro Pro Lys Lys Gly Leu Leu Gln
Arg Leu Phe Ser Arg Gln Arg545 550 555
56081683DNAHomo sapiens 8atggagctcg agaacatcgt agcgaacacg
gtgctactca aggcccggga aggtggcggt 60ggaaatcgca aaggcaaaag caagaaatgg
cggcagatgc tccagttccc tcacatcagc 120cagtgcgaag agctgcggct cagcctcgag
cgtgactatc acagcctgtg cgagcggcag 180cccattgggc gcctgctgtt ccgagagttc
tgtgccacga ggccggagct gagccgctgc 240gtcgccttcc tggatggggt ggccgagtat
gaagtgaccc cggatgacaa gcggaaggca 300tgtgggcggc agctaacgca gaattttctg
agccacacgg gtcctgacct catccctgag 360gtcccccggc agctggtgac gaactgcacc
cagcggctgg agcagggtcc ctgcaaagac 420cttttccagg aactcacccg gctgacccac
gagtacctga gcgtggcccc ttttgccgac 480tacctcgaca gcatctactt caaccgtttc
ctgcagtgga agtggctgga aaggcagcca 540gtgaccaaaa acaccttcag gcaataccga
gtcctgggca aaggtggctt tggggaggtg 600tgcgcctgcc aggtgcgggc cacaggtaag
atgtatgcct gcaagaagct agagaaaaag 660cggatcaaga agcggaaagg ggaggccatg
gcgctgaacg agaagcagat cctggagaaa 720gtgaacagta ggtttgtagt gagcttggcc
tacgcctatg agaccaagga cgcgctgtgc 780ctggtgctga cactgatgaa cgggggcgac
ctcaagttcc acatctacca catgggccag 840gctggcttcc ccgaagcgcg ggccgtcttc
tacgccgccg agatctgctg tggcctggag 900gacctgcacc gggagcgcat cgtgtacagg
gacctgaagc ccgagaacat cttgctggat 960gaccacggcc acatccgcat ctctgacctg
ggactagctg tgcatgtgcc cgagggccag 1020accatcaaag ggcgtgtggg caccgtgggt
tacatggctc cggaggtggt gaagaatgaa 1080cggtacacgt tcagccctga ctggtgggcg
ctcggctgcc tcctgtacga gatgatcgca 1140ggccagtcgc ccttccagca gaggaagaag
aagatcaagc gggaggaggt ggagcggctg 1200gtgaaggagg tccccgagga gtattccgag
cgcttttccc cgcaggcccg ctcactttgc 1260tcacagctcc tctgcaagga ccctgccgaa
cgcctggggt gtcgtggggg cagtgcccgc 1320gaggtgaagg agcaccccct ctttaagaag
ctgaacttca agcggctggg agctggcatg 1380ctggagccgc cgttcaagcc tgacccccag
gccatttact gcaaggatgt tctggacatt 1440gaacagttct ctacggtcaa gggcgtggag
ctggagccta ccgaccagga cttctaccag 1500aagtttgcca caggcagtgt gcccatcccc
tggcagaacg agatggtgga gaccgagtgc 1560ttccaagagc tgaatgtctt tgggctggat
ggctcagttc ccccagacct ggactggaag 1620ggccagccac ctgcacctcc taaaaaggga
ctgctgcaga gactcttcag tcgccaaagg 1680tga
16839576PRTHomo sapiens 9Met Glu Leu Glu
Asn Ile Val Ala Asn Thr Val Leu Leu Lys Ala Arg1 5
10 15Glu Gly Gly Gly Gly Asn Arg Lys Gly Lys Ser
Lys Lys Trp Arg Gln20 25 30Met Leu Gln
Phe Pro His Ile Ser Gln Cys Glu Glu Leu Arg Leu Ser35 40
45Leu Glu Arg Asp Tyr His Ser Leu Cys Glu Arg His Ala
Ile Gly Arg50 55 60Leu Leu Phe Arg Glu
Phe Cys Ala Thr Arg Pro Glu Leu Ser Arg Cys65 70
75 80Val Ala Phe Leu Asp Gly Val Ala Glu Tyr
Glu Val Thr Pro Asp Asp85 90 95Lys Arg
Lys Ala Cys Gly Arg His Val Thr Gln Asn Phe Leu Ser His100
105 110Thr Gly Pro Asp Leu Ile Pro Glu Val Pro Arg Gln
Leu Val Thr Asn115 120 125Cys Thr Gln Arg
Leu Glu Gln Gly Pro Cys Lys Asp Leu Phe Gln Glu130 135
140Leu Thr Arg Leu Thr His Glu Tyr Leu Ser Val Ala Pro Phe
Ala Asp145 150 155 160Tyr
Leu Asp Ser Ile Tyr Phe Asn Arg Phe Leu Gln Trp Lys Trp Leu165
170 175Glu Arg Gln Pro Val Thr Lys Asn Thr Phe Arg
Gln Tyr Arg Val Leu180 185 190Gly Lys Gly
Gly Phe Gly Glu Val Cys Ala Cys Gln Val Arg Ala Thr195
200 205Gly Lys Met Tyr Ala Cys Lys Lys Leu Glu Lys Lys
Arg Ile Lys Lys210 215 220Arg Lys Gly Glu
Ala Met Ala Leu Asn Glu Lys Gln Ile Leu Glu Lys225 230
235 240Val Asn Ser Arg Phe Val Val Ser Leu
Ala Tyr Ala Tyr Glu Thr Lys245 250 255Asp
Ala Leu Cys Leu Val Leu Thr Leu Met Asn Gly Gly Asp Leu Lys260
265 270Phe His Ile Tyr His Met Gly Gln Ala Gly Phe
Pro Glu Ala Arg Ala275 280 285Val Phe Tyr
Ala Ala Glu Ile Cys Cys Gly Leu Glu Asp Leu His Arg290
295 300Glu Arg Ile Val Tyr Arg Asp Leu Lys Pro Glu Asn
Ile Leu Leu Asp305 310 315
320Asp His Gly His Ile Arg Ile Ser Asp Leu Gly Leu Ala Val His Val325
330 335Pro Glu Gly Gln Thr Ile Lys Gly Arg
Val Gly Thr Val Gly Tyr Met340 345 350Ala
Pro Glu Val Val Lys Asn Glu Arg Tyr Thr Phe Ser Pro Asp Trp355
360 365Trp Ala Leu Gly Cys Leu Leu Tyr Glu Met Ile
Ala Gly Gln Ser Pro370 375 380Phe Gln Gln
Arg Lys Lys Lys Ile Lys Arg Glu Glu Val Glu Arg Leu385
390 395 400Val Lys Glu Val Pro Glu Glu
Tyr Ser Glu Arg Phe Ser Pro Gln Ala405 410
415Arg Ser Leu Cys Ser Gln Leu Leu Cys Lys Asp Pro Ala Glu Arg Leu420
425 430Gly Cys Arg Gly Gly Ser Ala Arg Glu
Val Lys Glu His Pro Leu Phe435 440 445Lys
Lys Leu Asn Phe Lys Arg Leu Gly Ala Gly Met Leu Glu Pro Pro450
455 460Phe Lys Pro Asp Pro Gln Ala Ile Tyr Cys Lys
Asp Val Leu Asp Ile465 470 475
480Glu Gln Phe Ser Thr Val Lys Gly Val Glu Leu Glu Pro Thr Asp
Gln485 490 495Asp Phe Tyr Gln Lys Phe Ala
Thr Gly Ser Val Pro Ile Pro Trp Gln500 505
510Asn Glu Met Val Glu Thr Glu Cys Phe Gln Glu Leu Asn Val Phe Gly515
520 525Leu Asp Gly Ser Val Pro Pro Asp Leu
Asp Trp Lys Gly Gln Pro Pro530 535 540Ala
Pro Pro Lys Lys Gly Leu Leu Gln Arg Leu Phe Ser Arg Gln Asp545
550 555 560Cys Cys Gly Asn Cys Ser
Asp Ser Glu Glu Glu Leu Pro Thr Arg Leu565 570
575101738DNAHomo sapiens 10atggagctcg agaacatcgt agcgaacacg
gtgctactca aggcccggga aggtggcggt 60ggaaatcgca aaggcaaaag caagaaatgg
cggcagatgc tccagttccc tcacatcagc 120cagtgcgaag agctgcggct cagcctcgag
cgtgactatc acagcctgtg cgagcggcac 180gccattgggc gcctgctgtt ccgagagttc
tgtgccacga ggccggagct gagccgctgc 240gtcgccttcc tggatggggt ggccgagtat
gaagtgaccc cggatgacaa gcggaaggca 300tgtgggcggc acgtaacgca gaattttctg
agccacacgg gtcctgacct catccctgag 360gtcccccggc agctggtgac gaactgcacc
cagcggctgg agcagggtcc ctgcaaagac 420cttttccagg aactcacccg gctgacccac
gagtacctga gcgtggcccc ttttgccgac 480tacctcgaca gcatctactt caaccgtttc
ctgcagtgga agtggctgga aaggcagcca 540gtgaccaaaa acaccttcag gcaataccga
gtcctgggca aaggtggctt tggggaggtg 600tgcgcctgcc aggtgcgggc cacaggtaag
atgtatgcct gcaagaagct agagaaaaag 660cggatcaaga agcggaaagg ggaggccatg
gcgctgaacg agaagcagat cctggagaaa 720gtgaacagta ggtttgtagt gagcttggcc
tacgcctatg agaccaagga cgcgctgtgc 780ctggtgctga cactgatgaa cgggggcgac
ctcaagttcc acatctacca catgggccag 840gctggcttcc ccgaagcgcg ggccgtcttc
tacgccgccg agatctgctg tggcctggag 900gacctgcacc gggagcgcat cgtgtacagg
gacctgaagc ccgagaacat cttgctggat 960gaccacggcc acatccgcat ctctgacctg
ggactagctg tgcatgtgcc cgagggccag 1020accatcaaag ggcgtgtggg caccgtgggt
tacatggctc cggaggtggt gaagaatgaa 1080cggtacacgt tcagccctga ctggtgggcg
ctcggctgcc tcctgtacga gatgatcgca 1140ggccagtcgc ccttccagca gaggaagaag
aagatcaagc gggaggaggt ggagcggctg 1200gtgaaggagg tccccgagga gtattccgag
cgcttttccc cgcaggcccg ctcactttgc 1260tcacagctcc tctgcaagga ccctgccgaa
cgcctggggt gtcgtggggg cagtgcccgc 1320gaggtgaagg agcaccccct ctttaagaag
ctgaacttca agcggctggg agctggcatg 1380ctggagccgc cgttcaagcc tgacccccag
gccatttact gcaaggatgt tctggacatt 1440gaacagttct ctacggtcaa gggcgtggag
ctggagccta ccgaccagga cttctaccag 1500aagtttgcca caggcagtgt gcccatcccc
tggcagaacg agatggtgga gaccgagtgc 1560ttccaagagc tgaatgtctt tgggctggat
ggctcagttc ccccagacct ggactggaag 1620ggccagccac ctgcacctcc taaaaaggga
ctgctgcaga gactcttcag tcgccaagat 1680tgctgtggaa actgcagcga cagcgaggaa
gagctcccca cccgcctcta gcccccag 173811590PRTHomo sapiens 11Met Glu Leu
Glu Asn Ile Val Ala Asn Thr Val Leu Leu Lys Ala Arg1 5
10 15Glu Gly Gly Gly Gly Lys Arg Lys Gly Lys
Ser Lys Lys Trp Lys Glu20 25 30Ile Leu
Lys Phe Pro His Ile Ser Gln Cys Glu Asp Leu Arg Arg Thr35
40 45Ile Asp Arg Asp Tyr Cys Ser Leu Cys Asp Lys Gln
Pro Ile Gly Arg50 55 60Leu Leu Phe Arg
Gln Phe Cys Glu Thr Arg Pro Gly Leu Glu Cys Tyr65 70
75 80Ile Gln Phe Leu Asp Ser Val Ala Glu
Tyr Glu Val Thr Pro Asp Glu85 90 95Lys
Leu Gly Glu Lys Gly Lys Glu Ile Met Thr Lys Tyr Leu Thr Pro100
105 110Lys Ser Pro Val Phe Ile Ala Gln Val Gly Gln
Asp Leu Val Ser Gln115 120 125Thr Glu Glu
Lys Leu Leu Gln Lys Pro Cys Lys Glu Leu Phe Ser Ala130
135 140Cys Ala Gln Ser Val His Glu Tyr Leu Arg Gly Glu
Pro Phe His Glu145 150 155
160Tyr Leu Asp Ser Met Phe Phe Asp Arg Phe Leu Gln Trp Lys Trp Leu165
170 175Glu Arg Gln Pro Val Thr Lys Asn Thr
Phe Arg Gln Tyr Arg Val Leu180 185 190Gly
Lys Gly Gly Phe Gly Glu Val Cys Ala Cys Gln Val Arg Ala Thr195
200 205Gly Lys Met Tyr Ala Cys Lys Arg Leu Glu Lys
Lys Arg Ile Lys Lys210 215 220Arg Lys Gly
Glu Ser Met Ala Leu Asn Glu Lys Gln Ile Leu Glu Lys225
230 235 240Val Asn Ser Gln Phe Val Val
Asn Leu Ala Tyr Ala Tyr Glu Thr Lys245 250
255Asp Ala Leu Cys Leu Val Leu Thr Ile Met Asn Gly Gly Asp Leu Lys260
265 270Phe His Ile Tyr Asn Met Gly Asn Pro
Gly Phe Glu Glu Glu Arg Ala275 280 285Leu
Phe Tyr Ala Ala Glu Ile Leu Cys Gly Leu Glu Asp Leu His Arg290
295 300Glu Asn Thr Val Tyr Arg Asp Leu Lys Pro Glu
Asn Ile Leu Leu Asp305 310 315
320Asp Tyr Gly His Ile Arg Ile Ser Asp Leu Gly Leu Ala Val Lys
Ile325 330 335Pro Glu Gly Asp Leu Ile Arg
Gly Arg Val Gly Thr Val Gly Tyr Met340 345
350Ala Pro Glu Val Leu Asn Asn Gln Arg Tyr Gly Leu Ser Pro Asp Tyr355
360 365Trp Gly Leu Gly Cys Leu Ile Tyr Glu
Met Ile Glu Gly Gln Ser Pro370 375 380Phe
Arg Gly Arg Lys Glu Lys Val Lys Arg Glu Glu Val Asp Arg Arg385
390 395 400Val Leu Glu Thr Glu Glu
Val Tyr Ser His Lys Phe Ser Glu Glu Ala405 410
415Lys Ser Ile Cys Lys Met Leu Leu Thr Lys Asp Ala Lys Gln Arg
Leu420 425 430Gly Cys Gln Glu Glu Gly Ala
Ala Glu Val Lys Arg His Pro Phe Phe435 440
445Arg Asn Met Asn Phe Lys Arg Leu Glu Ala Gly Met Leu Asp Pro Pro450
455 460Phe Val Pro Asp Pro Arg Ala Val Tyr
Cys Lys Asp Val Leu Asp Ile465 470 475
480Glu Gln Phe Ser Thr Val Lys Gly Val Asn Leu Asp His Thr
Asp Asp485 490 495Asp Phe Tyr Ser Lys Phe
Ser Thr Gly Ser Val Ser Ile Pro Trp Gln500 505
510Asn Glu Met Ile Glu Thr Glu Cys Phe Lys Glu Leu Asn Val Phe
Gly515 520 525Pro Asn Gly Thr Leu Pro Pro
Asp Leu Asn Arg Asn His Pro Pro Glu530 535
540Pro Pro Lys Lys Gly Leu Leu Gln Arg Leu Phe Lys Arg Gln His Gln545
550 555 560Asn Asn Ser Lys
Ser Ser Pro Ser Ser Lys Thr Ser Phe Asn His His565 570
575Ile Asn Ser Asn His Val Ser Ser Asn Ser Thr Gly Ser
Ser580 585 590121773DNAHomo sapiens
12atggagctgg aaaacatcgt ggccaacacg gtcttgctga aagccaggga agggggcgga
60ggaaagcgca aagggaaaag caagaagtgg aaagaaatcc tgaagttccc tcacattagc
120cagtgtgaag acctccgaag gaccatagac agagattact gcagtttatg tgacaagcag
180ccaatcggga ggctgctttt ccggcagttt tgtgaaacca ggcctgggct ggagtgttac
240attcagttcc tggactccgt ggcagaatat gaagttactc cagatgaaaa actgggagag
300aaagggaagg aaattatgac caagtacctc accccaaagt cccctgtttt catagcccaa
360gttggccaag acctggtctc ccagacggag gagaagctcc tacagaagcc gtgcaaagaa
420ctcttttctg cctgtgcaca gtctgtccac gagtacctga ggggagaacc attccacgaa
480tatctggaca gcatgttttt tgaccgcttt ctccagtgga agtggttgga aaggcaaccg
540gtgaccaaaa acactttcag gcagtatcga gtgctaggaa aagggggctt cggggaggtc
600tgtgcctgcc aggttcgggc cacgggtaaa atgtatgcct gcaagcgctt ggagaagaag
660aggatcaaaa agaggaaagg ggagtccatg gccctcaatg agaagcagat cctcgagaag
720gtcaacagtc agtttgtggt caacctggcc tatgcctacg agaccaagga tgcactgtgc
780ttggtcctga ccatcatgaa tgggggtgac ctgaagttcc acatctacaa catgggcaac
840cctggcttcg aggaggagcg ggccttgttt tatgcggcag agatcctctg cggcttagaa
900gacctccacc gtgagaacac cgtctaccga gatctgaaac ctgaaaacat cctgttagat
960gattatggcc acattaggat ctcagacctg ggcttggctg tgaagatccc cgagggagac
1020ctgatccgcg gccgggtggg cactgttggc tacatggctc cagaggtcct gaacaaccag
1080aggtacggcc tgagccccga ctactggggc cttggctgcc tcatctatga gatgatcgag
1140ggccagtcgc cgttccgcgg ccgcaaggag aaggtgaagc gggaggaggt ggaccgccgg
1200gtcctggaga cggaggaggt gtactcccac aagttctccg aggaggccaa gtccatctgc
1260aagatgctgc tcacgaaaga tgcgaagcag aggctgggct gccaggagga gggggctgca
1320gaggtcaaga gacacccctt cttcaggaac atgaacttca agcgcttaga agccgggatg
1380ttggaccctc ccttcgttcc agacccccgc gctgtgtact gtaaggacgt gctggacatc
1440gagcagttct ccactgtgaa gggcgtcaat ctggaccaca cagacgacga cttctactcc
1500aagttctcca cgggctctgt gtccatccca tggcaaaacg agatgataga aacagaatgc
1560tttaaggagc tgaacgtgtt tggacctaat ggtaccctcc cgccagatct gaacagaaac
1620caccctccgg aaccgcccaa gaaagggctg ctccagagac tcttcaagcg gcagcatcag
1680aacaattcca agagttcgcc cagctccaag accagtttta accaccacat aaactcaaac
1740catgtcagct cgaactccac cggaagcagc tag
177313500PRTHomo sapiens 13Met Glu Leu Glu Asn Ile Val Ala Asn Ser Leu
Leu Leu Lys Ala Arg1 5 10
15Gln Glu Lys Asp Tyr Ser Ser Leu Cys Asp Lys Gln Pro Ile Gly Arg20
25 30Arg Leu Phe Arg Gln Phe Cys Asp Thr Lys
Pro Thr Leu Lys Arg His35 40 45Ile Glu
Phe Leu Asp Ala Val Ala Glu Tyr Glu Val Ala Asp Asp Glu50
55 60Asp Arg Ser Asp Cys Gly Leu Ser Ile Leu Asp Arg
Phe Phe Asn Asp65 70 75
80Lys Leu Ala Ala Pro Leu Pro Glu Ile Pro Pro Asp Val Val Thr Glu85
90 95Cys Arg Leu Gly Leu Lys Glu Glu Asn Pro
Ser Lys Lys Ala Phe Glu100 105 110Glu Cys
Thr Arg Val Ala His Asn Tyr Leu Arg Gly Glu Pro Phe Glu115
120 125Glu Tyr Gln Glu Ser Ser Tyr Phe Ser Gln Phe Leu
Gln Trp Lys Trp130 135 140Leu Glu Arg Gln
Pro Val Thr Lys Asn Thr Phe Arg His Tyr Arg Val145 150
155 160Leu Gly Lys Gly Gly Phe Gly Glu Val
Cys Ala Cys Gln Val Arg Ala165 170 175Thr
Gly Lys Met Tyr Ala Cys Lys Lys Leu Gln Lys Lys Arg Ile Lys180
185 190Lys Arg Lys Gly Glu Ala Met Ala Leu Asn Glu
Lys Arg Ile Leu Glu195 200 205Lys Val Gln
Ser Arg Phe Val Val Ser Leu Ala Tyr Ala Tyr Glu Thr210
215 220Lys Asp Ala Leu Cys Leu Val Leu Thr Ile Met Asn
Gly Gly Asp Leu225 230 235
240Lys Phe His Ile Tyr Asn Leu Gly Asn Pro Gly Phe Asp Glu Gln Arg245
250 255Ala Val Phe Tyr Ala Ala Glu Leu Cys
Cys Gly Leu Glu Asp Leu Gln260 265 270Arg
Glu Arg Ile Val Tyr Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu275
280 285Asp Asp Arg Gly His Ile Arg Ile Ser Asp Leu
Gly Leu Ala Thr Glu290 295 300Ile Pro Glu
Gly Gln Arg Val Arg Gly Arg Val Gly Thr Val Gly Tyr305
310 315 320Met Ala Pro Glu Val Val Asn
Asn Glu Lys Tyr Thr Phe Ser Pro Asp325 330
335Trp Trp Gly Leu Gly Cys Leu Ile Tyr Glu Met Ile Gln Gly His Ser340
345 350Pro Phe Lys Lys Tyr Lys Glu Lys Val
Lys Trp Glu Glu Val Asp Gln355 360 365Arg
Ile Lys Asn Asp Thr Glu Glu Tyr Ser Glu Lys Phe Ser Glu Asp370
375 380Ala Lys Ser Ile Cys Arg Met Leu Leu Thr Lys
Asn Pro Ser Lys Arg385 390 395
400Leu Gly Cys Arg Gly Glu Gly Ala Ala Gly Val Lys Gln His Pro
Val405 410 415Phe Lys Asp Ile Asn Phe Arg
Arg Leu Glu Ala Asn Met Leu Glu Pro420 425
430Pro Phe Cys Pro Asp Pro His Ala Val Tyr Cys Lys Asp Val Leu Asp435
440 445Ile Glu Gln Phe Ser Ala Val Lys Gly
Ile Tyr Leu Asp Thr Ala Asp450 455 460Glu
Asp Phe Tyr Ala Arg Phe Ala Thr Gly Cys Val Ser Ile Pro Trp465
470 475 480Gln Asn Glu Asp Cys Leu
Thr Met Val Pro Ser Glu Lys Glu Val Glu485 490
495Pro Lys Gln Cys500141503DNAHomo sapiens 14atggagctcg agaacatcgt
ggccaactcg ctgctgctga aagcgcgtca agaaaaggat 60tatagcagtc tttgtgacaa
gcaaccgata ggaagacgtc tcttcaggca gttctgtgat 120accaaaccca ctctaaagag
gcacattgaa ttcttggatg cagtggcaga atatgaagtt 180gccgatgatg aggaccgaag
tgattgtgga ctgtcaatct tagatagatt cttcaatgat 240aagttggcag cccctttacc
agaaatacct ccagatgttg tgacagaatg tagattggga 300ctgaaggagg agaacccttc
caaaaaagcc tttgaggaat gtactagagt tgcccataac 360tacctaagag gggaaccatt
tgaagaatac caagaaagct catatttttc tcagttttta 420caatggaaat ggctggaaag
gcaacccgta acaaagaaca catttagaca ttacagagtt 480ctaggaaaag gcggatttgg
agaggtttgc gcctgtcaag tgcgagccac aggaaaaatg 540tatgcctgca aaaagctaca
aaaaaaaaga ataaagaaga ggaaaggtga agctatggct 600ctaaatgaga aaagaattct
ggagaaagtg caaagtagat tcgtagttag tttagcctac 660gcttatgaaa ccaaagatgc
cttgtgcttg gtgctcacca ttatgaatgg aggggatttg 720aagtttcaca tttacaacct
gggcaatccc ggctttgatg agcagagagc cgttttctat 780gctgcagagc tgtgttgcgg
cttggaagat ttacagaggg aaagaattgt atacagagac 840ttgaagcctg agaatattct
ccttgatgat cgtggacaca tccggatttc agacctcggt 900ttggccacag agatcccaga
aggacagagg gttcgaggaa gagttggaac agtcggctac 960atggcacctg aagttgtcaa
taatgaaaag tatacgttta gtcccgattg gtggggactt 1020ggctgtctga tctatgaaat
gattcaggga cattctccat tcaaaaaata caaagagaaa 1080gtcaaatggg aggaggtcga
tcaaagaatc aagaatgata ccgaggagta ttctgagaag 1140ttttcagagg atgccaaatc
tatctgcagg atgttactca ccaagaatcc aagcaagcgg 1200ctgggctgca ggggcgaggg
agcggctggg gtgaagcagc accccgtgtt caaggacatc 1260aacttcagga ggctggaggc
aaacatgctg gagccccctt tctgtcctga tcctcatgcc 1320gtttactgta aggacgtcct
ggatatcgag cagttctcgg cggtgaaagg gatctacctg 1380gacaccgcag atgaagactt
ctatgctcgg tttgctaccg ggtgtgtctc catcccctgg 1440cagaatgagg actgcctgac
catggtcccc agtgagaagg aagtggaacc caagcaatgc 1500tga
150315546PRTHomo sapiens
15Met Glu Leu Glu Asn Ile Val Ala Asn Ser Leu Leu Leu Lys Ala Arg1
5 10 15Gln Glu Lys Asp Tyr Ser
Ser Leu Cys Asp Lys Gln Pro Ile Gly Arg20 25
30Arg Leu Phe Arg Gln Phe Cys Asp Thr Lys Pro Thr Leu Lys Arg His35
40 45Ile Glu Phe Leu Asp Ala Val Ala Glu
Tyr Glu Val Ala Asp Asp Glu50 55 60Asp
Arg Ser Asp Cys Gly Leu Ser Ile Leu Asp Arg Phe Phe Asn Asp65
70 75 80Lys Leu Ala Ala Pro Leu
Pro Glu Ile Pro Pro Asp Val Val Thr Glu85 90
95Cys Arg Leu Gly Leu Lys Glu Glu Asn Pro Ser Lys Lys Ala Phe Glu100
105 110Glu Cys Thr Arg Val Ala His Asn
Tyr Leu Arg Gly Glu Pro Phe Glu115 120
125Glu Tyr Gln Glu Ser Ser Tyr Phe Ser Gln Phe Leu Gln Trp Lys Trp130
135 140Leu Glu Arg Gln Pro Val Thr Lys Asn
Thr Phe Arg His Tyr Arg Val145 150 155
160Leu Gly Lys Gly Gly Phe Gly Glu Val Cys Ala Cys Gln Val
Arg Ala165 170 175Thr Gly Lys Met Tyr Ala
Cys Lys Lys Leu Gln Lys Lys Arg Ile Lys180 185
190Lys Arg Lys Gly Glu Ala Met Ala Leu Asn Glu Lys Arg Ile Leu
Glu195 200 205Lys Val Gln Ser Arg Phe Val
Val Ser Leu Ala Tyr Ala Tyr Glu Thr210 215
220Lys Asp Ala Leu Cys Leu Val Leu Thr Ile Met Asn Gly Gly Asp Leu225
230 235 240Lys Phe His Ile
Tyr Asn Leu Gly Asn Pro Gly Phe Asp Glu Gln Arg245 250
255Ala Val Phe Tyr Ala Ala Glu Leu Cys Cys Gly Leu Glu Asp
Leu Gln260 265 270Arg Glu Arg Ile Val Tyr
Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu275 280
285Asp Asp Arg Gly His Ile Arg Ile Ser Asp Leu Gly Leu Ala Thr
Glu290 295 300Ile Pro Glu Gly Gln Arg Val
Arg Gly Arg Val Gly Thr Val Gly Tyr305 310
315 320Met Ala Pro Glu Val Val Asn Asn Glu Lys Tyr Thr
Phe Ser Pro Asp325 330 335Trp Trp Gly Leu
Gly Cys Leu Ile Tyr Glu Met Ile Gln Gly His Ser340 345
350Pro Phe Lys Lys Tyr Lys Glu Lys Val Lys Trp Glu Glu Val
Asp Gln355 360 365Arg Ile Lys Asn Asp Thr
Glu Glu Tyr Ser Glu Lys Phe Ser Glu Asp370 375
380Ala Lys Ser Ile Cys Arg Met Leu Leu Thr Lys Asn Pro Ser Lys
Arg385 390 395 400Leu Gly
Cys Arg Gly Glu Gly Ala Ala Gly Val Lys Gln His Pro Val405
410 415Phe Lys Asp Ile Asn Phe Arg Arg Leu Glu Ala Asn
Met Leu Glu Pro420 425 430Pro Phe Cys Pro
Asp Pro His Ala Val Tyr Cys Lys Asp Val Leu Asp435 440
445Ile Glu Gln Phe Ser Ala Val Lys Gly Ile Tyr Leu Asp Thr
Ala Asp450 455 460Glu Asp Phe Tyr Ala Arg
Phe Ala Thr Gly Cys Val Ser Ile Pro Trp465 470
475 480Gln Asn Glu Met Ile Glu Ser Gly Cys Phe Lys
Asp Ile Asn Lys Ser485 490 495Glu Ser Glu
Glu Ala Leu Pro Leu Asp Leu Asp Lys Asn Ile His Thr500
505 510Pro Val Ser Arg Pro Asn Arg Gly Phe Phe Tyr Arg
Leu Phe Arg Arg515 520 525Gly Gly Cys Leu
Thr Met Val Pro Ser Glu Lys Glu Val Glu Pro Lys530 535
540Gln Cys545161641DNAHomo sapiens 16atggagctcg agaacatcgt
ggccaactcg ctgctgctga aagcgcgtca agaaaaggat 60tatagcagtc tttgtgacaa
gcaaccgata ggaagacgtc tcttcaggca gttctgtgat 120accaaaccca ctctaaagag
gcacattgaa ttcttggatg cagtggcaga atatgaagtt 180gccgatgatg aggaccgaag
tgattgtgga ctgtcaatct tagatagatt cttcaatgat 240aagttggcag cccctttacc
agaaatacct ccagatgttg tgacagaatg tagattggga 300ctgaaggagg agaacccttc
caaaaaagcc tttgaggaat gtactagagt tgcccataac 360tacctaagag gggaaccatt
tgaagaatac caagaaagct catatttttc tcagttttta 420caatggaaat ggctggaaag
gcaacccgta acaaagaaca catttagaca ttacagagtt 480ctaggaaaag gcggatttgg
agaggtttgc gcctgtcaag tgcgagccac aggaaaaatg 540tatgcctgca aaaagctaca
aaaaaaaaga ataaagaaga ggaaaggtga agctatggct 600ctaaatgaga aaagaattct
ggagaaagtg caaagtagat tcgtagttag tttagcctac 660gcttatgaaa ccaaagatgc
cttgtgcttg gtgctcacca ttatgaatgg aggggatttg 720aagtttcaca tttacaacct
gggcaatccc ggctttgatg agcagagagc cgttttctat 780gctgcagagc tgtgttgcgg
cttggaagat ttacagaggg aaagaattgt atacagagac 840ttgaagcctg agaatattct
ccttgatgat cgtggacaca tccggatttc agacctcggt 900ttggccacag agatcccaga
aggacagagg gttcgaggaa gagttggaac agtcggctac 960atggcacctg aagttgtcaa
taatgaaaag tatacgttta gtcccgattg gtggggactt 1020ggctgtctga tctatgaaat
gattcaggga cattctccat tcaaaaaata caaagagaaa 1080gtcaaatggg aggaggtcga
tcaaagaatc aagaatgata ccgaggagta ttctgagaag 1140ttttcagagg atgccaaatc
tatctgcagg atgttactca ccaagaatcc aagcaagcgg 1200ctgggctgca ggggcgaggg
agcggctggg gtgaagcagc accccgtgtt caaggacatc 1260aacttcagga ggctggaggc
aaacatgctg gagccccctt tctgtcctga tcctcatgcc 1320gtttactgta aggacgtcct
ggatatcgag cagttctcgg cggtgaaagg gatctacctg 1380gacaccgcag atgaagactt
ctatgctcgg tttgctaccg ggtgtgtctc catcccctgg 1440cagaatgaga tgatcgaatc
cgggtgtttc aaagacatca acaaaagtga aagtgaggaa 1500gctttgccat tagatctaga
caagaacata cataccccgg tttccagacc aaacagaggc 1560ttcttctata gactcttcag
aagagggggc tgcctgacca tggtccccag tgagaaggaa 1620gtggaaccca agcaatgctg a
164117578PRTHomo sapiens
17Met Glu Leu Glu Asn Ile Val Ala Asn Ser Leu Leu Leu Lys Ala Arg1
5 10 15Gln Gly Gly Tyr Gly Lys
Lys Ser Gly Arg Ser Lys Lys Trp Lys Glu20 25
30Ile Leu Thr Leu Pro Pro Val Ser Gln Cys Ser Glu Leu Arg His Ser35
40 45Ile Glu Lys Asp Tyr Ser Ser Leu Cys
Asp Lys Gln Pro Ile Gly Arg50 55 60Arg
Leu Phe Arg Gln Phe Cys Asp Thr Lys Pro Thr Leu Lys Arg His65
70 75 80Ile Glu Phe Leu Asp Ala
Val Ala Glu Tyr Glu Val Ala Asp Asp Glu85 90
95Asp Arg Ser Asp Cys Gly Leu Ser Ile Leu Asp Arg Phe Phe Asn Asp100
105 110Lys Leu Ala Ala Pro Leu Pro Glu
Ile Pro Pro Asp Val Val Thr Glu115 120
125Cys Arg Leu Gly Leu Lys Glu Glu Asn Pro Ser Lys Lys Ala Phe Glu130
135 140Glu Cys Thr Arg Val Ala His Asn Tyr
Leu Arg Gly Glu Pro Phe Glu145 150 155
160Glu Tyr Gln Glu Ser Ser Tyr Phe Ser Gln Phe Leu Gln Trp
Lys Trp165 170 175Leu Glu Arg Gln Pro Val
Thr Lys Asn Thr Phe Arg His Tyr Arg Val180 185
190Leu Gly Lys Gly Gly Phe Gly Glu Val Cys Ala Cys Gln Val Arg
Ala195 200 205Thr Gly Lys Met Tyr Ala Cys
Lys Lys Leu Gln Lys Lys Arg Ile Lys210 215
220Lys Arg Lys Gly Glu Ala Met Ala Leu Asn Glu Lys Arg Ile Leu Glu225
230 235 240Lys Val Gln Ser
Arg Phe Val Val Ser Leu Ala Tyr Ala Tyr Glu Thr245 250
255Lys Asp Ala Leu Cys Leu Val Leu Thr Ile Met Asn Gly Gly
Asp Leu260 265 270Lys Phe His Ile Tyr Asn
Leu Gly Asn Pro Gly Phe Asp Glu Gln Arg275 280
285Ala Val Phe Tyr Ala Ala Glu Leu Cys Cys Gly Leu Glu Asp Leu
Gln290 295 300Arg Glu Arg Ile Val Tyr Arg
Asp Leu Lys Pro Glu Asn Ile Leu Leu305 310
315 320Asp Asp Arg Gly His Ile Arg Ile Ser Asp Leu Gly
Leu Ala Thr Glu325 330 335Ile Pro Glu Gly
Gln Arg Val Arg Gly Arg Val Gly Thr Val Gly Tyr340 345
350Met Ala Pro Glu Val Val Asn Asn Glu Lys Tyr Thr Phe Ser
Pro Asp355 360 365Trp Trp Gly Leu Gly Cys
Leu Ile Tyr Glu Met Ile Gln Gly His Ser370 375
380Pro Phe Lys Lys Tyr Lys Glu Lys Val Lys Trp Glu Glu Val Asp
Gln385 390 395 400Arg Ile
Lys Asn Asp Thr Glu Glu Tyr Ser Glu Lys Phe Ser Glu Asp405
410 415Ala Lys Ser Ile Cys Arg Met Leu Leu Thr Lys Asn
Pro Ser Lys Arg420 425 430Leu Gly Cys Arg
Gly Glu Gly Ala Ala Gly Val Lys Gln His Pro Val435 440
445Phe Lys Asp Ile Asn Phe Arg Arg Leu Glu Ala Asn Met Leu
Glu Pro450 455 460Pro Phe Cys Pro Asp Pro
His Ala Val Tyr Cys Lys Asp Val Leu Asp465 470
475 480Ile Glu Gln Phe Ser Ala Val Lys Gly Ile Tyr
Leu Asp Thr Ala Asp485 490 495Glu Asp Phe
Tyr Ala Arg Phe Ala Thr Gly Cys Val Ser Ile Pro Trp500
505 510Gln Asn Glu Met Ile Glu Ser Gly Cys Phe Lys Asp
Ile Asn Lys Ser515 520 525Glu Ser Glu Glu
Ala Leu Pro Leu Asp Leu Asp Lys Asn Ile His Thr530 535
540Pro Val Ser Arg Pro Asn Arg Gly Phe Phe Tyr Arg Leu Phe
Arg Arg545 550 555 560Gly
Gly Cys Leu Thr Met Val Pro Ser Glu Lys Glu Val Glu Pro Lys565
570 575Gln Cys181737DNAHomo sapiens 18atggagctcg
agaacatcgt ggccaactcg ctgctgctga aagcgcgtca aggaggatat 60ggcaaaaaaa
gtggtcgtag taaaaaatgg aaggagatac tgacactgcc tcctgtcagc 120cagtgcagtg
agcttagaca ttccattgaa aaggattata gcagtctttg tgacaagcaa 180ccgataggaa
gacgtctctt caggcagttc tgtgatacca aacccactct aaagaggcac 240attgaattct
tggatgcagt ggcagaatat gaagttgccg atgatgagga ccgaagtgat 300tgtggactgt
caatcttaga tagattcttc aatgataagt tggcagcccc tttaccagaa 360atacctccag
atgttgtgac agaatgtaga ttgggactga aggaggagaa cccttccaaa 420aaagcctttg
aggaatgtac tagagttgcc cataactacc taagagggga accatttgaa 480gaataccaag
aaagctcata tttttctcag tttttacaat ggaaatggct ggaaaggcaa 540cccgtaacaa
agaacacatt tagacattac agagttctag gaaaaggcgg atttggagag 600gtttgcgcct
gtcaagtgcg agccacagga aaaatgtatg cctgcaaaaa gctacaaaaa 660aaaagaataa
agaagaggaa aggtgaagct atggctctaa atgagaaaag aattctggag 720aaagtgcaaa
gtagattcgt agttagttta gcctacgctt atgaaaccaa agatgccttg 780tgcttggtgc
tcaccattat gaatggaggg gatttgaagt ttcacattta caacctgggc 840aatcccggct
ttgatgagca gagagccgtt ttctatgctg cagagctgtg ttgcggcttg 900gaagatttac
agagggaaag aattgtatac agagacttga agcctgagaa tattctcctt 960gatgatcgtg
gacacatccg gatttcagac ctcggtttgg ccacagagat cccagaagga 1020cagagggttc
gaggaagagt tggaacagtc ggctacatgg cacctgaagt tgtcaataat 1080gaaaagtata
cgtttagtcc cgattggtgg ggacttggct gtctgatcta tgaaatgatt 1140cagggacatt
ctccattcaa aaaatacaaa gagaaagtca aatgggagga ggtcgatcaa 1200agaatcaaga
atgataccga ggagtattct gagaagtttt cagaggatgc caaatctatc 1260tgcaggatgt
tactcaccaa gaatccaagc aagcggctgg gctgcagggg cgagggagcg 1320gctggggtga
agcagcaccc cgtgttcaag gacatcaact tcaggaggct ggaggcaaac 1380atgctggagc
cccctttctg tcctgatcct catgccgttt actgtaagga cgtcctggat 1440atcgagcagt
tctcggcggt gaaagggatc tacctggaca ccgcagatga agacttctat 1500gctcggtttg
ctaccgggtg tgtctccatc ccctggcaga atgagatgat cgaatccggg 1560tgtttcaaag
acatcaacaa aagtgaaagt gaggaagctt tgccattaga tctagacaag 1620aacatacata
ccccggtttc cagaccaaac agaggcttct tctatagact cttcagaaga 1680gggggctgcc
tgaccatggt ccccagtgag aaggaagtgg aacccaagca atgctga 173719532PRTHomo
sapiens 19Met Glu Leu Glu Asn Ile Val Ala Asn Ser Leu Leu Leu Lys Ala
Arg1 5 10 15Gln Gly Gly
Tyr Gly Lys Lys Ser Gly Arg Ser Lys Lys Trp Lys Glu20 25
30Ile Leu Thr Leu Pro Pro Val Ser Gln Cys Ser Glu Leu
Arg His Ser35 40 45Ile Glu Lys Asp Tyr
Ser Ser Leu Cys Asp Lys Gln Pro Ile Gly Arg50 55
60Arg Leu Phe Arg Gln Phe Cys Asp Thr Lys Pro Thr Leu Lys Arg
His65 70 75 80Ile Glu
Phe Leu Asp Ala Val Ala Glu Tyr Glu Val Ala Asp Asp Glu85
90 95Asp Arg Ser Asp Cys Gly Leu Ser Ile Leu Asp Arg
Phe Phe Asn Asp100 105 110Lys Leu Ala Ala
Pro Leu Pro Glu Ile Pro Pro Asp Val Val Thr Glu115 120
125Cys Arg Leu Gly Leu Lys Glu Glu Asn Pro Ser Lys Lys Ala
Phe Glu130 135 140Glu Cys Thr Arg Val Ala
His Asn Tyr Leu Arg Gly Glu Pro Phe Glu145 150
155 160Glu Tyr Gln Glu Ser Ser Tyr Phe Ser Gln Phe
Leu Gln Trp Lys Trp165 170 175Leu Glu Arg
Gln Pro Val Thr Lys Asn Thr Phe Arg His Tyr Arg Val180
185 190Leu Gly Lys Gly Gly Phe Gly Glu Val Cys Ala Cys
Gln Val Arg Ala195 200 205Thr Gly Lys Met
Tyr Ala Cys Lys Lys Leu Gln Lys Lys Arg Ile Lys210 215
220Lys Arg Lys Gly Glu Ala Met Ala Leu Asn Glu Lys Arg Ile
Leu Glu225 230 235 240Lys
Val Gln Ser Arg Phe Val Val Ser Leu Ala Tyr Ala Tyr Glu Thr245
250 255Lys Asp Ala Leu Cys Leu Val Leu Thr Ile Met
Asn Gly Gly Asp Leu260 265 270Lys Phe His
Ile Tyr Asn Leu Gly Asn Pro Gly Phe Asp Glu Gln Arg275
280 285Ala Val Phe Tyr Ala Ala Glu Leu Cys Cys Gly Leu
Glu Asp Leu Gln290 295 300Arg Glu Arg Ile
Val Tyr Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu305 310
315 320Asp Asp Arg Gly His Ile Arg Ile Ser
Asp Leu Gly Leu Ala Thr Glu325 330 335Ile
Pro Glu Gly Gln Arg Val Arg Gly Arg Val Gly Thr Val Gly Tyr340
345 350Met Ala Pro Glu Val Val Asn Asn Glu Lys Tyr
Thr Phe Ser Pro Asp355 360 365Trp Trp Gly
Leu Gly Cys Leu Ile Tyr Glu Met Ile Gln Gly His Ser370
375 380Pro Phe Lys Lys Tyr Lys Glu Lys Val Lys Trp Glu
Glu Val Asp Gln385 390 395
400Arg Ile Lys Asn Asp Thr Glu Glu Tyr Ser Glu Lys Phe Ser Glu Asp405
410 415Ala Lys Ser Ile Cys Arg Met Leu Leu
Thr Lys Asn Pro Ser Lys Arg420 425 430Leu
Gly Cys Arg Gly Glu Gly Ala Ala Gly Val Lys Gln His Pro Val435
440 445Phe Lys Asp Ile Asn Phe Arg Arg Leu Glu Ala
Asn Met Leu Glu Pro450 455 460Pro Phe Cys
Pro Asp Pro His Ala Val Tyr Cys Lys Asp Val Leu Asp465
470 475 480Ile Glu Gln Phe Ser Ala Val
Lys Gly Ile Tyr Leu Asp Thr Ala Asp485 490
495Glu Asp Phe Tyr Ala Arg Phe Ala Thr Gly Cys Val Ser Ile Pro Trp500
505 510Gln Asn Glu Gly Cys Leu Thr Met Val
Pro Ser Glu Lys Glu Val Glu515 520 525Pro
Lys Gln Cys530201599DNAHomo sapiens 20atggagctcg agaacatcgt ggccaactcg
ctgctgctga aagcgcgtca aggaggatat 60ggcaaaaaaa gtggtcgtag taaaaaatgg
aaggagatac tgacactgcc tcctgtcagc 120cagtgcagtg agcttagaca ttccattgaa
aaggattata gcagtctttg tgacaagcaa 180ccgataggaa gacgtctctt caggcagttc
tgtgatacca aacccactct aaagaggcac 240attgaattct tggatgcagt ggcagaatat
gaagttgccg atgatgagga ccgaagtgat 300tgtggactgt caatcttaga tagattcttc
aatgataagt tggcagcccc tttaccagaa 360atacctccag atgttgtgac agaatgtaga
ttgggactga aggaggagaa cccttccaaa 420aaagcctttg aggaatgtac tagagttgcc
cataactacc taagagggga accatttgaa 480gaataccaag aaagctcata tttttctcag
tttttacaat ggaaatggct ggaaaggcaa 540cccgtaacaa agaacacatt tagacattac
agagttctag gaaaaggcgg atttggagag 600gtttgcgcct gtcaagtgcg agccacagga
aaaatgtatg cctgcaaaaa gctacaaaaa 660aaaagaataa agaagaggaa aggtgaagct
atggctctaa atgagaaaag aattctggag 720aaagtgcaaa gtagattcgt agttagttta
gcctacgctt atgaaaccaa agatgccttg 780tgcttggtgc tcaccattat gaatggaggg
gatttgaagt ttcacattta caacctgggc 840aatcccggct ttgatgagca gagagccgtt
ttctatgctg cagagctgtg ttgcggcttg 900gaagatttac agagggaaag aattgtatac
agagacttga agcctgagaa tattctcctt 960gatgatcgtg gacacatccg gatttcagac
ctcggtttgg ccacagagat cccagaagga 1020cagagggttc gaggaagagt tggaacagtc
ggctacatgg cacctgaagt tgtcaataat 1080gaaaagtata cgtttagtcc cgattggtgg
ggacttggct gtctgatcta tgaaatgatt 1140cagggacatt ctccattcaa aaaatacaaa
gagaaagtca aatgggagga ggtcgatcaa 1200agaatcaaga atgataccga ggagtattct
gagaagtttt cagaggatgc caaatctatc 1260tgcaggatgt tactcaccaa gaatccaagc
aagcggctgg gctgcagggg cgagggagcg 1320gctggggtga agcagcaccc cgtgttcaag
gacatcaact tcaggaggct ggaggcaaac 1380atgctggagc cccctttctg tcctgatcct
catgccgttt actgtaagga cgtcctggat 1440atcgagcagt tctcggcggt gaaagggatc
tacctggaca ccgcagatga agacttctat 1500gctcggtttg ctaccgggtg tgtctccatc
ccctggcaga atgagggctg cctgaccatg 1560gtccccagtg agaaggaagt ggaacccaag
caatgctga 159921553PRTHomo sapiens 21Met Val Asp
Met Gly Ala Leu Asp Asn Leu Ile Ala Asn Thr Ala Tyr1 5
10 15Leu Gln Ala Arg Lys Pro Ser Asp Cys Asp
Ser Lys Glu Leu Gln Arg20 25 30Arg Arg
Arg Ser Leu Ala Leu Pro Gly Leu Gln Gly Cys Ala Glu Leu35
40 45Arg Gln Lys Leu Ser Leu Asn Phe His Ser Leu Cys
Glu Gln Gln Pro50 55 60Ile Gly Arg Arg
Leu Phe Arg Asp Phe Leu Ala Thr Val Pro Thr Phe65 70
75 80Arg Lys Ala Ala Thr Phe Leu Glu Asp
Val Gln Asn Trp Glu Leu Ala85 90 95Glu
Glu Gly Pro Thr Lys Asp Ser Ala Leu Gln Gly Leu Val Ala Thr100
105 110Cys Ala Ser Ala Pro Ala Pro Gly Asn Pro Gln
Pro Phe Leu Ser Gln115 120 125Ala Val Ala
Thr Lys Cys Gln Ala Ala Thr Thr Glu Glu Glu Arg Val130
135 140Ala Ala Val Thr Leu Ala Lys Ala Glu Ala Met Ala
Phe Leu Gln Glu145 150 155
160Gln Pro Phe Lys Asp Phe Val Thr Ser Ala Phe Tyr Asp Lys Phe Leu165
170 175Gln Trp Lys Leu Phe Glu Met Gln Pro
Val Ser Asp Lys Tyr Phe Thr180 185 190Glu
Phe Arg Val Leu Gly Lys Gly Gly Phe Gly Glu Val Cys Ala Val195
200 205Gln Val Lys Asn Thr Gly Lys Met Tyr Ala Cys
Lys Lys Leu Asp Lys210 215 220Lys Arg Leu
Lys Lys Lys Gly Gly Glu Lys Met Ala Leu Leu Glu Lys225
230 235 240Glu Ile Leu Glu Lys Val Ser
Ser Pro Phe Ile Val Ser Leu Ala Tyr245 250
255Ala Phe Glu Ser Lys Thr His Leu Cys Leu Val Met Ser Leu Met Asn260
265 270Gly Gly Asp Leu Lys Phe His Ile Tyr
Asn Val Gly Thr Arg Gly Leu275 280 285Asp
Met Ser Arg Val Ile Phe Tyr Ser Ala Gln Ile Ala Cys Gly Met290
295 300Leu His Leu His Glu Leu Gly Ile Val Tyr Arg
Asp Met Lys Pro Glu305 310 315
320Asn Val Leu Leu Asp Asp Leu Gly Asn Cys Arg Leu Ser Asp Leu
Gly325 330 335Leu Ala Val Glu Met Lys Gly
Gly Lys Pro Ile Thr Gln Arg Ala Gly340 345
350Thr Asn Gly Tyr Met Ala Pro Glu Ile Leu Met Glu Lys Val Ser Tyr355
360 365Ser Tyr Pro Val Asp Trp Phe Ala Met
Gly Cys Ser Ile Tyr Glu Met370 375 380Val
Ala Gly Arg Thr Pro Phe Lys Asp Tyr Lys Glu Lys Val Ser Lys385
390 395 400Glu Asp Leu Lys Gln Arg
Thr Leu Gln Asp Glu Val Lys Phe Gln His405 410
415Asp Asn Phe Thr Glu Glu Ala Lys Asp Ile Cys Arg Leu Phe Leu
Ala420 425 430Lys Lys Pro Glu Gln Arg Leu
Gly Ser Arg Glu Lys Ser Asp Asp Pro435 440
445Arg Lys His His Phe Phe Lys Thr Ile Asn Phe Pro Arg Leu Glu Ala450
455 460Gly Leu Ile Glu Pro Pro Phe Val Pro
Asp Pro Ser Val Val Tyr Ala465 470 475
480Lys Asp Ile Ala Glu Ile Asp Asp Phe Ser Glu Val Arg Gly
Val Glu485 490 495Phe Asp Asp Lys Asp Lys
Gln Phe Phe Lys Asn Phe Ala Thr Gly Ala500 505
510Val Pro Ile Ala Trp Gln Glu Glu Ile Ile Glu Thr Gly Leu Phe
Glu515 520 525Glu Leu Asn Asp Pro Asn Arg
Pro Thr Gly Cys Glu Glu Gly Asn Ser530 535
540Ser Lys Ser Gly Val Cys Leu Leu Leu545
550221662DNAHomo sapiens 22atggtggaca tgggggccct ggacaacctg atcgccaaca
ccgcctacct gcaggcccgg 60aagccctcgg actgcgacag caaagagctg cagcggcggc
ggcgtagcct ggccctgccc 120gggctgcagg gctgcgcgga gctccgccag aagctgtccc
tgaacttcca cagcctgtgt 180gagcagcagc ccatcggtcg ccgcctcttc cgtgacttcc
tagccacagt gcccacgttc 240cgcaaggcgg caaccttcct agaggacgtg cagaactggg
agctggccga ggagggaccc 300accaaagaca gcgcgctgca ggggctggtg gccacttgtg
cgagtgcccc tgccccgggg 360aacccgcaac ccttcctcag ccaggccgtg gccaccaagt
gccaagcagc caccactgag 420gaagagcgag tggctgcagt gacgctggcc aaggctgagg
ccatggcttt cttgcaagag 480cagcccttta aggatttcgt gaccagcgcc ttctacgaca
agtttctgca gtggaaactc 540ttcgagatgc aaccagtgtc agacaagtac ttcactgagt
tcagagtgct ggggaaaggt 600ggttttgggg aggtatgtgc cgtccaggtg aaaaacactg
ggaagatgta tgcctgtaag 660aaactggaca agaagcggct gaagaagaaa ggtggcgaga
agatggctct cttggaaaag 720gaaatcttgg agaaggtcag cagccctttc attgtctctc
tggcctatgc ctttgagagc 780aagacccatc tctgccttgt catgagcctg atgaatgggg
gagacctcaa gttccacatc 840tacaacgtgg gcacgcgtgg cctggacatg agccgggtga
tcttttactc ggcccagata 900gcctgtggga tgctgcacct ccatgaactc ggcatcgtct
atcgggacat gaagcctgag 960aatgtgcttc tggatgacct cggcaactgc aggttatctg
acctggggct ggccgtggag 1020atgaagggtg gcaagcccat cacccagagg gctggaacca
atggttacat ggctcctgag 1080atcctaatgg aaaaggtaag ttattcctat cctgtggact
ggtttgccat gggatgcagc 1140atttatgaaa tggttgctgg acgaacacca ttcaaagatt
acaaggaaaa ggtcagtaaa 1200gaggatctga agcaaagaac tctgcaagac gaggtcaaat
tccagcatga taacttcaca 1260gaggaagcaa aagatatttg caggctcttc ttggctaaga
aaccagagca acgcttagga 1320agcagagaaa agtctgatga tcccaggaaa catcatttct
ttaaaacgat caactttcct 1380cgcctggaag ctggcctaat tgaaccccca tttgtgccag
acccttcagt ggtttatgcc 1440aaagacatcg ctgaaattga tgatttctct gaggttcggg
gggtggaatt tgatgacaaa 1500gataagcagt tcttcaaaaa ctttgcgaca ggtgctgttc
ctatagcatg gcaggaagaa 1560attatagaaa cgggactgtt tgaggaactg aatgacccca
acagacctac gggttgtgag 1620gagggtaatt catccaagtc tggcgtgtgt ttgttattgt
aa 166223563PRTHomo sapiens 23Met Asp Phe Gly Ser
Leu Glu Thr Val Val Ala Asn Ser Ala Phe Ile1 5
10 15Ala Ala Arg Gly Ser Phe Asp Gly Ser Ser Ser Gln
Pro Ser Arg Asp20 25 30Lys Lys Tyr Leu
Ala Lys Leu Lys Leu Pro Pro Leu Ser Lys Cys Glu35 40
45Ser Leu Arg Asp Ser Leu Ser Leu Glu Phe Glu Ser Val Cys
Leu Glu50 55 60Gln Pro Ile Gly Lys Lys
Leu Phe Gln Gln Phe Leu Gln Ser Ala Glu65 70
75 80Lys His Leu Pro Ala Leu Glu Leu Trp Lys Asp
Ile Glu Asp Tyr Asp85 90 95Thr Ala Asp
Asn Asp Leu Gln Pro Gln Lys Ala Gln Thr Ile Leu Ala100
105 110Gln Tyr Leu Asp Pro Gln Ala Lys Leu Phe Cys Ser
Phe Leu Asp Glu115 120 125Gly Ile Val Ala
Lys Phe Lys Glu Gly Pro Val Glu Ile Gln Asp Gly130 135
140Leu Phe Gln Pro Leu Leu Gln Ala Thr Leu Ala His Leu Gly
Gln Ala145 150 155 160Pro
Phe Gln Glu Tyr Leu Gly Ser Leu Tyr Phe Leu Arg Phe Leu Gln165
170 175Trp Lys Trp Leu Glu Ala Gln Pro Met Gly Glu
Asp Trp Phe Leu Asp180 185 190Phe Arg Val
Leu Gly Lys Gly Gly Phe Gly Glu Val Ser Ala Cys Gln195
200 205Met Lys Ala Thr Gly Lys Leu Tyr Ala Cys Lys Lys
Leu Asn Lys Lys210 215 220Arg Leu Lys Lys
Arg Lys Gly Tyr Gln Gly Ala Met Val Glu Lys Lys225 230
235 240Ile Leu Met Lys Val His Ser Arg Phe
Ile Val Ser Leu Ala Tyr Ala245 250 255Phe
Glu Thr Lys Ala Asp Leu Cys Leu Val Met Thr Ile Met Asn Gly260
265 270Gly Asp Ile Arg Tyr His Ile Tyr Asn Val Asn
Glu Glu Asn Pro Gly275 280 285Phe Pro Glu
Pro Arg Ala Leu Phe Tyr Thr Ala Gln Ile Ile Cys Gly290
295 300Leu Glu His Leu His Gln Arg Arg Ile Val Tyr Arg
Asp Leu Lys Pro305 310 315
320Glu Asn Val Leu Leu Asp Asn Asp Gly Asn Val Arg Ile Ser Asp Leu325
330 335Gly Leu Ala Val Glu Leu Leu Asp Gly
Gln Ser Lys Thr Lys Gly Tyr340 345 350Ala
Gly Thr Pro Gly Phe Met Ala Pro Glu Leu Leu Gln Gly Glu Glu355
360 365Tyr Asp Phe Ser Val Asp Tyr Phe Ala Leu Gly
Val Thr Leu Tyr Glu370 375 380Met Ile Ala
Ala Arg Gly Pro Phe Arg Ala Arg Gly Glu Lys Val Glu385
390 395 400Asn Lys Glu Leu Lys His Arg
Ile Ile Ser Glu Pro Val Lys Tyr Pro405 410
415Asp Lys Phe Ser Gln Ala Ser Lys Asp Phe Cys Glu Ala Leu Leu Glu420
425 430Lys Asp Pro Glu Lys Arg Leu Gly Phe
Arg Asp Glu Thr Cys Asp Lys435 440 445Leu
Arg Ala His Pro Leu Phe Lys Asp Leu Asn Trp Arg Gln Leu Glu450
455 460Ala Gly Met Leu Met Pro Pro Phe Ile Pro Asp
Ser Lys Thr Val Tyr465 470 475
480Ala Lys Asp Ile Gln Asp Val Gly Ala Phe Ser Thr Val Lys Gly
Val485 490 495Ala Phe Asp Lys Thr Asp Thr
Glu Phe Phe Gln Glu Phe Ala Thr Gly500 505
510Asn Cys Pro Ile Pro Trp Gln Glu Glu Met Ile Glu Thr Gly Ile Phe515
520 525Gly Glu Leu Asn Val Trp Arg Ser Asp
Gly Gln Met Pro Asp Asp Met530 535 540Lys
Gly Ile Ser Gly Gly Ser Ser Ser Ser Ser Lys Ser Gly Met Cys545
550 555 560Leu Val Ser241692DNAHomo
sapiens 24atggatttcg ggtctttgga gaccgtggtg gccaactctg ccttcatcgc
cgcccgaggc 60agctttgacg gcagcagctc ccaaccctcc cgggacaaga agtacctggc
caagctcaag 120ctgcccccgc tgtccaagtg tgagtccctc cgcgacagcc tcagcctgga
gtttgagagt 180gtgtgcttgg agcagcccat cggcaagaag ctctttcagc agttcctaca
atcggcagag 240aagcacctgc cggccctgga gctctggaaa gacatcgagg actatgacac
ggcagacaat 300gacctccagc cacagaaggc ccagaccatc ctggcccagt acctggaccc
ccaggccaaa 360ctcttctgca gcttcctgga tgaggggata gtggcgaagt ttaaggaggg
gcctgtggag 420atccaggacg ggctcttcca gcccctgctg caggccaccc tggcacacct
gggccaagcc 480cccttccagg agtacctggg cagcctgtac ttcctgaggt tcctgcagtg
gaagtggctg 540gaagcccagc ccatggggga ggactggttc ctggacttca gggtcctggg
gaaagggggc 600ttcggggagg tgtcggcctg ccagatgaag gcgaccggca agctgtatgc
ctgcaagaag 660ctgaacaaga agcggctgaa gaagaggaag ggctaccagg gtgctatggt
ggagaagaag 720attctgatga aagtacacag caggttcatc gtgtctctgg cctatgcgtt
tgaaaccaaa 780gccgacctct gtctggtgat gaccatcatg aacggaggtg acatcaggta
ccacatctac 840aacgtgaatg aggagaaccc tggcttcccg gagccgcgcg ccctcttcta
cacggcgcag 900atcatctgcg gcctggagca cctgcaccag aggcggatcg tctaccgcga
cctcaagccc 960gagaacgtgc tgctggacaa tgacggcaat gtccggatct ctgaccttgg
gctggccgtg 1020gagctgctgg acggacagag caagaccaag ggctacgcag ggaccccagg
tttcatggcc 1080cccgagctcc tgcagggcga ggagtacgac ttctccgtgg actactttgc
cctgggggtc 1140accctgtatg agatgattgc ggccagagga cccttccgag cccgtggaga
gaaggtggag 1200aacaaggagc tgaagcaccg gatcatctca gagcccgtga agtaccctga
taagttcagc 1260caggccagca aggacttctg cgaggcgctg ctggagaagg acccggagaa
gcgcctgggg 1320ttcagagatg agacctgcga caagctccgt gcccaccccc tcttcaagga
ccttaactgg 1380aggcagctgg aggctgggat gctgatgccc cctttcatcc cagactccaa
aactgtctac 1440gcaaaggata ttcaggacgt gggtgccttt tccaccgtca aaggtgtggc
ctttgacaaa 1500acagacacag aattctttca ggaatttgcc actggcaact gccccatccc
ctggcaggag 1560gagatgatcg agacgggcat ctttggcgag ctgaacgtgt ggcgctcgga
cggtcagatg 1620ccggacgaca tgaagggcat ctccgggggc tccagctcct cgtccaagtc
agggatgtgt 1680ctggtttcct ag
169225688PRTHomo sapiens 25Met Ala Asp Leu Glu Ala Val Leu Ala
Asp Val Ser Tyr Leu Met Ala1 5 10
15Met Glu Lys Ser Lys Ala Thr Pro Ala Ala Arg Ala Ser Lys Arg
Ile20 25 30Val Leu Pro Glu Pro Ser Ile
Arg Ser Val Met Gln Lys Tyr Leu Ala35 40
45Glu Arg Asn Glu Ile Thr Phe Asp Lys Ile Phe Asn Gln Lys Ile Gly50
55 60Phe Leu Leu Phe Lys Asp Phe Cys Leu Asn
Glu Ile Asn Glu Ala Val65 70 75
80Pro Gln Val Lys Phe Tyr Glu Glu Ile Lys Glu Tyr Glu Lys Leu
Asp85 90 95Asn Glu Glu Asp Arg Leu Cys
Arg Ser Arg Gln Ile Tyr Asp Ala Tyr100 105
110Ile Met Lys Glu Leu Leu Ser Cys Ser His Pro Phe Ser Lys Gln Ala115
120 125Val Glu His Val Gln Ser His Leu Ser
Lys Lys Gln Val Thr Ser Thr130 135 140Leu
Phe Gln Pro Tyr Ile Glu Glu Ile Cys Glu Ser Leu Arg Gly Asp145
150 155 160Ile Phe Gln Lys Phe Met
Glu Ser Asp Lys Phe Thr Arg Phe Cys Gln165 170
175Trp Lys Asn Val Glu Leu Asn Ile His Leu Thr Met Asn Glu Phe
Ser180 185 190Val His Arg Ile Ile Gly Arg
Gly Gly Phe Gly Glu Val Tyr Gly Cys195 200
205Arg Lys Ala Asp Thr Gly Lys Met Tyr Ala Met Lys Cys Leu Asp Lys210
215 220Lys Arg Ile Lys Met Lys Gln Gly Glu
Thr Leu Ala Leu Asn Glu Arg225 230 235
240Ile Met Leu Ser Leu Val Ser Thr Gly Asp Cys Pro Phe Ile
Val Cys245 250 255Met Thr Tyr Ala Phe His
Thr Pro Asp Lys Leu Cys Phe Ile Leu Asp260 265
270Leu Met Asn Gly Gly Asp Leu His Tyr His Leu Ser Gln His Gly
Val275 280 285Phe Ser Glu Lys Glu Met Arg
Phe Tyr Ala Thr Glu Ile Ile Leu Gly290 295
300Leu Glu His Met His Asn Arg Phe Val Val Tyr Arg Asp Leu Lys Pro305
310 315 320Ala Asn Ile Leu
Leu Asp Glu His Gly His Ala Arg Ile Ser Asp Leu325 330
335Gly Leu Ala Cys Asp Phe Ser Lys Lys Lys Pro His Ala Ser
Val Gly340 345 350Thr His Gly Tyr Met Ala
Pro Glu Val Leu Gln Lys Gly Thr Ala Tyr355 360
365Asp Ser Ser Ala Asp Trp Phe Ser Leu Gly Cys Met Leu Phe Lys
Leu370 375 380Leu Arg Gly His Ser Pro Phe
Arg Gln His Lys Thr Lys Asp Lys His385 390
395 400Glu Ile Asp Arg Met Thr Leu Thr Val Asn Val Glu
Leu Pro Asp Thr405 410 415Phe Ser Pro Glu
Leu Lys Ser Leu Leu Glu Gly Leu Leu Gln Arg Asp420 425
430Val Ser Lys Arg Leu Gly Cys His Gly Gly Gly Ser Gln Glu
Val Lys435 440 445Glu His Ser Phe Phe Lys
Gly Val Asp Trp Gln His Val Tyr Leu Gln450 455
460Lys Tyr Pro Pro Pro Leu Ile Pro Pro Arg Gly Glu Val Asn Ala
Ala465 470 475 480Asp Ala
Phe Asp Ile Gly Ser Phe Asp Glu Glu Asp Thr Lys Gly Ile485
490 495Lys Leu Leu Asp Cys Asp Gln Glu Leu Tyr Lys Asn
Phe Pro Leu Val500 505 510Ile Ser Glu Arg
Trp Gln Gln Glu Val Thr Glu Thr Val Tyr Glu Ala515 520
525Val Asn Ala Asp Thr Asp Lys Ile Glu Ala Arg Lys Arg Ala
Lys Asn530 535 540Lys Gln Leu Gly His Glu
Glu Asp Tyr Ala Leu Gly Lys Asp Cys Ile545 550
555 560Met His Gly Tyr Met Leu Lys Leu Gly Asn Pro
Phe Leu Thr Gln Trp565 570 575Gln Arg Arg
Tyr Phe Tyr Leu Phe Pro Asn Arg Leu Glu Trp Arg Gly580
585 590Glu Gly Glu Ser Arg Gln Asn Leu Leu Thr Met Glu
Gln Ile Leu Ser595 600 605Val Glu Glu Thr
Gln Ile Lys Asp Lys Lys Cys Ile Leu Phe Arg Ile610 615
620Lys Gly Gly Lys Gln Phe Val Leu Gln Cys Glu Ser Asp Pro
Glu Phe625 630 635 640Val
Gln Trp Lys Lys Glu Leu Asn Glu Thr Phe Lys Glu Ala Gln Arg645
650 655Leu Leu Arg Arg Ala Pro Lys Phe Leu Asn Lys
Pro Arg Ser Gly Thr660 665 670Val Glu Leu
Pro Lys Pro Ser Leu Cys His Arg Asn Ser Asn Gly Leu675
680 685262067DNAHomo sapiens 26atggcggacc tggaggctgt
gctggccgat gtcagttacc tgatggccat ggagaagagc 60aaggcgaccc cggccgcccg
cgccagcaag aggatcgtcc tgccggagcc cagtatccgg 120agtgtgatgc agaagtacct
tgcagagaga aatgaaataa cctttgacaa gattttcaat 180cagaaaattg gtttcttgct
atttaaagat ttttgtttga atgaaattaa tgaagctgta 240cctcaggtga agttttatga
agagataaag gaatatgaaa aacttgataa tgaggaagac 300cgcctttgca gaagtcgaca
aatttatgat gcctacatca tgaaggaact tctttcctgt 360tcacatcctt tctcaaagca
agctgtagaa cacgtacaaa gtcatttatc caagaaacaa 420gtgacatcaa ctctttttca
gccatacata gaagaaattt gtgaaagcct tcgaggtgac 480atttttcaaa aatttatgga
aagtgacaag ttcactagat tttgtcagtg gaaaaacgtt 540gaattaaata tccatttgac
catgaatgag ttcagtgtgc ataggattat tggacgagga 600ggattcgggg aagtttatgg
ttgcaggaaa gcagacactg gaaaaatgta tgcaatgaaa 660tgcttagata agaagaggat
caaaatgaaa caaggagaaa cattagcctt aaatgaaaga 720atcatgttgt ctcttgtcag
cacaggagac tgtcctttca ttgtatgtat gacctatgcc 780ttccataccc cagataaact
ctgcttcatc ctggatctga tgaacggggg cgatttgcac 840taccaccttt cacaacacgg
tgtgttctct gagaaggaga tgcggtttta tgccactgaa 900atcattctgg gtctggaaca
catgcacaat cggtttgttg tctacagaga tttgaagcca 960gcaaatattc tcttggatga
acatggacac gcaagaatat cagatcttgg tcttgcctgc 1020gatttttcca aaaagaagcc
tcatgcgagt gttggcaccc atgggtacat ggctcccgag 1080gtgctgcaga aggggacggc
ctatgacagc agtgccgact ggttctccct gggctgcatg 1140cttttcaaac ttctgagagg
tcacagccct ttcagacaac ataaaaccaa agacaagcat 1200gaaattgacc gaatgacact
caccgtgaat gtggaacttc cagacacctt ctctcctgaa 1260ctgaagtccc ttttggaggg
cttgcttcag cgagacgtta gcaagcggct gggctgtcac 1320ggaggcggct cacaggaagt
aaaagagcac agctttttca aaggtgttga ctggcagcat 1380gtctacttac aaaagtaccc
accacccttg attcctcccc ggggagaagt caatgctgct 1440gatgcctttg atattggctc
atttgatgaa gaggatacca aagggattaa gctacttgat 1500tgcgaccaag aactctacaa
gaacttccct ttggtcatct ctgaacgctg gcagcaagaa 1560gtaacggaaa cagtttatga
agcagtaaat gcagacacag ataaaatcga ggccaggaag 1620agagctaaaa ataagcaact
tggccacgaa gaagattacg ctctggggaa ggactgtatt 1680atgcacgggt acatgctgaa
actgggaaac ccatttctga ctcagtggca gcgtcgctat 1740ttttacctct ttccaaatag
acttgaatgg agaggagagg gagagtcccg gcaaaattta 1800ctgacaatgg aacagattct
ctctgtggaa gaaactcaaa ttaaagacaa aaaatgcatt 1860ttgttcagaa taaaaggagg
gaaacaattt gtcttgcaat gtgagagtga tccagagttt 1920gtgcagtgga agaaagagtt
gaacgaaacc ttcaaggagg cccagcggct attgcgtcgt 1980gccccgaagt tcctcaacaa
acctcggtca ggtactgtgg agctcccaaa gccatccctc 2040tgtcacagaa acagcaacgg
cctctag 206727688PRTBos taurus
27Met Ala Asp Leu Glu Ala Val Leu Ala Asp Val Ser Tyr Leu Met Ala1
5 10 15Met Glu Lys Ser Lys Ala
Thr Pro Ala Ala Arg Ala Ser Lys Lys Ile20 25
30Val Leu Pro Glu Pro Ser Ile Arg Ser Val Met Gln Lys Tyr Leu Glu35
40 45Glu Arg His Glu Ile Thr Phe Asp Lys
Ile Phe Asn Gln Arg Ile Gly50 55 60Phe
Leu Leu Phe Lys Asp Phe Cys Leu Asn Glu Ile Asn Glu Ala Val65
70 75 80Pro Gln Val Lys Phe Tyr
Glu Glu Ile Lys Glu Tyr Glu Lys Leu Glu85 90
95Asn Glu Glu Asp Arg Leu Cys Arg Ser Arg Gln Ile Tyr Asp Thr Tyr100
105 110Ile Met Lys Glu Leu Leu Ser Cys
Ser His Pro Phe Ser Lys Gln Ala115 120
125Val Glu His Val Gln Ser His Leu Ser Lys Lys Gln Val Thr Ser Thr130
135 140Leu Phe Gln Pro Tyr Ile Glu Glu Ile
Cys Glu Ser Leu Arg Gly Ser145 150 155
160Ile Phe Gln Lys Phe Met Glu Ser Asp Lys Phe Thr Arg Phe
Cys Gln165 170 175Trp Lys Asn Val Glu Leu
Asn Ile His Leu Thr Met Asn Asp Phe Ser180 185
190Val His Arg Ile Ile Gly Arg Gly Gly Phe Gly Glu Val Tyr Gly
Cys195 200 205Arg Lys Ala Asp Thr Gly Lys
Met Tyr Ala Met Lys Cys Leu Asp Lys210 215
220Lys Arg Ile Lys Met Lys Gln Gly Glu Thr Leu Ala Leu Asn Glu Arg225
230 235 240Ile Met Leu Ser
Leu Val Ser Thr Gly Asp Cys Pro Phe Ile Val Cys245 250
255Met Thr Tyr Ala Phe His Thr Pro Asp Lys Leu Cys Phe Ile
Leu Asp260 265 270Leu Met Asn Gly Gly Asp
Leu His Tyr His Leu Ser Gln His Gly Val275 280
285Phe Ser Glu Lys Glu Met Arg Phe Tyr Ala Thr Glu Ile Ile Leu
Gly290 295 300Leu Glu His Met His Asn Arg
Phe Val Val Tyr Arg Asp Leu Lys Pro305 310
315 320Ala Asn Ile Leu Leu Asp Glu His Gly His Val Arg
Ile Ser Asp Leu325 330 335Gly Leu Ala Cys
Asp Phe Ser Lys Lys Lys Pro His Ala Ser Val Gly340 345
350Thr His Gly Tyr Met Ala Pro Glu Val Leu Gln Lys Gly Thr
Ala Tyr355 360 365Asp Ser Ser Ala Asp Trp
Phe Ser Leu Gly Cys Met Leu Phe Lys Leu370 375
380Leu Arg Gly His Ser Pro Phe Arg Gln His Lys Thr Lys Asp Lys
His385 390 395 400Glu Ile
Asp Arg Met Thr Leu Thr Met Asn Val Glu Leu Pro Asp Val405
410 415Phe Ser Pro Glu Leu Lys Ser Leu Leu Glu Gly Leu
Leu Gln Arg Asp420 425 430Val Ser Lys Arg
Leu Gly Cys His Gly Gly Ser Ala Gln Glu Leu Lys435 440
445Thr His Asp Phe Phe Arg Gly Ile Asp Trp Gln His Val Tyr
Leu Gln450 455 460Lys Tyr Pro Pro Pro Leu
Ile Pro Pro Arg Gly Glu Val Asn Ala Ala465 470
475 480Asp Ala Phe Asp Ile Gly Ser Phe Asp Glu Glu
Asp Thr Lys Gly Ile485 490 495Lys Leu Leu
Asp Cys Asp Gln Glu Leu Tyr Lys Asn Phe Pro Leu Val500
505 510Ile Ser Glu Arg Trp Gln Gln Glu Val Ala Glu Thr
Val Tyr Glu Ala515 520 525Val Asn Ala Asp
Thr Asp Lys Ile Glu Ala Arg Lys Arg Ala Lys Asn530 535
540Lys Gln Leu Gly His Glu Glu Asp Tyr Ala Leu Gly Arg Asp
Cys Ile545 550 555 560Val
His Gly Tyr Met Leu Lys Leu Gly Asn Pro Phe Leu Thr Gln Trp565
570 575Gln Arg Arg Tyr Phe Tyr Leu Phe Pro Asn Arg
Leu Glu Trp Arg Gly580 585 590Glu Gly Glu
Ser Arg Gln Ser Leu Leu Thr Met Glu Gln Ile Val Ser595
600 605Val Glu Glu Thr Gln Ile Lys Asp Lys Lys Cys Ile
Leu Leu Arg Ile610 615 620Lys Gly Gly Lys
Gln Phe Val Leu Gln Cys Glu Ser Asp Pro Glu Phe625 630
635 640Val Gln Trp Lys Lys Glu Leu Thr Glu
Thr Phe Met Glu Ala Gln Arg645 650 655Leu
Leu Arg Arg Ala Pro Lys Phe Leu Asn Lys Ser Arg Ser Ala Val660
665 670Val Glu Leu Ser Lys Pro Pro Leu Cys His Arg
Asn Ser Asn Gly Leu675 680
685282067DNABos taurus 28atggcggacc tggaggccgt gctggccgat gtcagctacc
tgatggcgat ggagaagagc 60aaggcgaccc cggccgcccg cgccagcaag aagatcgtcc
tgcccgagcc cagtatccgg 120agcgtgatgc agaagtatct tgaggagaga cacgaaatca
cctttgacaa gatttttaat 180cagagaattg gtttcttgct atttaaagat ttttgtttga
atgaaattaa tgaagctgta 240cctcaggtga agttttatga agagataaaa gaatatgaaa
agcttgagaa tgaggaagat 300cgcctttgta gaagtcgaca gatttatgac acttacatca
tgaaggagct gctgtcgtgt 360tcacatccat tctcaaagca agccgtagaa cacgtacaaa
gtcatctgtc caagaaacaa 420gtgacatcaa ctctttttca gccatacata gaagaaattt
gtgaaagtct ccgaggcagc 480atttttcaaa aattcatgga aagtgacaag tttactagat
tttgtcagtg gaaaaacgtg 540gaattaaata tccatttgac catgaatgat ttcagcgtgc
atcggatcat tggacgagga 600ggattcggtg aagtatacgg ttgcaggaaa gcagacactg
gaaagatgta tgcaatgaaa 660tgcttggata agaagagaat caagatgaaa cagggagaaa
ccttagcctt aaatgaaagg 720atcatgttgt ccctggtgag cacaggagat tgccctttca
tcgtctgtat gacctatgcc 780ttccacactc cagataaact gtgcttcatc ttggatctga
tgaacggggg tgacctgcac 840tatcaccttt cgcagcacgg ggtgttttct gagaaggaga
tgcggtttta cgccacagaa 900atcatcctgg ggctggaaca catgcacaat cggtttgttg
tttacagaga cttgaagccc 960gccaatatcc tcctggatga gcacggacat gtgaggatat
cagaccttgg tcttgcctgc 1020gatttttcca aaaagaagcc gcacgcgagc gtgggcaccc
acgggtacat ggcgcccgaa 1080gttctgcaga aggggaccgc ctacgacagc agtgccgact
ggttctccct gggctgtatg 1140cttttcaaac ttctgagagg tcacagccct ttcagacaac
ataaaaccaa agataagcat 1200gagatagacc gaatgactct caccatgaac gtggaacttc
cagacgtctt ctcccctgag 1260ctcaagtccc ttctggaagg cctgcttcag cgagatgtca
gtaagcgcct cggctgccat 1320ggaggcagcg cacaggagct aaaaacgcac gacttcttca
gaggcatcga ctggcagcac 1380gtctacctgc agaagtaccc tccacccttg atccctcccc
gaggggaagt caatgcagcc 1440gacgcctttg acatcggctc atttgatgaa gaggatacca
aaggcatcaa gcttcttgat 1500tgcgaccaag aactctacaa gaacttccct ctggtgatct
ctgagcgctg gcagcaggaa 1560gtggcggaaa cagtttatga agcagtaaat gcagacacgg
ataaaatcga ggccaggaag 1620agagctaaaa ataagcagct tggccacgaa gaagattacg
ccctgggaag agactgcatc 1680gtgcacgggt acatgctgaa gctggggaac cctttcctga
cccagtggca gcgccgctat 1740ttttacctct ttccgaacag acttgagtgg agaggagaag
gcgagtcgcg acaaagttta 1800ctgacaatgg aacagattgt gtccgtggaa gaaactcaga
ttaaagacaa aaagtgcatt 1860ttgttgagaa taaaaggagg gaagcagttc gttttgcagt
gtgagagtga cccagagttt 1920gtgcagtgga agaaagagct gacggagaca ttcatggagg
cccagcggct gctacggcga 1980gcccccaagt tcctcaacaa atcccgctca gccgtcgtgg
aactctcaaa gcctcccctc 2040tgccatagga acagcaacgg cctctga
206729689PRTHomo sapiens 29Met Ala Asp Leu Glu Ala
Val Leu Ala Asp Val Ser Tyr Leu Met Ala1 5
10 15Met Glu Lys Ser Lys Ala Thr Pro Ala Ala Arg Ala Ser
Lys Lys Ile20 25 30Leu Leu Pro Glu Pro
Ser Ile Arg Ser Val Met Gln Lys Tyr Leu Glu35 40
45Asp Arg Gly Glu Val Thr Phe Glu Lys Ile Phe Ser Gln Lys Leu
Gly50 55 60Tyr Leu Leu Phe Arg Asp Phe
Cys Leu Asn His Leu Glu Glu Ala Arg65 70
75 80Pro Leu Val Glu Phe Tyr Glu Glu Ile Lys Lys Tyr
Glu Lys Leu Glu85 90 95Thr Glu Glu Glu
Arg Val Ala Arg Ser Arg Glu Ile Phe Asp Ser Tyr100 105
110Ile Met Lys Glu Leu Leu Ala Cys Ser His Pro Phe Ser Lys
Ser Ala115 120 125Thr Glu His Val Gln Gly
His Leu Gly Lys Lys Gln Val Pro Pro Asp130 135
140Leu Phe Gln Pro Tyr Ile Glu Glu Ile Cys Gln Asn Leu Arg Gly
Asp145 150 155 160Val Phe
Gln Lys Phe Ile Glu Ser Asp Lys Phe Thr Arg Phe Cys Gln165
170 175Trp Lys Asn Val Glu Leu Asn Ile His Leu Thr Met
Asn Asp Phe Ser180 185 190Val His Arg Ile
Ile Gly Arg Gly Gly Phe Gly Glu Val Tyr Gly Cys195 200
205Arg Lys Ala Asp Thr Gly Lys Met Tyr Ala Met Lys Cys Leu
Asp Lys210 215 220Lys Arg Ile Lys Met Lys
Gln Gly Glu Thr Leu Ala Leu Asn Glu Arg225 230
235 240Ile Met Leu Ser Leu Val Ser Thr Gly Asp Cys
Pro Phe Ile Val Cys245 250 255Met Ser Tyr
Ala Phe His Thr Pro Asp Lys Leu Ser Phe Ile Leu Asp260
265 270Leu Met Asn Gly Gly Asp Leu His Tyr His Leu Ser
Gln His Gly Val275 280 285Phe Ser Glu Ala
Asp Met Arg Phe Tyr Ala Ala Glu Ile Ile Leu Gly290 295
300Leu Glu His Met His Asn Arg Phe Val Val Tyr Arg Asp Leu
Lys Pro305 310 315 320Ala
Asn Ile Leu Leu Asp Glu His Gly His Val Arg Ile Ser Asp Leu325
330 335Gly Leu Ala Cys Asp Phe Ser Lys Lys Lys Pro
His Ala Ser Val Gly340 345 350Thr His Gly
Tyr Met Ala Pro Glu Val Leu Gln Lys Gly Val Ala Tyr355
360 365Asp Ser Ser Ala Asp Trp Phe Ser Leu Gly Cys Met
Leu Phe Lys Leu370 375 380Leu Arg Gly His
Ser Pro Phe Arg Gln His Lys Thr Lys Asp Lys His385 390
395 400Glu Ile Asp Arg Met Thr Leu Thr Met
Ala Val Glu Leu Pro Asp Ser405 410 415Phe
Ser Pro Glu Leu Arg Ser Leu Leu Glu Gly Leu Leu Gln Arg Asp420
425 430Val Asn Arg Arg Leu Gly Cys Leu Gly Arg Gly
Ala Gln Glu Val Lys435 440 445Glu Ser Pro
Phe Phe Arg Ser Leu Asp Trp Gln Met Val Phe Leu Gln450
455 460Lys Tyr Pro Pro Pro Leu Ile Pro Pro Arg Gly Glu
Val Asn Ala Ala465 470 475
480Asp Ala Phe Asp Ile Gly Ser Phe Asp Glu Glu Asp Thr Lys Gly Ile485
490 495Lys Leu Leu Asp Ser Asp Gln Glu Leu
Tyr Arg Asn Phe Pro Leu Thr500 505 510Ile
Ser Glu Arg Trp Gln Gln Glu Val Ala Glu Thr Val Phe Asp Thr515
520 525Ile Asn Ala Glu Thr Asp Arg Leu Glu Ala Arg
Lys Lys Ala Lys Asn530 535 540Lys Gln Leu
Gly His Glu Glu Asp Tyr Ala Leu Gly Lys Asp Cys Ile545
550 555 560Met His Gly Tyr Met Ser Lys
Met Gly Asn Pro Phe Leu Thr Gln Trp565 570
575Gln Arg Arg Tyr Phe Tyr Leu Phe Pro Asn Arg Leu Glu Trp Arg Gly580
585 590Glu Gly Glu Ala Pro Gln Ser Leu Leu
Thr Met Glu Glu Ile Gln Ser595 600 605Val
Glu Glu Thr Gln Ile Lys Glu Arg Lys Cys Leu Leu Leu Lys Ile610
615 620Arg Gly Gly Lys Gln Phe Ile Leu Gln Cys Asp
Ser Asp Pro Glu Leu625 630 635
640Val Gln Trp Lys Lys Glu Leu Arg Asp Ala Tyr Arg Glu Ala Gln
Gln645 650 655Leu Val Gln Arg Val Pro Lys
Met Lys Asn Lys Pro Arg Ser Pro Val660 665
670Val Glu Leu Ser Lys Val Pro Leu Val Gln Arg Gly Ser Ala Asn Gly675
680 685Leu302070DNAHomo sapiens 30atggcggacc
tggaggcggt gctggccgac gtgagctacc tgatggccat ggagaagagc 60aaggccacgc
cggccgcgcg cgccagcaag aagatactgc tgcccgagcc cagcatccgc 120agtgtcatgc
agaagtacct ggaggaccgg ggcgaggtga cctttgagaa gatcttttcc 180cagaagctgg
ggtacctgct cttccgagac ttctgcctga accacctgga ggaggccagg 240cccttggtgg
aattctatga ggagatcaag aagtacgaga agctggagac ggaggaggag 300cgtgtggccc
gcagccggga gatcttcgac tcatacatca tgaaggagct gctggcctgc 360tcgcatccct
tctcgaagag tgccactgag catgtccaag gccacctggg gaagaagcag 420gtgcctccgg
atctcttcca gccatacatc gaagagattt gtcaaaacct ccgaggggac 480gtgttccaga
aattcattga gagcgataag ttcacacggt tttgccagtg gaagaatgtg 540gagctcaaca
tccacctgac catgaatgac ttcagcgtgc atcgcatcat tgggcgcggg 600ggctttggcg
aggtctatgg gtgccggaag gctgacacag gcaagatgta cgccatgaag 660tgcctggaca
aaaagcgcat caagatgaag cagggggaga ccctggccct gaacgagcgc 720atcatgctct
cgctcgtcag cactggggac tgcccattca ttgtctgcat gtcatacgcg 780ttccacacgc
cagacaagct cagcttcatc ctggacctca tgaacggtgg ggacctgcac 840taccacctct
cccagcacgg ggtcttctca gaggctgaca tgcgcttcta tgcggccgag 900atcatcctgg
gcctggagca catgcacaac cgcttcgtgg tctaccggga cctgaagcca 960gccaacatcc
ttctggacga gcatggccac gtgcggatct cggacctggg cctggcctgt 1020gacttctcca
agaagaagcc ccatgccagc gtgggcaccc acgggtacat ggctccggag 1080gtcctgcaga
agggcgtggc ctacgacagc agtgccgact ggttctctct ggggtgcatg 1140ctcttcaagt
tgctgcgggg gcacagcccc ttccggcagc acaagaccaa agacaagcat 1200gagatcgacc
gcatgacgct gacgatggcc gtggagctgc ccgactcctt ctcccctgaa 1260ctacgctccc
tgctggaggg gttgctgcag agggatgtca accggagatt gggctgcctg 1320ggccgagggg
ctcaggaggt gaaagagagc ccctttttcc gctccctgga ctggcagatg 1380gtcttcttgc
agaagtaccc tcccccgctg atccccccac gaggggaggt gaacgcggcc 1440gacgccttcg
acattggctc cttcgatgag gaggacacaa aaggaatcaa gttactggac 1500agtgatcagg
agctctaccg caacttcccc ctcaccatct cggagcggtg gcagcaggag 1560gtggcagaga
ctgtcttcga caccatcaac gctgagacag accggctgga ggctcgcaag 1620aaagccaaga
acaagcagct gggccatgag gaagactacg ccctgggcaa ggactgcatc 1680atgcatggct
acatgtccaa gatgggcaac cccttcctga cccagtggca gcggcggtac 1740ttctacctgt
tccccaaccg cctcgagtgg cggggcgagg gcgaggcccc gcagagcctg 1800ctgaccatgg
aggagatcca gtcggtggag gagacgcaga tcaaggagcg caagtgcctg 1860ctcctcaaga
tccgcggtgg gaaacagttc attttgcagt gcgatagcga ccctgagctg 1920gtgcagtgga
agaaggagct gcgcgacgcc taccgcgagg cccagcagct ggtgcagcgg 1980gtgcccaaga
tgaagaacaa gccgcgctcg cccgtggtgg agctgagcaa ggtgccgctg 2040gtccagcgcg
gcagtgccaa cggcctctga 207031689PRTHomo
sapiens 31Met Ala Asp Leu Glu Ala Val Leu Ala Asp Val Ser Tyr Leu Met
Ala1 5 10 15Met Glu Lys
Ser Lys Ala Thr Pro Ala Ala Arg Ala Ser Lys Lys Ile20 25
30Leu Leu Pro Glu Pro Ser Ile Arg Ser Val Met Gln Lys
Tyr Leu Glu35 40 45Asp Arg Gly Glu Val
Thr Phe Glu Lys Ile Phe Ser Gln Lys Leu Gly50 55
60Tyr Leu Leu Phe Arg Asp Phe Cys Leu Asn His Leu Glu Glu Ala
Arg65 70 75 80Pro Leu
Val Glu Phe Tyr Glu Glu Ile Lys Lys Tyr Glu Lys Leu Glu85
90 95Thr Glu Glu Glu Arg Val Ala Arg Ser Arg Glu Ile
Phe Asp Ser Tyr100 105 110Ile Met Lys Glu
Leu Leu Ala Cys Ser His Pro Phe Ser Lys Ser Ala115 120
125Thr Glu His Val Gln Gly His Leu Gly Lys Lys Gln Val Pro
Pro Asp130 135 140Leu Phe Gln Pro Tyr Ile
Glu Glu Ile Cys Gln Asn Leu Arg Gly Asp145 150
155 160Val Phe Gln Lys Phe Ile Glu Ser Asp Lys Phe
Thr Arg Phe Cys Gln165 170 175Trp Lys Asn
Val Glu Leu Asn Ile His Leu Thr Met Asn Asp Phe Ser180
185 190Val His Arg Ile Ile Gly Arg Gly Gly Phe Gly Glu
Val Tyr Gly Cys195 200 205Arg Lys Ala Asp
Thr Gly Lys Met Tyr Ala Met Lys Cys Leu Asp Lys210 215
220Lys Arg Ile Lys Met Lys Gln Gly Glu Thr Leu Ala Leu Asn
Glu Arg225 230 235 240Ile
Met Leu Ser Leu Val Ser Thr Gly Asp Cys Pro Phe Ile Val Cys245
250 255Met Ser Tyr Ala Phe His Thr Pro Asp Lys Leu
Ser Phe Ile Leu Asp260 265 270Leu Met Asn
Gly Gly Asp Leu His Tyr His Leu Ser Gln His Gly Val275
280 285Phe Ser Glu Ala Asp Met Arg Phe Tyr Ala Ala Glu
Ile Ile Leu Gly290 295 300Leu Glu His Met
His Asn Arg Phe Val Val Tyr Arg Asp Leu Lys Pro305 310
315 320Ala Asn Ile Leu Leu Asp Glu His Gly
His Val Arg Ile Ser Asp Leu325 330 335Gly
Leu Ala Cys Asp Phe Ser Lys Lys Lys Pro His Ala Ser Val Gly340
345 350Thr His Gly Tyr Met Ala Pro Glu Val Leu Gln
Lys Gly Val Ala Tyr355 360 365Asp Ser Ser
Ala Asp Trp Phe Ser Leu Gly Cys Met Leu Phe Lys Leu370
375 380Leu Arg Gly His Ser Pro Phe Arg Gln His Lys Thr
Lys Asp Lys His385 390 395
400Glu Ile Asp Arg Met Thr Leu Thr Met Ala Val Glu Leu Pro Asp Ser405
410 415Phe Ser Pro Glu Leu Arg Ser Leu Leu
Glu Gly Leu Leu Gln Arg Asp420 425 430Val
Asn Arg Arg Leu Gly Cys Leu Gly Arg Gly Ala Gln Glu Val Lys435
440 445Glu Ser Pro Phe Phe Arg Ser Leu Asp Trp Gln
Met Val Phe Leu Gln450 455 460Lys Tyr Pro
Pro Pro Leu Ile Pro Pro Arg Gly Glu Val Asn Ala Ala465
470 475 480Asp Ala Phe Asp Ile Gly Ser
Phe Asp Glu Glu Asp Thr Lys Gly Ile485 490
495Lys Leu Leu Asp Ser Asp Gln Glu Leu Tyr Arg Asn Phe Pro Leu Thr500
505 510Ile Ser Glu Arg Trp Gln Gln Glu Val
Ala Glu Thr Val Phe Asp Thr515 520 525Ile
Asn Ala Glu Thr Asp Arg Leu Glu Ala Arg Lys Lys Ala Lys Asn530
535 540Lys Gln Leu Gly His Glu Glu Asp Tyr Ala Leu
Gly Lys Asp Cys Ile545 550 555
560Met His Gly Tyr Met Ser Lys Met Gly Asn Pro Phe Leu Thr Gln
Trp565 570 575Gln Arg Arg Tyr Phe Tyr Leu
Phe Pro Asn Arg Leu Glu Trp Arg Gly580 585
590Glu Gly Glu Ala Pro Gln Ser Leu Leu Thr Met Glu Glu Ile Gln Ser595
600 605Val Glu Glu Thr Gln Ile Lys Glu Arg
Lys Cys Leu Leu Leu Lys Ile610 615 620Arg
Gly Gly Lys Gln Phe Ile Leu Gln Cys Asp Ser Asp Pro Glu Leu625
630 635 640Val Gln Trp Lys Lys Glu
Leu Arg Asp Ala Tyr Arg Glu Ala Gln Gln645 650
655Leu Val Gln Arg Val Pro Lys Met Lys Asn Lys Pro Arg Ser Pro
Val660 665 670Val Glu Leu Ser Lys Val Pro
Leu Val Gln Arg Gly Ser Cys Val Leu675 680
685Leu322070DNAHomo sapiens 32atggcggacc tggaggcggt gctggccgac
gtgagctacc tgatggccat ggagaagagc 60aaggccacgc cggccgcgcg cgccagcaag
aagatactgc tgcccgagcc cagcatccgc 120agtgtcatgc agaagtacct ggaggaccgg
ggcgaggtga cctttgagaa gatcttttcc 180cagaagctgg ggtacctgct cttccgagac
ttctgcctga accacctgga ggaggccagg 240cccttggtgg aattctatga ggagatcaag
aagtacgaga agctggagac ggaggaggag 300cgtgtggccc gcagccggga gatcttcgac
tcatacatca tgaaggagct gctggcctgc 360tcgcatccct tctcgaagag tgccactgag
catgtccaag gccacctggg gaagaagcag 420gtgcctccgg atctcttcca gccatacatc
gaagagattt gtcaaaacct ccgaggggac 480gtgttccaga aattcattga gagcgataag
ttcacacggt tttgccagtg gaagaatgtg 540gagctcaaca tccacctgac catgaatgac
ttcagcgtgc atcgcatcat tgggcgcggg 600ggctttggcg aggtctatgg gtgccggaag
gctgacacag gcaagatgta cgccatgaag 660tgcctggaca aaaagcgcat caagatgaag
cagggggaga ccctggccct gaacgagcgc 720atcatgctct cgctcgtcag cactggggac
tgcccattca ttgtctgcat gtcatacgcg 780ttccacacgc cagacaagct cagcttcatc
ctggacctca tgaacggtgg ggacctgcac 840taccacctct cccagcacgg ggtcttctca
gaggctgaca tgcgcttcta tgcggccgag 900atcatcctgg gcctggagca catgcacaac
cgcttcgtgg tctaccggga cctgaagcca 960gccaacatcc ttctggacga gcatggccac
gtgcggatct cggacctggg cctggcctgt 1020gacttctcca agaagaagcc ccatgccagc
gtgggcaccc acgggtacat ggctccggag 1080gtcctgcaga agggcgtggc ctacgacagc
agtgccgact ggttctctct ggggtgcatg 1140ctcttcaagt tgctgcgggg gcacagcccc
ttccggcagc acaagaccaa agacaagcat 1200gagatcgacc gcatgacgct gacgatggcc
gtggagctgc ccgactcctt ctcccctgaa 1260ctacgctccc tgctggaggg gttgctgcag
agggatgtca accggagatt gggctgcctg 1320ggccgagggg ctcaggaggt gaaagagagc
ccctttttcc gctccctgga ctggcagatg 1380gtcttcttgc agaagtaccc tcccccgctg
atccccccac gaggggaggt gaacgcggcc 1440gacgccttcg acattggctc cttcgatgag
gaggacacaa aaggaatcaa gttactggac 1500agtgatcagg agctctaccg caacttcccc
ctcaccatct cggagcggtg gcagcaggag 1560gtggcagaga ctgtcttcga caccatcaac
gctgagacag accggctgga ggctcgcaag 1620aaagccaaga acaagcagct gggccatgag
gaagactacg ccctgggcaa ggactgcatc 1680atgcatggct acatgtccaa gatgggcaac
cccttcctga cccagtggca gcggcggtac 1740ttctacctgt tccccaaccg cctcgagtgg
cggggcgagg gcgaggcccc gcagagcctg 1800ctgaccatgg aggagatcca gtcggtggag
gagacgcaga tcaaggagcg caagtgcctg 1860ctcctcaaga tccgcggtgg gaaacagttc
attttgcagt gcgatagcga ccctgagctg 1920gtgcagtgga agaaggagct gcgcgacgcc
taccgcgagg cccagcagct ggtgcagcgg 1980gtgcccaaga tgaagaacaa gccgcgctcg
cccgtggtgg agctgagcaa ggtgccgctg 2040gtccagcgcg gcagttgtgt gcttctttag
207033689PRTBos taurus 33Met Ala Asp Leu
Glu Ala Val Leu Ala Asp Val Ser Tyr Leu Met Ala1 5
10 15Met Glu Lys Ser Lys Ala Thr Pro Ala Ala Arg
Ala Ser Lys Lys Ile20 25 30Leu Leu Pro
Glu Pro Ser Ile Arg Ser Val Met Gln Lys Tyr Leu Glu35 40
45Asp Arg Gly Glu Val Thr Phe Glu Lys Ile Phe Ser Gln
Lys Leu Gly50 55 60Tyr Leu Leu Phe Arg
Asp Phe Cys Leu Lys His Leu Glu Glu Ala Lys65 70
75 80Pro Leu Val Glu Phe Tyr Glu Glu Ile Lys
Lys Tyr Glu Lys Leu Glu85 90 95Thr Glu
Glu Glu Arg Leu Val Cys Ser Arg Glu Ile Phe Asp Thr Tyr100
105 110Ile Met Lys Glu Leu Leu Ala Cys Ser His Pro Phe
Ser Lys Ser Ala115 120 125Ile Glu His Val
Gln Gly His Leu Val Lys Lys Gln Val Pro Pro Asp130 135
140Leu Phe Gln Pro Tyr Ile Glu Glu Ile Cys Gln Asn Leu Arg
Gly Asp145 150 155 160Val
Phe Gln Lys Phe Ile Glu Ser Asp Lys Phe Thr Arg Phe Cys Gln165
170 175Trp Lys Asn Val Glu Leu Asn Ile His Leu Thr
Met Asn Asp Phe Ser180 185 190Val His Arg
Ile Ile Gly Arg Gly Gly Phe Gly Glu Val Tyr Gly Cys195
200 205Arg Lys Ala Asp Thr Gly Lys Met Tyr Ala Met Lys
Cys Leu Asp Lys210 215 220Lys Arg Ile Lys
Met Lys Gln Gly Glu Thr Leu Ala Leu Asn Glu Arg225 230
235 240Ile Met Leu Ser Leu Val Ser Thr Gly
Asp Cys Pro Phe Ile Val Cys245 250 255Met
Ser Tyr Ala Phe His Thr Pro Asp Lys Leu Ser Phe Ile Leu Asp260
265 270Leu Met Asn Gly Gly Asp Leu His Tyr His Leu
Ser Gln His Gly Val275 280 285Phe Ser Glu
Ala Asp Met Arg Phe Tyr Ala Ala Glu Ile Ile Leu Gly290
295 300Leu Glu His Met His Asn Arg Phe Val Val Tyr Arg
Asp Leu Lys Pro305 310 315
320Ala Asn Ile Leu Leu Asp Glu His Gly His Val Arg Ile Ser Asp Leu325
330 335Gly Leu Ala Cys Asp Phe Ser Lys Lys
Lys Pro His Ala Ser Val Gly340 345 350Thr
His Gly Tyr Met Ala Pro Glu Val Leu Gln Lys Gly Val Ala Tyr355
360 365Asp Ser Ser Ala Asp Trp Phe Ser Leu Gly Cys
Met Leu Phe Lys Leu370 375 380Leu Arg Gly
His Ser Pro Phe Arg Gln His Lys Thr Lys Asp Lys His385
390 395 400Glu Ile Asp Arg Met Thr Leu
Thr Met Ala Val Glu Leu Pro Asp Ser405 410
415Phe Ser Pro Glu Leu Arg Ser Leu Leu Glu Gly Leu Leu Gln Arg Asp420
425 430Val Asn Arg Arg Leu Gly Cys Leu Gly
Arg Gly Ala Gln Glu Val Lys435 440 445Glu
Ser Pro Phe Phe Arg Ser Leu Asp Trp Gln Met Val Phe Leu Gln450
455 460Lys Tyr Pro Pro Pro Leu Ile Pro Pro Arg Gly
Glu Val Asn Ala Ala465 470 475
480Asp Ala Phe Asp Ile Gly Ser Phe Asp Glu Glu Asp Thr Lys Gly
Ile485 490 495Lys Leu Leu Asp Ser Asp Gln
Glu Leu Tyr Arg Asn Phe Pro Leu Thr500 505
510Ile Ser Glu Arg Trp Gln Gln Glu Val Ala Glu Thr Val Phe Asp Thr515
520 525Ile Asn Ala Glu Thr Asp Arg Leu Glu
Ala Arg Lys Lys Thr Lys Asn530 535 540Lys
Gln Leu Gly His Glu Glu Asp Tyr Ala Leu Gly Lys Asp Cys Ile545
550 555 560Met His Gly Tyr Met Ser
Lys Met Gly Asn Pro Phe Leu Thr Gln Trp565 570
575Gln Arg Arg Tyr Phe Tyr Leu Phe Pro Asn Arg Leu Glu Trp Arg
Gly580 585 590Glu Gly Glu Ala Pro Gln Ser
Leu Leu Thr Met Glu Glu Ile Gln Ser595 600
605Val Glu Glu Thr Gln Ile Lys Glu Arg Lys Cys Leu Leu Leu Lys Ile610
615 620Arg Gly Gly Lys Gln Phe Val Leu Gln
Cys Asp Ser Asp Pro Glu Leu625 630 635
640Val Gln Trp Lys Lys Glu Leu Arg Asp Ala Tyr Arg Glu Ala
Gln Gln645 650 655Leu Val Gln Arg Val Pro
Lys Met Lys Asn Lys Pro Arg Ser Pro Val660 665
670Val Glu Leu Ser Lys Val Pro Leu Ile Gln Arg Gly Ser Ala Asn
Gly675 680 685Leu342070DNABos taurus
34atggcggacc tggaggcggt gctggccgac gtgagctacc tgatggccat ggagaagagc
60aaggccacgc cggcggcgcg cgccagcaag aagatcctgc tgcccgagcc cagcatccgc
120agcgtcatgc agaagtacct ggaggaccgg ggcgaggtga cttttgagaa gatcttctcc
180cagaagctgg ggtacctgct tttccgagac ttctgcctga agcacctgga ggaggccaag
240cccttggtag agttctacga ggagatcaag aaatacgaga agctggagac agaggaggag
300cgcctggtct gcagccgaga gatcttcgac acgtacatca tgaaggagct gctggcctgc
360tcacatcctt tctcgaagag cgccattgag cacgtccagg gccatctggt gaagaagcag
420gtgcctccgg atctcttcca gccatatatt gaagaaattt gccagaacct ccgaggagac
480gtgttccaga aattcatcga gagcgataaa ttcacacgat tttgccagtg gaagaatgta
540gagctcaaca tccacctgac catgaacgac ttcagtgtgc accgcatcat cgggcgaggc
600ggcttcggtg aggtctacgg ctgccggaag gccgacacgg gcaagatgta cgccatgaag
660tgtctggaca agaagcgcat caagatgaag caaggggaga ctctggccct gaatgagcgc
720atcatgctgt cgctcgtcag caccggggac tgcccgttca tcgtctgcat gtcatacgcc
780ttccacacac cggacaagct cagcttcatc ctggatctca tgaacggcgg ggacctgcac
840taccacctgt cccagcacgg ggtcttctcc gaggccgaca tgcgtttcta cgccgccgag
900atcatcctgg gcctggagca catgcacaac cgcttcgtgg tctaccggga cctgaagccg
960gccaacatcc tgctggacga gcacggccac gtgcgcatct cagacctggg cctggcctgt
1020gacttctcca agaagaagcc tcacgccagt gtgggcaccc acgggtacat ggctcccgag
1080gttctacaga agggtgtggc ctacgacagc agcgccgact ggttctccct gggctgcatg
1140ctcttcaagc tgctgcgagg gcatagccct ttccggcagc acaagaccaa agacaagcat
1200gagatcgaca gaatgacatt gacaatggct gtggagctgc ctgactcctt ctcccctgag
1260ctccgctcct tgctggaggg gctgctgcag agggatgtca accggaggct aggctgcctg
1320ggccgagggg cccaggaggt gaaggagagc cccttcttcc gttccctgga ctggcagatg
1380gtctttttac aaaagtaccc tcccccgttg atccccccac gaggggaggt gaatgcagcc
1440gacgcctttg acattggctc cttcgatgag gaggacacaa aaggaatcaa gctactggac
1500agtgaccagg agctctaccg caacttcccc ctgaccatct cggagcggtg gcagcaggag
1560gtagcagaga ctgtctttga caccatcaat gctgagacgg accggctgga ggcccgcaag
1620aaaaccaaaa acaagcagtt gggccacgag gaagactacg ccctgggcaa ggactgcatc
1680atgcatggct acatgtccaa gatgggcaac cccttcctga cccagtggca gcggcggtac
1740ttctacctgt tccctaaccg gctcgagtgg cggggcgagg gcgaggcccc gcagagcctg
1800ctgaccatgg aggagatcca gtcggtggag gagacgcaga tcaaggagcg aaagtgcctc
1860ctcctcaaga tccgaggtgg caagcagttt gtcctgcagt gcgatagtga cccagagctg
1920gtgcagtgga agaaggagct tcgagacgcc taccgcgagg cccagcagct agtgcagcgg
1980gtgcccaaga tgaagaacaa gccgcgctcg cccgtcgtgg agctgagcaa ggtgccactg
2040atccagcgcg gcagtgccaa cggcctctga
207035330PRTHomo sapiens 35Met Met Trp Gly Ala Gly Ser Pro Leu Ala Trp
Leu Ser Ala Gly Ser1 5 10
15Gly Asn Val Asn Val Ser Ser Val Gly Pro Ala Glu Gly Pro Thr Gly20
25 30Pro Ala Ala Pro Leu Pro Ser Pro Lys Ala
Trp Asp Val Val Leu Cys35 40 45Ile Ser
Gly Thr Leu Val Ser Cys Glu Asn Ala Leu Val Val Ala Ile50
55 60Ile Val Gly Thr Pro Ala Phe Arg Ala Pro Met Phe
Leu Leu Val Gly65 70 75
80Ser Leu Ala Val Ala Asp Leu Leu Ala Gly Leu Gly Leu Val Leu His85
90 95Phe Ala Ala Val Phe Cys Ile Gly Ser Ala
Glu Met Ser Leu Val Leu100 105 110Val Gly
Val Leu Ala Met Ala Phe Thr Ala Ser Ile Gly Ser Leu Leu115
120 125Ala Ile Thr Val Asp Arg Tyr Leu Ser Leu Tyr Asn
Ala Leu Thr Tyr130 135 140Tyr Ser Glu Thr
Thr Val Thr Arg Thr Tyr Val Met Leu Ala Leu Val145 150
155 160Trp Gly Gly Ala Leu Gly Leu Gly Leu
Leu Pro Val Leu Ala Trp Asn165 170 175Cys
Leu Asp Gly Leu Thr Thr Cys Gly Val Val Tyr Pro Leu Ser Lys180
185 190Asn His Leu Val Val Leu Ala Ile Ala Phe Phe
Met Val Phe Gly Ile195 200 205Met Leu Gln
Leu Tyr Ala Gln Ile Cys Arg Ile Val Cys Arg His Ala210
215 220Gln Gln Ile Ala Leu Gln Arg His Leu Leu Pro Ala
Ser His Tyr Val225 230 235
240Ala Thr Arg Lys Gly Ile Ala Thr Leu Ala Val Val Leu Gly Ala Phe245
250 255Ala Ala Cys Trp Leu Pro Phe Thr Val
Tyr Cys Leu Leu Gly Asp Ala260 265 270His
Ser Pro Pro Leu Tyr Thr Tyr Leu Thr Leu Leu Pro Ala Thr Tyr275
280 285Asn Ser Met Ile Asn Pro Ile Ile Tyr Ala Phe
Arg Asn Gln Asp Val290 295 300Gln Lys Val
Leu Trp Ala Val Cys Cys Cys Cys Ser Ser Ser Lys Ile305
310 315 320Pro Phe Arg Ser Arg Ser Pro
Ser Asp Val325 33036993DNAHomo sapiens 36atgatgtggg
gtgcaggcag ccctctggcc tggctctcag ctggctcagg caacgtgaat 60gtaagcagcg
tgggcccagc agaggggccc acaggtccag ccgcaccact gccctcgcct 120aaggcctggg
atgtggtgct ctgcatctca ggcaccctgg tgtcctgcga gaatgcgcta 180gtggtggcca
tcatcgtggg cactcctgcc ttccgtgccc ccatgttcct gctggtgggc 240agcctggccg
tggcagacct gctggcaggc ctgggcctgg tcctgcactt tgctgctgtc 300ttctgcatcg
gctcagcgga gatgagcctg gtgctggttg gcgtgctggc aatggccttt 360accgccagca
tcggcagtct actggccatc actgtcgacc gctacctttc tctgtacaat 420gccctcacct
actattcaga gacaacagtg acacggacct atgtgatgct ggccttagtg 480tggggaggtg
ccctgggcct ggggctgctg cctgtgctgg cctggaactg cctggatggc 540ctgaccacat
gtggcgtggt ttatccactc tccaagaacc atctggtagt tctggccatt 600gccttcttca
tggtgtttgg catcatgctg cagctctacg cccaaatctg ccgcatcgtc 660tgccgccatg
cccagcagat tgcccttcag cggcacctgc tgcctgcctc ccactatgtg 720gccacccgca
agggcattgc cacactggcc gtggtgcttg gagcctttgc cgcctgctgg 780ttgcccttca
ctgtctactg cctgctgggt gatgcccact ctccacctct ctacacctat 840cttaccttgc
tccctgccac ctacaactcc atgatcaacc ctatcatcta cgccttccgc 900aaccaggatg
tgcagaaagt gctgtgggct gtctgctgct gctgttcctc ttccaagatc 960cccttccgat
cccgctcccc cagtgatgtc tag 99337344PRTHomo
sapiens 37Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ala Ala Ala Ala Met
Met1 5 10 15Trp Gly Ala
Gly Ser Pro Leu Ala Trp Leu Ser Ala Gly Ser Gly Asn20 25
30Val Asn Val Ser Ser Val Gly Pro Ala Glu Gly Pro Thr
Gly Pro Ala35 40 45Ala Pro Leu Pro Ser
Pro Lys Ala Trp Asp Val Val Leu Cys Ile Ser50 55
60Gly Thr Leu Val Ser Cys Glu Asn Ala Leu Val Val Ala Ile Ile
Val65 70 75 80Gly Thr
Pro Ala Phe Arg Ala Pro Met Phe Leu Leu Val Gly Ser Leu85
90 95Ala Val Ala Asp Leu Leu Ala Gly Leu Gly Leu Val
Leu His Phe Ala100 105 110Ala Val Phe Cys
Ile Gly Ser Ala Glu Met Ser Leu Val Leu Val Gly115 120
125Val Leu Ala Met Ala Phe Thr Ala Ser Ile Gly Ser Leu Leu
Ala Ile130 135 140Thr Val Asp Arg Tyr Leu
Ser Leu Tyr Asn Ala Leu Thr Tyr Tyr Ser145 150
155 160Glu Thr Thr Val Thr Arg Thr Tyr Val Met Leu
Ala Leu Val Trp Gly165 170 175Gly Ala Leu
Gly Leu Gly Leu Leu Pro Val Leu Ala Trp Asn Cys Leu180
185 190Asp Gly Leu Thr Thr Cys Gly Val Val Tyr Pro Leu
Ser Lys Asn His195 200 205Leu Val Val Leu
Ala Ile Ala Phe Phe Met Val Phe Gly Ile Met Leu210 215
220Gln Leu Tyr Ala Gln Ile Cys Arg Ile Val Cys Arg His Ala
Gln Gln225 230 235 240Ile
Ala Leu Gln Arg His Leu Leu Pro Ala Ser His Tyr Val Ala Thr245
250 255Arg Lys Gly Ile Ala Thr Leu Ala Val Val Leu
Gly Ala Phe Ala Ala260 265 270Cys Trp Leu
Pro Phe Thr Val Tyr Cys Leu Leu Gly Asp Ala His Ser275
280 285Pro Pro Leu Tyr Thr Tyr Leu Thr Leu Leu Pro Ala
Thr Tyr Asn Ser290 295 300Met Ile Asn Pro
Ile Ile Tyr Ala Phe Arg Asn Gln Asp Val Gln Lys305 310
315 320Val Leu Trp Ala Val Cys Cys Cys Cys
Ser Ser Ser Lys Ile Pro Phe325 330 335Arg
Ser Arg Ser Pro Ser Asp Val340381035DNAHomo sapiens 38atgtacccat
acgacgtacc tgattacgca gcagcagcag caatgatgtg gggtgcaggc 60agccctctgg
cctggctctc agctggctca ggcaacgtga atgtaagcag cgtgggccca 120gcagaggggc
ccacaggtcc agccgcacca ctgccctcgc ctaaggcctg ggatgtggtg 180ctctgcatct
caggcaccct ggtgtcctgc gagaatgcgc tagtggtggc catcatcgtg 240ggcactcctg
ccttccgtgc ccccatgttc ctgctggtgg gcagcctggc cgtggcagac 300ctgctggcag
gcctgggcct ggtcctgcac tttgctgctg tcttctgcat cggctcagcg 360gagatgagcc
tggtgctggt tggcgtgctg gcaatggcct ttaccgccag catcggcagt 420ctactggcca
tcactgtcga ccgctacctt tctctgtaca atgccctcac ctactattca 480gagacaacag
tgacacggac ctatgtgatg ctggccttag tgtggggagg tgccctgggc 540ctggggctgc
tgcctgtgct ggcctggaac tgcctggatg gcctgaccac atgtggcgtg 600gtttatccac
tctccaagaa ccatctggta gttctggcca ttgccttctt catggtgttt 660ggcatcatgc
tgcagctcta cgcccaaatc tgccgcatcg tctgccgcca tgcccagcag 720attgcccttc
agcggcacct gctgcctgcc tcccactatg tggccacccg caagggcatt 780gccacactgg
ccgtggtgct tggagccttt gccgcctgct ggttgccctt cactgtctac 840tgcctgctgg
gtgatgccca ctctccacct ctctacacct atcttacctt gctccctgcc 900acctacaact
ccatgatcaa ccctatcatc tacgccttcc gcaaccagga tgtgcagaaa 960gtgctgtggg
ctgtctgctg ctgctgttcc tcttccaaga tccccttccg atcccgctcc 1020cccagtgatg
tctag 103539346PRTHomo
sapiens 39Met Met Trp Gly Ala Gly Ser Pro Leu Ala Trp Leu Ser Ala Gly
Ser1 5 10 15Gly Asn Val
Asn Val Ser Ser Val Gly Pro Ala Glu Gly Pro Thr Gly20 25
30Pro Ala Ala Pro Leu Pro Ser Pro Lys Ala Trp Asp Val
Val Leu Cys35 40 45Ile Ser Gly Thr Leu
Val Ser Cys Glu Asn Ala Leu Val Val Ala Ile50 55
60Ile Val Gly Thr Pro Ala Phe Arg Ala Pro Met Phe Leu Leu Val
Gly65 70 75 80Ser Leu
Ala Val Ala Asp Leu Leu Ala Gly Leu Gly Leu Val Leu His85
90 95Phe Ala Ala Val Phe Cys Ile Gly Ser Ala Glu Met
Ser Leu Val Leu100 105 110Val Gly Val Leu
Ala Met Ala Phe Thr Ala Ser Ile Gly Ser Leu Leu115 120
125Ala Ile Thr Val Asp Arg Tyr Leu Ser Leu Tyr Asn Ala Leu
Thr Tyr130 135 140Tyr Ser Glu Thr Thr Val
Thr Arg Thr Tyr Val Met Leu Ala Leu Val145 150
155 160Trp Gly Gly Ala Leu Gly Leu Gly Leu Leu Pro
Val Leu Ala Trp Asn165 170 175Cys Leu Asp
Gly Leu Thr Thr Cys Gly Val Val Tyr Pro Leu Ser Lys180
185 190Asn His Leu Val Val Leu Ala Ile Ala Phe Phe Met
Val Phe Gly Ile195 200 205Met Leu Gln Leu
Tyr Ala Gln Ile Cys Arg Ile Val Cys Arg His Ala210 215
220Gln Gln Ile Ala Leu Gln Arg His Leu Leu Pro Ala Ser His
Tyr Val225 230 235 240Ala
Thr Arg Lys Gly Ile Ala Thr Leu Ala Val Val Leu Gly Ala Phe245
250 255Ala Ala Cys Trp Leu Pro Phe Thr Val Tyr Cys
Leu Leu Gly Asp Ala260 265 270His Ser Pro
Pro Leu Tyr Thr Tyr Leu Thr Leu Leu Pro Ala Thr Tyr275
280 285Asn Ser Met Ile Asn Pro Ile Ile Tyr Ala Phe Arg
Asn Gln Asp Val290 295 300Gln Lys Val Leu
Trp Ala Val Cys Cys Cys Cys Ala Ala Ala Arg Gly305 310
315 320Arg Thr Pro Pro Ser Leu Gly Pro Gln
Asp Glu Ser Cys Thr Thr Ala325 330 335Ser
Ser Ser Leu Ala Lys Asp Thr Ser Ser340 345401041DNAHomo
sapiens 40atgatgtggg gtgcaggcag ccctctggcc tggctctcag ctggctcagg
caacgtgaat 60gtaagcagcg tgggcccagc agaggggccc acaggtccag ccgcaccact
gccctcgcct 120aaggcctggg atgtggtgct ctgcatctca ggcaccctgg tgtcctgcga
gaatgcgcta 180gtggtggcca tcatcgtggg cactcctgcc ttccgtgccc ccatgttcct
gctggtgggc 240agcctggccg tggcagacct gctggcaggc ctgggcctgg tcctgcactt
tgctgctgtc 300ttctgcatcg gctcagcgga gatgagcctg gtgctggttg gcgtgctggc
aatggccttt 360acygccagca tcggcagtct actggccatc actgtcgacc gctacctttc
tctgtacaat 420gccctcacct actattcaga gacaacagtg acacggacct atgtgatgct
ggccttagtg 480tggggaggtg ccctgggcct ggggctgctg cctgtgctgg cctggaactg
cctggatggc 540ctgaccacat gtggcgtggt ttatccactc tccaagaacc atctggtagt
tctggccatt 600gccttcttca tggtgtttgg catcatgctg cagctctacg cccaaatctg
ccgcatcgtc 660tgccgccatg cccagcagat tgcccttcag cggcacctgc tgcctgcctc
ccactatgtg 720gccacccgca agggcattgc cacactggcc gtggtgcttg gagcctttgc
cgcctgctgg 780ttgcccttca ctgtctactg cctgctgggt gatgcccact ctccacctct
ctacacctat 840cttaccttgc tccctgccac ctacaactcc atgatcaacc ctatcatcta
cgccttccgc 900aaccaggatg tgcagaaagt gctgtgggct gtctgctgct gctgtgcggc
cgcacgggga 960cgcaccccac ccagcctggg tccccaagat gagtcctgca ccaccgccag
ctcctccctg 1020gccaaggaca cttcatcgtg a
104141360PRTHomo sapiens 41Met Tyr Pro Tyr Asp Val Pro Asp Tyr
Ala Ala Ala Ala Ala Met Met1 5 10
15Trp Gly Ala Gly Ser Pro Leu Ala Trp Leu Ser Ala Gly Ser Gly
Asn20 25 30Val Asn Val Ser Ser Val Gly
Pro Ala Glu Gly Pro Thr Gly Pro Ala35 40
45Ala Pro Leu Pro Ser Pro Lys Ala Trp Asp Val Val Leu Cys Ile Ser50
55 60Gly Thr Leu Val Ser Cys Glu Asn Ala Leu
Val Val Ala Ile Ile Val65 70 75
80Gly Thr Pro Ala Phe Arg Ala Pro Met Phe Leu Leu Val Gly Ser
Leu85 90 95Ala Val Ala Asp Leu Leu Ala
Gly Leu Gly Leu Val Leu His Phe Ala100 105
110Ala Val Phe Cys Ile Gly Ser Ala Glu Met Ser Leu Val Leu Val Gly115
120 125Val Leu Ala Met Ala Phe Thr Ala Ser
Ile Gly Ser Leu Leu Ala Ile130 135 140Thr
Val Asp Arg Tyr Leu Ser Leu Tyr Asn Ala Leu Thr Tyr Tyr Ser145
150 155 160Glu Thr Thr Val Thr Arg
Thr Tyr Val Met Leu Ala Leu Val Trp Gly165 170
175Gly Ala Leu Gly Leu Gly Leu Leu Pro Val Leu Ala Trp Asn Cys
Leu180 185 190Asp Gly Leu Thr Thr Cys Gly
Val Val Tyr Pro Leu Ser Lys Asn His195 200
205Leu Val Val Leu Ala Ile Ala Phe Phe Met Val Phe Gly Ile Met Leu210
215 220Gln Leu Tyr Ala Gln Ile Cys Arg Ile
Val Cys Arg His Ala Gln Gln225 230 235
240Ile Ala Leu Gln Arg His Leu Leu Pro Ala Ser His Tyr Val
Ala Thr245 250 255Arg Lys Gly Ile Ala Thr
Leu Ala Val Val Leu Gly Ala Phe Ala Ala260 265
270Cys Trp Leu Pro Phe Thr Val Tyr Cys Leu Leu Gly Asp Ala His
Ser275 280 285Pro Pro Leu Tyr Thr Tyr Leu
Thr Leu Leu Pro Ala Thr Tyr Asn Ser290 295
300Met Ile Asn Pro Ile Ile Tyr Ala Phe Arg Asn Gln Asp Val Gln Lys305
310 315 320Val Leu Trp Ala
Val Cys Cys Cys Cys Ala Ala Ala Arg Gly Arg Thr325 330
335Pro Pro Ser Leu Gly Pro Gln Asp Glu Ser Cys Thr Thr Ala
Ser Ser340 345 350Ser Leu Ala Lys Asp Thr
Ser Ser355 360421083DNAHomo sapiens 42atgtacccat
acgacgtacc tgattacgca gcagcagcag caatgatgtg gggtgcaggc 60agccctctgg
cctggctctc agctggctca ggcaacgtga atgtaagcag cgtgggccca 120gcagaggggc
ccacaggtcc agccgcacca ctgccctcgc ctaaggcctg ggatgtggtg 180ctctgcatct
caggcaccct ggtgtcctgc gagaatgcgc tagtggtggc catcatcgtg 240ggcactcctg
ccttccgtgc ccccatgttc ctgctggtgg gcagcctggc cgtggcagac 300ctgctggcag
gcctgggcct ggtcctgcac tttgctgctg tcttctgcat cggctcagcg 360gagatgagcc
tggtgctggt tggcgtgctg gcaatggcct ttactgccag catcggcagt 420ctactggcca
tcactgtcga ccgctacctt tctctgtaca atgccctcac ctactattca 480gagacaacag
tgacacggac ctatgtgatg ctggccttag tgtggggagg tgccctgggc 540ctggggctgc
tgcctgtgct ggcctggaac tgcctggatg gcctgaccac atgtggcgtg 600gtttatccac
tctccaagaa ccatctggta gttctggcca ttgccttctt catggtgttt 660ggcatcatgc
tgcagctcta cgcccaaatc tgccgcatcg tctgccgcca tgcccagcag 720attgcccttc
agcggcacct gctgcctgcc tcccactatg tggccacccg caagggcatt 780gccacactgg
ccgtggtgct tggagccttt gccgcctgct ggttgccctt cactgtctac 840tgcctgctgg
gtgatgccca ctctccacct ctctacacct atcttacctt gctccctgcc 900acctacaact
ccatgatcaa ccctatcatc tacgccttcc gcaaccagga tgtgcagaaa 960gtgctgtggg
ctgtctgctg ctgctgtgcg gccgcacggg gacgcacccc acccagcctg 1020ggtccccaag
atgagtcctg caccaccgcc agctcctccc tggccaagga cacttcatcg 1080tga
108343362PRTHomo
sapiens 43Met Asn Ala Ser Ala Ala Ser Leu Asn Asp Ser Gln Val Val Val
Val1 5 10 15Ala Ala Glu
Gly Ala Ala Ala Ala Ala Thr Ala Ala Gly Gly Pro Asp20 25
30Thr Gly Glu Trp Gly Pro Pro Ala Ala Ala Ala Leu Gly
Ala Gly Gly35 40 45Gly Ala Asn Gly Ser
Leu Glu Leu Ser Ser Gln Leu Ser Ala Gly Pro50 55
60Pro Gly Leu Leu Leu Pro Ala Val Asn Pro Trp Asp Val Leu Leu
Cys65 70 75 80Val Ser
Gly Thr Val Ile Ala Gly Glu Asn Ala Leu Val Val Ala Leu85
90 95Ile Ala Ser Thr Pro Ala Leu Arg Thr Pro Met Phe
Val Leu Val Gly100 105 110Ser Leu Ala Thr
Ala Asp Leu Leu Ala Gly Cys Gly Leu Ile Leu His115 120
125Phe Val Phe Gln Tyr Leu Val Pro Ser Glu Thr Val Ser Leu
Leu Thr130 135 140Val Gly Phe Leu Val Ala
Ser Phe Ala Ala Ser Val Ser Ser Leu Leu145 150
155 160Ala Ile Thr Val Asp Arg Tyr Leu Ser Leu Tyr
Asn Ala Leu Thr Tyr165 170 175Tyr Ser Arg
Arg Thr Leu Leu Gly Val His Leu Leu Leu Ala Ala Thr180
185 190Trp Thr Val Ser Leu Gly Leu Gly Leu Leu Pro Val
Leu Gly Trp Asn195 200 205Cys Leu Ala Glu
Arg Ala Ala Cys Ser Val Val Arg Pro Leu Ala Arg210 215
220Ser His Val Ala Leu Leu Ser Ala Ala Phe Phe Met Val Phe
Gly Ile225 230 235 240Met
Leu His Leu Tyr Val Arg Ile Cys Gln Val Val Trp Arg His Ala245
250 255His Gln Ile Ala Leu Gln Gln His Cys Leu Ala
Pro Pro His Leu Ala260 265 270Ala Thr Arg
Lys Gly Val Gly Thr Leu Ala Val Val Leu Gly Thr Phe275
280 285Gly Ala Ser Trp Leu Pro Phe Ala Ile Tyr Cys Val
Val Gly Ser His290 295 300Glu Asp Pro Ala
Val Tyr Thr Tyr Ala Thr Leu Leu Pro Ala Thr Tyr305 310
315 320Asn Ser Met Ile Asn Pro Ile Ile Tyr
Ala Phe Arg Asn Gln Glu Ile325 330 335Gln
Arg Ala Leu Trp Leu Leu Leu Cys Gly Cys Phe Gln Ser Lys Val340
345 350Pro Phe Arg Ser Arg Ser Pro Ser Glu Val355
360441089DNAHomo sapiens 44atgaacgcga gcgccgcctc gctcaacgac
tcccaggtgg tggtagtggc ggccgaagga 60gcggcggcgg cggccacagc agcagggggg
ccggacacgg gcgaatgggg accccctgct 120gcggcggctc taggagccgg cggcggagct
aatgggtctc tggagctgtc ctcgcagctg 180tcggctgggc caccgggact cctgctgcca
gcggtgaatc cgtgggacgt gctcctgtgc 240gtgtcgggga cagtgatcgc tggagaaaac
gcgctggtgg tggcgctcat cgcgtccact 300ccggcgctgc gcacgcccat gttcgtgctg
gtaggcagcc tggccaccgc tgacctgttg 360gcgggctgtg gcctcatctt gcactttgtg
ttccagtact tggtgccctc ggagactgtg 420agtctgctca cggtgggctt cctcgtggcc
tccttcgccg cctctgtcag cagcctgctg 480gccattacgg tggaccgcta cctgtccctg
tataacgcgc tcacctatta ctcgcgccgg 540accctgttgg gcgtgcacct cctgcttgcc
gccacttgga ccgtgtccct aggcctgggg 600ctgctgcccg tgctgggctg gaactgcctg
gcagagcgcg ccgcctgcag cgtggtgcgc 660ccgctggcgc gcagccacgt ggctctgctc
tccgccgcct tcttcatggt cttcggcatc 720atgctgcacc tgtacgtgcg catctgccag
gtggtctggc gccacgcgca ccagatcgcg 780ctgcagcagc actgcctggc gccaccccat
ctcgctgcca ccagaaaggg tgtgggtaca 840ctggctgtgg tgctgggcac tttcggcgcc
agctggctgc ccttcgccat ctattgcgtg 900gtgggcagcc atgaggaccc ggcggtctac
acttacgcca ccctgctgcc cgccacctac 960aactccatga tcaatcccat catctatgcc
ttccgcaacc aggagatcca gcgcgccctg 1020tggctcctgc tctgtggctg tttccagtcc
aaagtgccct ttcgttccag gtctcccagc 1080gaggtctga
108945376PRTHomo sapiens 45Met Tyr Pro
Tyr Asp Val Pro Asp Tyr Ala Ala Ala Ala Ala Met Asn1 5
10 15Ala Ser Ala Ala Ser Leu Asn Asp Ser Gln
Val Val Val Val Ala Ala20 25 30Glu Gly
Ala Ala Ala Ala Ala Thr Ala Ala Gly Gly Pro Asp Thr Gly35
40 45Glu Trp Gly Pro Pro Ala Ala Ala Ala Leu Gly Ala
Gly Gly Gly Ala50 55 60Asn Gly Ser Leu
Glu Leu Ser Ser Gln Leu Ser Ala Gly Pro Pro Gly65 70
75 80Leu Leu Leu Pro Ala Val Asn Pro Trp
Asp Val Leu Leu Cys Val Ser85 90 95Gly
Thr Val Ile Ala Gly Glu Asn Ala Leu Val Val Ala Leu Ile Ala100
105 110Ser Thr Pro Ala Leu Arg Thr Pro Met Phe Val
Leu Val Gly Ser Leu115 120 125Ala Thr Ala
Asp Leu Leu Ala Gly Cys Gly Leu Ile Leu His Phe Val130
135 140Phe Gln Tyr Leu Val Pro Ser Glu Thr Val Ser Leu
Leu Thr Val Gly145 150 155
160Phe Leu Val Ala Ser Phe Ala Ala Ser Val Ser Ser Leu Leu Ala Ile165
170 175Thr Val Asp Arg Tyr Leu Ser Leu Tyr
Asn Ala Leu Thr Tyr Tyr Ser180 185 190Arg
Arg Thr Leu Leu Gly Val His Leu Leu Leu Ala Ala Thr Trp Thr195
200 205Val Ser Leu Gly Leu Gly Leu Leu Pro Val Leu
Gly Trp Asn Cys Leu210 215 220Ala Glu Arg
Ala Ala Cys Ser Val Val Arg Pro Leu Ala Arg Ser His225
230 235 240Val Ala Leu Leu Ser Ala Ala
Phe Phe Met Val Phe Gly Ile Met Leu245 250
255His Leu Tyr Val Arg Ile Cys Gln Val Val Trp Arg His Ala His Gln260
265 270Ile Ala Leu Gln Gln His Cys Leu Ala
Pro Pro His Leu Ala Ala Thr275 280 285Arg
Lys Gly Val Gly Thr Leu Ala Val Val Leu Gly Thr Phe Gly Ala290
295 300Ser Trp Leu Pro Phe Ala Ile Tyr Cys Val Val
Gly Ser His Glu Asp305 310 315
320Pro Ala Val Tyr Thr Tyr Ala Thr Leu Leu Pro Ala Thr Tyr Asn
Ser325 330 335Met Ile Asn Pro Ile Ile Tyr
Ala Phe Arg Asn Gln Glu Ile Gln Arg340 345
350Ala Leu Trp Leu Leu Leu Cys Gly Cys Phe Gln Ser Lys Val Pro Phe355
360 365Arg Ser Arg Ser Pro Ser Glu Val370
375461131DNAHomo sapiens 46atgtacccat acgacgtacc tgattacgca
gcagcagcag caatgaacgc gagcgccgcc 60tcgctcaacg actcccaggt ggtggtagtg
gcggccgaag gagcggcggc ggcggccaca 120gcagcagggg ggccggacac gggcgaatgg
ggaccccctg ctgcggcggc tctaggagcc 180ggcggcggag ctaatgggtc tctggagctg
tcctcgcagc tgtcggctgg gccaccggga 240ctcctgctgc cagcggtgaa tccgtgggac
gtgctcctgt gcgtgtcggg gacagtgatc 300gctggagaaa acgcgctggt ggtggcgctc
atcgcgtcca ctccggcgct gcgcacgccc 360atgttcgtgc tggtaggcag cctggccacc
gctgacctgt tggcgggctg tggcctcatc 420ttgcactttg tgttccagta cttggtgccc
tcggagactg tgagtctgct cacggtgggc 480ttcctcgtgg cctccttcgc cgcctctgtc
agcagcctgc tggccattac ggtggaccgc 540tacctgtccc tgtataacgc gctcacctat
tactcgcgcc ggaccctgtt gggcgtgcac 600ctcctgcttg ccgccacttg gaccgtgtcc
ctaggcctgg ggctgctgcc cgtgctgggc 660tggaactgcc tggcagagcg cgccgcctgc
agcgtggtgc gcccgctggc gcgcagccac 720gtggctctgc tctccgccgc cttcttcatg
gtcttcggca tcatgctgca cctgtacgtg 780cgcatctgcc aggtggtctg gcgccacgcg
caccagatcg cgctgcagca gcactgcctg 840gcgccacccc atctcgctgc caccagaaag
ggtgtgggta cactggctgt ggtgctgggc 900actttcggcg ccagctggct gcccttcgcc
atctattgcg tggtgggcag ccatgaggac 960ccggcggtct acacttacgc caccctgctg
cccgccacct acaactccat gatcaatccc 1020atcatctatg ccttccgcaa ccaggagatc
cagcgcgccc tgtggctcct gctctgtggc 1080tgtttccagt ccaaagtgcc ctttcgttcc
aggtctccca gcgaggtctg a 113147378PRTHomo sapiens 47Met Asn Ala
Ser Ala Ala Ser Leu Asn Asp Ser Gln Val Val Val Val1 5
10 15Ala Ala Glu Gly Ala Ala Ala Ala Ala Thr
Ala Ala Gly Gly Pro Asp20 25 30Thr Gly
Glu Trp Gly Pro Pro Ala Ala Ala Ala Leu Gly Ala Gly Gly35
40 45Gly Ala Asn Gly Ser Leu Glu Leu Ser Ser Gln Leu
Ser Ala Gly Pro50 55 60Pro Gly Leu Leu
Leu Pro Ala Val Asn Pro Trp Asp Val Leu Leu Cys65 70
75 80Val Ser Gly Thr Val Ile Ala Gly Glu
Asn Ala Leu Val Val Ala Leu85 90 95Ile
Ala Ser Thr Pro Ala Leu Arg Thr Pro Met Phe Val Leu Val Gly100
105 110Ser Leu Ala Thr Ala Asp Leu Leu Ala Gly Cys
Gly Leu Ile Leu His115 120 125Phe Val Phe
Gln Tyr Leu Val Pro Ser Glu Thr Val Ser Leu Leu Thr130
135 140Val Gly Phe Leu Val Ala Ser Phe Ala Ala Ser Val
Ser Ser Leu Leu145 150 155
160Ala Ile Thr Val Asp Arg Tyr Leu Ser Leu Tyr Asn Ala Leu Thr Tyr165
170 175Tyr Ser Arg Arg Thr Leu Leu Gly Val
His Leu Leu Leu Ala Ala Thr180 185 190Trp
Thr Val Ser Leu Gly Leu Gly Leu Leu Pro Val Leu Gly Trp Asn195
200 205Cys Leu Ala Glu Arg Ala Ala Cys Ser Val Val
Arg Pro Leu Ala Arg210 215 220Ser His Val
Ala Leu Leu Ser Ala Ala Phe Phe Met Val Phe Gly Ile225
230 235 240Met Leu His Leu Tyr Val Arg
Ile Cys Gln Val Val Trp Arg His Ala245 250
255His Gln Ile Ala Leu Gln Gln His Cys Leu Ala Pro Pro His Leu Ala260
265 270Ala Thr Arg Lys Gly Val Gly Thr Leu
Ala Val Val Leu Gly Thr Phe275 280 285Gly
Ala Ser Trp Leu Pro Phe Ala Ile Tyr Cys Val Val Gly Ser His290
295 300Glu Asp Pro Ala Val Tyr Thr Tyr Ala Thr Leu
Leu Pro Ala Thr Tyr305 310 315
320Asn Ser Met Ile Asn Pro Ile Ile Tyr Ala Phe Arg Asn Gln Glu
Ile325 330 335Gln Arg Ala Leu Trp Leu Leu
Leu Cys Gly Cys Ala Ala Ala Arg Gly340 345
350Arg Thr Pro Pro Ser Leu Gly Pro Gln Asp Glu Ser Cys Thr Thr Ala355
360 365Ser Ser Ser Leu Ala Lys Asp Thr Ser
Ser370 375481137DNAHomo sapiens 48atgaacgcga gcgccgcctc
gctcaacgac tcccaggtgg tggtagtggc ggccgaagga 60gcggcggcgg cggccacagc
agcagggggg ccggacacgg gcgaatgggg accccctgct 120gcggcggctc taggagccgg
cggcggagct aatgggtctc tggagctgtc ctcgcagctg 180tcggctgggc caccgggact
cctgctgcca gcggtgaatc cgtgggacgt gctcctgtgc 240gtgtcgggga cagtgatcgc
tggagaaaac gcgctggtgg tggcgctcat cgcgtccact 300ccggcgctgc gcacgcccat
gttcgtgctg gtaggcagcc tggccaccgc tgacctgttg 360gcgggctgtg gcctcatctt
gcactttgtg ttccagtact tggtgccctc ggagactgtg 420agtctgctca cggtgggctt
cctcgtggcc tccttcgccg cctctgtcag cagcctgctg 480gccattacgg tggaccgcta
cctgtccctg tataacgcgc tcacctatta ctcgcgccgg 540accctgttgg gcgtgcacct
cctgcttgcc gccacttgga ccgtgtccct aggcctgggg 600ctgctgcccg tgctgggctg
gaactgcctg gcagagcgcg ccgcctgcag cgtggtgcgc 660ccgctggcgc gcagccacgt
ggctctgctc tccgccgcct tcttcatggt cttcggcatc 720atgctgcacc tgtacgtgcg
catctgccag gtggtctggc gccacgcgca ccagatcgcg 780ctgcagcagc actgcctggc
gccaccccat ctcgctgcca ccagaaaggg tgtgggtaca 840ctggctgtgg tgctgggcac
tttcggcgcc agctggctgc ccttcgccat ctattgcgtg 900gtgggcagcc atgaggaccc
ggcggtctac acttacgcca ccctgctgcc cgccacctac 960aactccatga tcaatcccat
catctatgcc ttccgcaacc aggagatcca gcgcgccctg 1020tggctcctgc tctgtggctg
tgcggccgca cggggacgca ccccacccag cctgggtccc 1080caagatgagt cctgcaccac
cgccagctcc tccctggcca aggacacttc atcgtga 113749392PRTHomo sapiens
49Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ala Ala Ala Ala Met Asn1
5 10 15Ala Ser Ala Ala Ser Leu
Asn Asp Ser Gln Val Val Val Val Ala Ala20 25
30Glu Gly Ala Ala Ala Ala Ala Thr Ala Ala Gly Gly Pro Asp Thr Gly35
40 45Glu Trp Gly Pro Pro Ala Ala Ala Ala
Leu Gly Ala Gly Gly Gly Ala50 55 60Asn
Gly Ser Leu Glu Leu Ser Ser Gln Leu Ser Ala Gly Pro Pro Gly65
70 75 80Leu Leu Leu Pro Ala Val
Asn Pro Trp Asp Val Leu Leu Cys Val Ser85 90
95Gly Thr Val Ile Ala Gly Glu Asn Ala Leu Val Val Ala Leu Ile Ala100
105 110Ser Thr Pro Ala Leu Arg Thr Pro
Met Phe Val Leu Val Gly Ser Leu115 120
125Ala Thr Ala Asp Leu Leu Ala Gly Cys Gly Leu Ile Leu His Phe Val130
135 140Phe Gln Tyr Leu Val Pro Ser Glu Thr
Val Ser Leu Leu Thr Val Gly145 150 155
160Phe Leu Val Ala Ser Phe Ala Ala Ser Val Ser Ser Leu Leu
Ala Ile165 170 175Thr Val Asp Arg Tyr Leu
Ser Leu Tyr Asn Ala Leu Thr Tyr Tyr Ser180 185
190Arg Arg Thr Leu Leu Gly Val His Leu Leu Leu Ala Ala Thr Trp
Thr195 200 205Val Ser Leu Gly Leu Gly Leu
Leu Pro Val Leu Gly Trp Asn Cys Leu210 215
220Ala Glu Arg Ala Ala Cys Ser Val Val Arg Pro Leu Ala Arg Ser His225
230 235 240Val Ala Leu Leu
Ser Ala Ala Phe Phe Met Val Phe Gly Ile Met Leu245 250
255His Leu Tyr Val Arg Ile Cys Gln Val Val Trp Arg His Ala
His Gln260 265 270Ile Ala Leu Gln Gln His
Cys Leu Ala Pro Pro His Leu Ala Ala Thr275 280
285Arg Lys Gly Val Gly Thr Leu Ala Val Val Leu Gly Thr Phe Gly
Ala290 295 300Ser Trp Leu Pro Phe Ala Ile
Tyr Cys Val Val Gly Ser His Glu Asp305 310
315 320Pro Ala Val Tyr Thr Tyr Ala Thr Leu Leu Pro Ala
Thr Tyr Asn Ser325 330 335Met Ile Asn Pro
Ile Ile Tyr Ala Phe Arg Asn Gln Glu Ile Gln Arg340 345
350Ala Leu Trp Leu Leu Leu Cys Gly Cys Ala Ala Ala Arg Gly
Arg Thr355 360 365Pro Pro Ser Leu Gly Pro
Gln Asp Glu Ser Cys Thr Thr Ala Ser Ser370 375
380Ser Leu Ala Lys Asp Thr Ser Ser385
390501179DNAHomo sapiens 50atgtacccat acgacgtacc tgattacgca gcagcagcag
caatgaacgc gagcgccgcc 60tcgctcaacg actcccaggt ggtggtagtg gcggccgaag
gagcggcggc ggcggccaca 120gcagcagggg ggccggacac gggcgaatgg ggaccccctg
ctgcggcggc tctaggagcc 180ggcggcggag ctaatgggtc tctggagctg tcctcgcagc
tgtcggctgg gccaccggga 240ctcctgctgc cagcggtgaa tccgtgggac gtgctcctgt
gcgtgtcggg gacagtgatc 300gctggagaaa acgcgctggt ggtggcgctc atcgcgtcca
ctccggcgct gcgcacgccc 360atgttcgtgc tggtaggcag cctggccacc gctgacctgt
tggcgggctg tggcctcatc 420ttgcactttg tgttccagta cttggtgccc tcggagactg
tgagtctgct cacggtgggc 480ttcctcgtgg cctccttcgc cgcctctgtc agcagcctgc
tggccattac ggtggaccgc 540tacctgtccc tgtataacgc gctcacctat tactcgcgcc
ggaccctgtt gggcgtgcac 600ctcctgcttg ccgccacttg gaccgtgtcc ctaggcctgg
ggctgctgcc cgtgctgggc 660tggaactgcc tggcagagcg cgccgcctgc agcgtggtgc
gcccgctggc gcgcagccac 720gtggctctgc tctccgccgc cttcttcatg gtcttcggca
tcatgctgca cctgtacgtg 780cgcatctgcc aggtggtctg gcgccacgcg caccagatcg
cgctgcagca gcactgcctg 840gcgccacccc atctcgctgc caccagaaag ggtgtgggta
cactggctgt ggtgctgggc 900actttcggcg ccagctggct gcccttcgcc atctattgcg
tggtgggcag ccatgaggac 960ccggcggtct acacttacgc caccctgctg cccgccacct
acaactccat gatcaatccc 1020atcatctatg ccttccgcaa ccaggagatc cagcgcgccc
tgtggctcct gctctgtggc 1080tgtgcggccg cacggggacg caccccaccc agcctgggtc
cccaagatga gtcctgcacc 1140accgccagct cctccctggc caaggacact tcatcgtga
117951334PRTHomo sapiens 51Met Asn Glu Asp Leu Lys
Val Asn Leu Ser Gly Leu Pro Arg Asp Tyr1 5
10 15Leu Asp Ala Ala Ala Ala Glu Asn Ile Ser Ala Ala Val
Ser Ser Arg20 25 30Val Pro Ala Val Glu
Pro Glu Pro Glu Leu Val Val Asn Pro Trp Asp35 40
45Ile Val Leu Cys Thr Ser Gly Thr Leu Ile Ser Cys Glu Asn Ala
Ile50 55 60Val Val Leu Ile Ile Phe His
Asn Pro Ser Leu Arg Ala Pro Met Phe65 70
75 80Leu Leu Ile Gly Ser Leu Ala Leu Ala Asp Leu Leu
Ala Gly Ile Gly85 90 95Leu Ile Thr Asn
Phe Val Phe Ala Tyr Leu Leu Gln Ser Glu Ala Thr100 105
110Lys Leu Val Thr Ile Gly Leu Ile Val Ala Ser Phe Ser Ala
Ser Val115 120 125Cys Ser Leu Leu Ala Ile
Thr Val Asp Arg Tyr Leu Ser Leu Tyr Tyr130 135
140Ala Leu Thr Tyr His Ser Glu Arg Thr Val Thr Phe Thr Tyr Val
Met145 150 155 160Leu Val
Met Leu Trp Gly Thr Ser Ile Cys Leu Gly Leu Leu Pro Val165
170 175Met Gly Trp Asn Cys Leu Arg Asp Glu Ser Thr Cys
Ser Val Val Arg180 185 190Pro Leu Thr Lys
Asn Asn Ala Ala Ile Leu Ser Val Ser Phe Leu Phe195 200
205Met Phe Ala Leu Met Leu Gln Leu Tyr Ile Gln Ile Cys Lys
Ile Val210 215 220Met Arg His Ala His Gln
Ile Ala Leu Gln His His Phe Leu Ala Thr225 230
235 240Ser His Tyr Val Thr Thr Arg Lys Gly Val Ser
Thr Leu Ala Ile Ile245 250 255Leu Gly Thr
Phe Ala Ala Cys Trp Met Pro Phe Thr Leu Tyr Ser Leu260
265 270Ile Ala Asp Tyr Thr Tyr Pro Ser Ile Tyr Thr Tyr
Ala Thr Leu Leu275 280 285Pro Ala Thr Tyr
Asn Ser Ile Ile Asn Pro Val Ile Tyr Ala Phe Arg290 295
300Asn Gln Glu Ile Gln Lys Ala Leu Cys Leu Ile Cys Cys Gly
Cys Ile305 310 315 320Pro
Ser Ser Leu Ala Gln Arg Ala Arg Ser Pro Ser Asp Val325
330521005DNAHomo sapiens 52atgaatgaag acctgaaggt caatttaagc gggctgcctc
gggattattt agatgccgct 60gctgcggaga acatctcggc tgctgtctcc tcccgggttc
ctgccgtaga gccagagcct 120gagctcgtag tcaacccctg ggacattgtc ttgtgtacct
cgggaaccct catctcctgt 180gaaaatgcca ttgtggtcct tatcatcttc cacaacccca
gcctgcgagc acccatgttc 240ctgctaatag gcagcctggc tcttgcagac ctgctggccg
gcattggact catcaccaat 300tttgtttttg cctacctgct tcagtcagaa gccaccaagc
tggtcacgat cggcctcatt 360gtcgcctctt tctctgcctc tgtctgcagc ttgctggcta
tcactgttga ccgctacctc 420tcactgtact acgctctgac gtaccattcg gagaggacgg
tcacgtttac ctatgtcatg 480ctcgtcatgc tctgggggac ctccatctgc ctggggctgc
tgcccgtcat gggctggaac 540tgcctccgag acgagtccac ctgcagcgtg gtcagaccgc
tcaccaagaa caacgcggcc 600atcctctcgg tgtccttcct cttcatgttt gcgctcatgc
ttcagctcta catccagatc 660tgtaagattg tgatgaggca cgcccatcag atagccctgc
agcaccactt cctggccacg 720tcgcactatg tgaccacccg gaaaggggtc tccaccctgg
ctatcatcct ggggacgttt 780gctgcttgct ggatgccttt caccctctat tccttgatag
cggattacac ctacccctcc 840atctatacct acgccaccct cctgcccgcc acctacaatt
ccatcatcaa ccctgtcata 900tatgctttca gaaaccaaga gatccagaaa gcgctctgtc
tcatttgctg cggctgcatc 960ccgtccagtc tcgcccagag agcgcgctcg cccagtgatg
tgtag 100553348PRTHomo sapiens 53Met Tyr Pro Tyr Asp
Val Pro Asp Tyr Ala Ala Ala Ala Ala Met Asn1 5
10 15Glu Asp Leu Lys Val Asn Leu Ser Gly Leu Pro Arg
Asp Tyr Leu Asp20 25 30Ala Ala Ala Ala
Glu Asn Ile Ser Ala Ala Val Ser Ser Arg Val Pro35 40
45Ala Val Glu Pro Glu Pro Glu Leu Val Val Asn Pro Trp Asp
Ile Val50 55 60Leu Cys Thr Ser Gly Thr
Leu Ile Ser Cys Glu Asn Ala Ile Val Val65 70
75 80Leu Ile Ile Phe His Asn Pro Ser Leu Arg Ala
Pro Met Phe Leu Leu85 90 95Ile Gly Ser
Leu Ala Leu Ala Asp Leu Leu Ala Gly Ile Gly Leu Ile100
105 110Thr Asn Phe Val Phe Ala Tyr Leu Leu Gln Ser Glu
Ala Thr Lys Leu115 120 125Val Thr Ile Gly
Leu Ile Val Ala Ser Phe Ser Ala Ser Val Cys Ser130 135
140Leu Leu Ala Ile Thr Val Asp Arg Tyr Leu Ser Leu Tyr Tyr
Ala Leu145 150 155 160Thr
Tyr His Ser Glu Arg Thr Val Thr Phe Thr Tyr Val Met Leu Val165
170 175Met Leu Trp Gly Thr Ser Ile Cys Leu Gly Leu
Leu Pro Val Met Gly180 185 190Trp Asn Cys
Leu Arg Asp Glu Ser Thr Cys Ser Val Val Arg Pro Leu195
200 205Thr Lys Asn Asn Ala Ala Ile Leu Ser Val Ser Phe
Leu Phe Met Phe210 215 220Ala Leu Met Leu
Gln Leu Tyr Ile Gln Ile Cys Lys Ile Val Met Arg225 230
235 240His Ala His Gln Ile Ala Leu Gln His
His Phe Leu Ala Thr Ser His245 250 255Tyr
Val Thr Thr Arg Lys Gly Val Ser Thr Leu Ala Ile Ile Leu Gly260
265 270Thr Phe Ala Ala Cys Trp Met Pro Phe Thr Leu
Tyr Ser Leu Ile Ala275 280 285Asp Tyr Thr
Tyr Pro Ser Ile Tyr Thr Tyr Ala Thr Leu Leu Pro Ala290
295 300Thr Tyr Asn Ser Ile Ile Asn Pro Val Ile Tyr Ala
Phe Arg Asn Gln305 310 315
320Glu Ile Gln Lys Ala Leu Cys Leu Ile Cys Cys Gly Cys Ile Pro Ser325
330 335Ser Leu Ala Gln Arg Ala Arg Ser Pro
Ser Asp Val340 345541047DNAHomo sapiens 54atgtacccat
acgacgtacc tgattacgca gcagcagcag caatgaatga agacctgaag 60gtcaatttaa
gcgggctgcc tcgggattat ttagatgccg ctgctgcgga gaacatctcg 120gctgctgtct
cctcccgggt tcctgccgta gagccagagc ctgagctcgt agtcaacccc 180tgggacattg
tcttgtgtac ctcgggaacc ctcatctcct gtgaaaatgc cattgtggtc 240cttatcatct
tccacaaccc cagcctgcga gcacccatgt tcctgctaat aggcagcctg 300gctcttgcag
acctgctggc cggcattgga ctcatcacca attttgtttt tgcctacctg 360cttcagtcag
aagccaccaa gctggtcacg atcggcctca ttgtcgcctc tttctctgcc 420tctgtctgca
gcttgctggc tatcactgtt gaccgctacc tctcactgta ctacgctctg 480acgtaccatt
cggagaggac ggtcacgttt acctatgtca tgctcgtcat gctctggggg 540acctccatct
gcctggggct gctgcccgtc atgggctgga actgcctccg agacgagtcc 600acctgcagcg
tggtcagacc gctcaccaag aacaacgcgg ccatcctctc ggtgtccttc 660ctcttcatgt
ttgcgctcat gcttcagctc tacatccaga tctgtaagat tgtgatgagg 720cacgcccatc
agatagccct gcagcaccac ttcctggcca cgtcgcacta tgtgaccacc 780cggaaagggg
tctccaccct ggctatcatc ctggggacgt ttgctgcttg ctggatgcct 840ttcaccctct
attccttgat agcggattac acctacccct ccatctatac ctacgccacc 900ctcctgcccg
ccacctacaa ttccatcatc aaccctgtca tatatgcttt cagaaaccaa 960gagatccaga
aagcgctctg tctcatttgc tgcggctgca tcccgtccag tctcgcccag 1020agagcgcgct
cgcccagtga tgtgtag 104755350PRTHomo
sapiens 55Met Asn Glu Asp Leu Lys Val Asn Leu Ser Gly Leu Pro Arg Asp
Tyr1 5 10 15Leu Asp Ala
Ala Ala Ala Glu Asn Ile Ser Ala Ala Val Ser Ser Arg20 25
30Val Pro Ala Val Glu Pro Glu Pro Glu Leu Val Val Asn
Pro Trp Asp35 40 45Ile Val Leu Cys Thr
Ser Gly Thr Leu Ile Ser Cys Glu Asn Ala Ile50 55
60Val Val Leu Ile Ile Phe His Asn Pro Ser Leu Arg Ala Pro Met
Phe65 70 75 80Leu Leu
Ile Gly Ser Leu Ala Leu Ala Asp Leu Leu Ala Gly Ile Gly85
90 95Leu Ile Thr Asn Phe Val Phe Ala Tyr Leu Leu Gln
Ser Glu Ala Thr100 105 110Lys Leu Val Thr
Ile Gly Leu Ile Val Ala Ser Phe Ser Ala Ser Val115 120
125Cys Ser Leu Leu Ala Ile Thr Val Asp Arg Tyr Leu Ser Leu
Tyr Tyr130 135 140Ala Leu Thr Tyr His Ser
Glu Arg Thr Val Thr Phe Thr Tyr Val Met145 150
155 160Leu Val Met Leu Trp Gly Thr Ser Ile Cys Leu
Gly Leu Leu Pro Val165 170 175Met Gly Trp
Asn Cys Leu Arg Asp Glu Ser Thr Cys Ser Val Val Arg180
185 190Pro Leu Thr Lys Asn Asn Ala Ala Ile Leu Ser Val
Ser Phe Leu Phe195 200 205Met Phe Ala Leu
Met Leu Gln Leu Tyr Ile Gln Ile Cys Lys Ile Val210 215
220Met Arg His Ala His Gln Ile Ala Leu Gln His His Phe Leu
Ala Thr225 230 235 240Ser
His Tyr Val Thr Thr Arg Lys Gly Val Ser Thr Leu Ala Ile Ile245
250 255Leu Gly Thr Phe Ala Ala Cys Trp Met Pro Phe
Thr Leu Tyr Ser Leu260 265 270Ile Ala Asp
Tyr Thr Tyr Pro Ser Ile Tyr Thr Tyr Ala Thr Leu Leu275
280 285Pro Ala Thr Tyr Asn Ser Ile Ile Asn Pro Val Ile
Tyr Ala Phe Arg290 295 300Asn Gln Glu Ile
Gln Lys Ala Leu Cys Leu Ile Cys Cys Gly Cys Ala305 310
315 320Ala Ala Arg Gly Arg Thr Pro Pro Ser
Leu Gly Pro Gln Asp Glu Ser325 330 335Cys
Thr Thr Ala Ser Ser Ser Leu Ala Lys Asp Thr Ser Ser340
345 350561053DNAHomo sapiens 56atgaatgaag acctgaaggt
caatttaagc gggctgcctc gggattattt agatgccgct 60gctgcggaga acatctcggc
tgctgtctcc tcccgggttc ctgccgtaga gccagagcct 120gagctcgtag tcaacccctg
ggacattgtc ttgtgtacct cgggaaccct catctcctgt 180gaaaatgcca ttgtggtcct
tatcatcttc cacaacccca gcctgcgagc acccatgttc 240ctgctaatag gcagcctggc
tcttgcagac ctgctggccg gcattggact catcaccaat 300tttgtttttg cctacctgct
tcagtcagaa gccaccaagc tggtcacgat cggcctcatt 360gtcgcctctt tctctgcctc
tgtctgcagc ttgctggcta tcactgttga ccgctacctc 420tcactgtact acgctctgac
gtaccattcg gagaggacgg tcacgtttac ctatgtcatg 480ctcgtcatgc tctgggggac
ctccatctgc ctggggctgc tgcccgtcat gggctggaac 540tgcctccgag acgagtccac
ctgcagcgtg gtcagaccgc tcaccaagaa caacgcggcc 600atcctctcgg tgtccttcct
cttcatgttt gcgctcatgc ttcagctcta catccagatc 660tgtaagattg tgatgaggca
cgcccatcag atagccctgc agcaccactt cctggccacg 720tcgcactatg tgaccacccg
gaaaggggtc tccaccctgg ctatcatcct ggggacgttt 780gctgcttgct ggatgccttt
caccctctat tccttgatag cggattacac ctacccctcc 840atctatacct acgccaccct
cctgcccgcc acctacaatt ccatcatcaa ccctgtcata 900tatgctttca gaaaccaaga
gatccagaaa gcgctctgtc tcatttgctg cggctgcgcg 960gccgcacggg gacgcacccc
acccagcctg ggtccccaag atgagtcctg caccaccgcc 1020agctcctccc tggccaagga
cacttcatcg tga 105357364PRTHomo sapiens
57Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ala Ala Ala Ala Met Asn1
5 10 15Glu Asp Leu Lys Val Asn
Leu Ser Gly Leu Pro Arg Asp Tyr Leu Asp20 25
30Ala Ala Ala Ala Glu Asn Ile Ser Ala Ala Val Ser Ser Arg Val Pro35
40 45Ala Val Glu Pro Glu Pro Glu Leu Val
Val Asn Pro Trp Asp Ile Val50 55 60Leu
Cys Thr Ser Gly Thr Leu Ile Ser Cys Glu Asn Ala Ile Val Val65
70 75 80Leu Ile Ile Phe His Asn
Pro Ser Leu Arg Ala Pro Met Phe Leu Leu85 90
95Ile Gly Ser Leu Ala Leu Ala Asp Leu Leu Ala Gly Ile Gly Leu Ile100
105 110Thr Asn Phe Val Phe Ala Tyr Leu
Leu Gln Ser Glu Ala Thr Lys Leu115 120
125Val Thr Ile Gly Leu Ile Val Ala Ser Phe Ser Ala Ser Val Cys Ser130
135 140Leu Leu Ala Ile Thr Val Asp Arg Tyr
Leu Ser Leu Tyr Tyr Ala Leu145 150 155
160Thr Tyr His Ser Glu Arg Thr Val Thr Phe Thr Tyr Val Met
Leu Val165 170 175Met Leu Trp Gly Thr Ser
Ile Cys Leu Gly Leu Leu Pro Val Met Gly180 185
190Trp Asn Cys Leu Arg Asp Glu Ser Thr Cys Ser Val Val Arg Pro
Leu195 200 205Thr Lys Asn Asn Ala Ala Ile
Leu Ser Val Ser Phe Leu Phe Met Phe210 215
220Ala Leu Met Leu Gln Leu Tyr Ile Gln Ile Cys Lys Ile Val Met Arg225
230 235 240His Ala His Gln
Ile Ala Leu Gln His His Phe Leu Ala Thr Ser His245 250
255Tyr Val Thr Thr Arg Lys Gly Val Ser Thr Leu Ala Ile Ile
Leu Gly260 265 270Thr Phe Ala Ala Cys Trp
Met Pro Phe Thr Leu Tyr Ser Leu Ile Ala275 280
285Asp Tyr Thr Tyr Pro Ser Ile Tyr Thr Tyr Ala Thr Leu Leu Pro
Ala290 295 300Thr Tyr Asn Ser Ile Ile Asn
Pro Val Ile Tyr Ala Phe Arg Asn Gln305 310
315 320Glu Ile Gln Lys Ala Leu Cys Leu Ile Cys Cys Gly
Cys Ala Ala Ala325 330 335Arg Gly Arg Thr
Pro Pro Ser Leu Gly Pro Gln Asp Glu Ser Cys Thr340 345
350Thr Ala Ser Ser Ser Leu Ala Lys Asp Thr Ser Ser355
360581095DNAHomo sapiens 58atgtacccat acgacgtacc tgattacgca
gcagcagcag caatgaatga agacctgaag 60gtcaatttaa gcgggctgcc tcgggattat
ttagatgccg ctgctgcgga gaacatctcg 120gctgctgtct cctcccgggt tcctgccgta
gagccagagc ctgagctcgt agtcaacccc 180tgggacattg tcttgtgtac ctcgggaacc
ctcatctcct gtgaaaatgc cattgtggtc 240cttatcatct tccacaaccc cagcctgcga
gcacccatgt tcctgctaat aggcagcctg 300gctcttgcag acctgctggc cggcattgga
ctcatcacca attttgtttt tgcctacctg 360cttcagtcag aagccaccaa gctggtcacg
atcggcctca ttgtcgcctc tttctctgcc 420tctgtctgca gcttgctggc tatcactgtt
gaccgctacc tctcactgta ctacgctctg 480acgtaccatt cggagaggac ggtcacgttt
acctatgtca tgctcgtcat gctctggggg 540acctccatct gcctggggct gctgcccgtc
atgggctgga actgcctccg agacgagtcc 600acctgcagcg tggtcagacc gctcaccaag
aacaacgcgg ccatcctctc ggtgtccttc 660ctcttcatgt ttgcgctcat gcttcagctc
tacatccaga tctgtaagat tgtgatgagg 720cacgcccatc agatagccct gcagcaccac
ttcctggcca cgtcgcacta tgtgaccacc 780cggaaagggg tctccaccct ggctatcatc
ctggggacgt ttgctgcttg ctggatgcct 840ttcaccctct attccttgat agcggattac
acctacccct ccatctatac ctacgccacc 900ctcctgcccg ccacctacaa ttccatcatc
aaccctgtca tatatgcttt cagaaaccaa 960gagatccaga aagcgctctg tctcatttgc
tgcggctgcg cggccgcacg gggacgcacc 1020ccacccagcc tgggtcccca agatgagtcc
tgcaccaccg ccagctcctc cctggccaag 1080gacacttcat cgtga
109559373PRTHomo sapiens 59Met Ala Asn
Thr Thr Gly Glu Pro Glu Glu Val Ser Gly Ala Leu Ser1 5
10 15Pro Pro Ser Ala Ser Ala Tyr Val Lys Leu
Val Leu Leu Gly Leu Ile20 25 30Met Cys
Val Ser Leu Ala Gly Asn Ala Ile Leu Ser Leu Leu Val Leu35
40 45Lys Glu Arg Ala Leu His Lys Ala Pro Tyr Tyr Phe
Leu Leu Asp Leu50 55 60Cys Leu Ala Asp
Gly Ile Arg Ser Ala Val Cys Phe Pro Phe Val Leu65 70
75 80Ala Ser Val Arg His Gly Ser Ser Trp
Thr Phe Ser Ala Leu Ser Cys85 90 95Lys
Ile Val Ala Phe Met Ala Val Leu Phe Cys Phe His Ala Ala Phe100
105 110Met Leu Phe Cys Ile Ser Val Thr Arg Tyr Met
Ala Ile Ala His His115 120 125Arg Phe Tyr
Ala Lys Arg Met Thr Leu Trp Thr Cys Ala Ala Val Ile130
135 140Cys Met Ala Trp Thr Leu Ser Val Ala Met Ala Phe
Pro Pro Val Phe145 150 155
160Asp Val Gly Thr Tyr Lys Phe Ile Arg Glu Glu Asp Gln Cys Ile Phe165
170 175Glu His Arg Tyr Phe Lys Ala Asn Asp
Thr Leu Gly Phe Met Leu Met180 185 190Leu
Ala Val Leu Met Ala Ala Thr His Ala Val Tyr Gly Lys Leu Leu195
200 205Leu Phe Glu Tyr Arg His Arg Lys Met Lys Pro
Val Gln Met Val Pro210 215 220Ala Ile Ser
Gln Asn Trp Thr Phe His Gly Pro Gly Ala Thr Gly Gln225
230 235 240Ala Ala Ala Asn Trp Ile Ala
Gly Phe Gly Arg Gly Pro Met Pro Pro245 250
255Thr Leu Leu Gly Ile Arg Gln Asn Gly His Ala Ala Ser Arg Arg Leu260
265 270Leu Gly Met Asp Glu Val Lys Gly Glu
Lys Gln Leu Gly Arg Met Phe275 280 285Tyr
Ala Ile Thr Leu Leu Phe Leu Leu Leu Trp Ser Pro Tyr Ile Val290
295 300Ala Cys Tyr Trp Arg Val Phe Val Lys Ala Cys
Ala Val Pro His Arg305 310 315
320Tyr Leu Ala Thr Ala Val Trp Met Ser Phe Ala Gln Ala Ala Val
Asn325 330 335Pro Ile Val Cys Phe Leu Leu
Asn Lys Asp Leu Lys Lys Cys Leu Arg340 345
350Thr His Ala Pro Cys Trp Gly Thr Gly Gly Ala Pro Ala Pro Arg Glu355
360 365Pro Tyr Cys Val Met370601122DNAHomo
sapiens 60atggccaaca ctaccggaga gcctgaggag gtgagcggcg ctctgtcccc
accgtccgca 60tcagcttatg tgaagctggt actgctggga ctgattatgt gcgtgagcct
ggcgggtaac 120gccatcttgt ccctgctggt gctcaaggag cgtgccctgc acaaggctcc
ttactacttc 180ctgctggacc tgtgcctggc cgatggcata cgctctgccg tctgcttccc
ctttgtgctg 240gcttctgtgc gccacggctc ttcatggacc ttcagtgcac tcagctgcaa
gattgtggcc 300tttatggccg tgctcttttg cttccatgcg gccttcatgc tgttctgcat
cagcgtcacc 360cgctacatgg ccatcgccca ccaccgcttc tacgccaagc gcatgacact
ctggacatgc 420gcggctgtca tctgcatggc ctggaccctg tctgtggcca tggccttccc
acctgtcttt 480gacgtgggca cctacaagtt tattcgggag gaggaccagt gcatctttga
gcatcgctac 540ttcaaggcca atgacacgct gggcttcatg cttatgttgg ctgtgctcat
ggcagctacc 600catgctgtct acggcaagct gctcctcttc gagtatcgtc accgcaagat
gaagccagtg 660cagatggtgc cagccatcag ccagaactgg acattccatg gtcccggggc
caccggccag 720gctgctgcca actggatcgc cggctttggc cgtgggccca tgccaccaac
cctgctgggt 780atccggcaga atgggcatgc agccagccgg cggctactgg gcatggacga
ggtcaagggt 840gaaaagcagc tgggccgcat gttctacgcg atcacactgc tctttctgct
cctctggtca 900ccctacatcg tggcctgcta ctggcgagtg tttgtgaaag cctgtgctgt
gccccaccgc 960tacctggcca ctgctgtttg gatgagcttc gcccaggctg ccgtcaaccc
aattgtctgc 1020ttcctgctca acaaggacct caagaagtgc ctgaggactc acgccccctg
ctggggcaca 1080ggaggtgccc cggctcccag agaaccctac tgtgtcatgt ga
112261387PRTHomo sapiens 61Met Tyr Pro Tyr Asp Val Pro Asp Tyr
Ala Ala Ala Ala Ala Met Ala1 5 10
15Asn Thr Thr Gly Glu Pro Glu Glu Val Ser Gly Ala Leu Ser Pro
Pro20 25 30Ser Ala Ser Ala Tyr Val Lys
Leu Val Leu Leu Gly Leu Ile Met Cys35 40
45Val Ser Leu Ala Gly Asn Ala Ile Leu Ser Leu Leu Val Leu Lys Glu50
55 60Arg Ala Leu His Lys Ala Pro Tyr Tyr Phe
Leu Leu Asp Leu Cys Leu65 70 75
80Ala Asp Gly Ile Arg Ser Ala Val Cys Phe Pro Phe Val Leu Ala
Ser85 90 95Val Arg His Gly Ser Ser Trp
Thr Phe Ser Ala Leu Ser Cys Lys Ile100 105
110Val Ala Phe Met Ala Val Leu Phe Cys Phe His Ala Ala Phe Met Leu115
120 125Phe Cys Ile Ser Val Thr Arg Tyr Met
Ala Ile Ala His His Arg Phe130 135 140Tyr
Ala Lys Arg Met Thr Leu Trp Thr Cys Ala Ala Val Ile Cys Met145
150 155 160Ala Trp Thr Leu Ser Val
Ala Met Ala Phe Pro Pro Val Phe Asp Val165 170
175Gly Thr Tyr Lys Phe Ile Arg Glu Glu Asp Gln Cys Ile Phe Glu
His180 185 190Arg Tyr Phe Lys Ala Asn Asp
Thr Leu Gly Phe Met Leu Met Leu Ala195 200
205Val Leu Met Ala Ala Thr His Ala Val Tyr Gly Lys Leu Leu Leu Phe210
215 220Glu Tyr Arg His Arg Lys Met Lys Pro
Val Gln Met Val Pro Ala Ile225 230 235
240Ser Gln Asn Trp Thr Phe His Gly Pro Gly Ala Thr Gly Gln
Ala Ala245 250 255Ala Asn Trp Ile Ala Gly
Phe Gly Arg Gly Pro Met Pro Pro Thr Leu260 265
270Leu Gly Ile Arg Gln Asn Gly His Ala Ala Ser Arg Arg Leu Leu
Gly275 280 285Met Asp Glu Val Lys Gly Glu
Lys Gln Leu Gly Arg Met Phe Tyr Ala290 295
300Ile Thr Leu Leu Phe Leu Leu Leu Trp Ser Pro Tyr Ile Val Ala Cys305
310 315 320Tyr Trp Arg Val
Phe Val Lys Ala Cys Ala Val Pro His Arg Tyr Leu325 330
335Ala Thr Ala Val Trp Met Ser Phe Ala Gln Ala Ala Val Asn
Pro Ile340 345 350Val Cys Phe Leu Leu Asn
Lys Asp Leu Lys Lys Cys Leu Arg Thr His355 360
365Ala Pro Cys Trp Gly Thr Gly Gly Ala Pro Ala Pro Arg Glu Pro
Tyr370 375 380Cys Val Met385621164DNAHomo
sapiens 62atgtacccat acgacgtacc tgattacgca gcagcagcag caatggccaa
cactaccgga 60gagcctgagg aggtgagcgg cgctctgtcc ccaccgtccg catcagctta
tgtgaagctg 120gtactgctgg gactgattat gtgcgtgagc ctggcgggta acgccatctt
gtccctgctg 180gtgctcaagg agcgtgccct gcacaaggct ccttactact tcctgctgga
cctgtgcctg 240gccgatggca tacgctctgc cgtctgcttc ccctttgtgc tggcttctgt
gcgccacggc 300tcttcatgga ccttcagtgc actcagctgc aagattgtgg cctttatggc
cgtgctcttt 360tgcttccatg cggccttcat gctgttctgc atcagcgtca cccgctacat
ggccatcgcc 420caccaccgct tctacgccaa gcgcatgaca ctctggacat gcgcggctgt
catctgcatg 480gcctggaccc tgtctgtggc catggccttc ccacctgtct ttgacgtggg
cacctacaag 540tttattcggg aggaggacca gtgcatcttt gagcatcgct acttcaaggc
caatgacacg 600ctgggcttca tgcttatgtt ggctgtgctc atggcagcta cccatgctgt
ctacggcaag 660ctgctcctct tcgagtatcg tcaccgcaag atgaagccag tgcagatggt
gccagccatc 720agccagaact ggacattcca tggtcccggg gccaccggcc aggctgctgc
caactggatc 780gccggctttg gccgtgggcc catgccacca accctgctgg gtatccggca
gaatgggcat 840gcagccagcc ggcggctact gggcatggac gaggtcaagg gtgaaaagca
gctgggccgc 900atgttctacg cgatcacact gctctttctg ctcctctggt caccctacat
cgtggcctgc 960tactggcgag tgtttgtgaa agcctgtgct gtgccccacc gctacctggc
cactgctgtt 1020tggatgagct tcgcccaggc tgccgtcaac ccaattgtct gcttcctgct
caacaaggac 1080ctcaagaagt gcctgaggac tcacgccccc tgctggggca caggaggtgc
cccggctccc 1140agagaaccct actgtgtcat gtga
116463388PRTHomo sapiens 63Met Ala Asn Thr Thr Gly Glu Pro Glu
Glu Val Ser Gly Ala Leu Ser1 5 10
15Pro Pro Ser Ala Ser Ala Tyr Val Lys Leu Val Leu Leu Gly Leu
Ile20 25 30Met Cys Val Ser Leu Ala Gly
Asn Ala Ile Leu Ser Leu Leu Val Leu35 40
45Lys Glu Arg Ala Leu His Lys Ala Pro Tyr Tyr Phe Leu Leu Asp Leu50
55 60Cys Leu Ala Asp Gly Ile Arg Ser Ala Val
Cys Phe Pro Phe Val Leu65 70 75
80Ala Ser Val Arg His Gly Ser Ser Trp Thr Phe Ser Ala Leu Ser
Cys85 90 95Lys Ile Val Ala Phe Met Ala
Val Leu Phe Cys Phe His Ala Ala Phe100 105
110Met Leu Phe Cys Ile Ser Val Thr Arg Tyr Met Ala Ile Ala His His115
120 125Arg Phe Tyr Ala Lys Arg Met Thr Leu
Trp Thr Cys Ala Ala Val Ile130 135 140Cys
Met Ala Trp Thr Leu Ser Val Ala Met Ala Phe Pro Pro Val Phe145
150 155 160Asp Val Gly Thr Tyr Lys
Phe Ile Arg Glu Glu Asp Gln Cys Ile Phe165 170
175Glu His Arg Tyr Phe Lys Ala Asn Asp Thr Leu Gly Phe Met Leu
Met180 185 190Leu Ala Val Leu Met Ala Ala
Thr His Ala Val Tyr Gly Lys Leu Leu195 200
205Leu Phe Glu Tyr Arg His Arg Lys Met Lys Pro Val Gln Met Val Pro210
215 220Ala Ile Ser Gln Asn Trp Thr Phe His
Gly Pro Gly Ala Thr Gly Gln225 230 235
240Ala Ala Ala Asn Trp Ile Ala Gly Phe Gly Arg Gly Pro Met
Pro Pro245 250 255Thr Leu Leu Gly Ile Arg
Gln Asn Gly His Ala Ala Ser Arg Arg Leu260 265
270Leu Gly Met Asp Glu Val Lys Gly Glu Lys Gln Leu Gly Arg Met
Phe275 280 285Tyr Ala Ile Thr Leu Leu Phe
Leu Leu Leu Trp Ser Pro Tyr Ile Val290 295
300Ala Cys Tyr Trp Arg Val Phe Val Lys Ala Cys Ala Val Pro His Arg305
310 315 320Tyr Leu Ala Thr
Ala Val Trp Met Ser Phe Ala Gln Ala Ala Val Asn325 330
335Pro Ile Val Cys Phe Leu Leu Asn Lys Asp Leu Lys Lys Cys
Leu Arg340 345 350Thr His Ala Pro Cys Ala
Ala Ala Arg Gly Arg Thr Pro Pro Ser Leu355 360
365Gly Pro Gln Asp Glu Ser Cys Thr Thr Ala Ser Ser Ser Leu Ala
Lys370 375 380Asp Thr Ser
Ser385641167DNAHomo sapiens 64atggccaaca ctaccggaga gcctgaggag gtgagcggcg
ctctgtcccc accgtccgca 60tcagcttatg tgaagctggt actgctggga ctgattatgt
gcgtgagcct ggcgggtaac 120gccatcttgt ccctgctggt gctcaaggag cgtgccctgc
acaaggctcc ttactacttc 180ctgctggacc tgtgcctggc cgatggcata cgctctgccg
tctgcttccc ctttgtgctg 240gcttctgtgc gccacggctc ttcatggacc ttcagtgcac
tcagctgcaa gattgtggcc 300tttatggccg tgctcttttg cttccatgcg gccttcatgc
tgttctgcat cagcgtcacc 360cgctacatgg ccatcgccca ccaccgcttc tacgccaagc
gcatgacact ctggacatgc 420gcggctgtca tctgcatggc ctggaccctg tctgtggcca
tggccttccc acctgtcttt 480gacgtgggca cctacaagtt tattcgggag gaggaccagt
gcatctttga gcatcgctac 540ttcaaggcca atgacacgct gggcttcatg cttatgttgg
ctgtgctcat ggcagctacc 600catgctgtct acggcaagct gctcctcttc gagtatcgtc
accgcaagat gaagccagtg 660cagatggtgc cagccatcag ccagaactgg acattccatg
gtcccggggc caccggccag 720gctgctgcca actggatcgc cggctttggc cgtgggccca
tgccaccaac cctgctgggt 780atccggcaga atgggcatgc agccagccgg cggctactgg
gcatggacga ggtcaagggt 840gaaaagcagc tgggccgcat gttctacgcg atcacactgc
tctttctgct cctctggtca 900ccctacatcg tggcctgcta ctggcgagtg tttgtgaaag
cctgtgctgt gccccaccgc 960tacctggcca ctgctgtttg gatgagcttc gcccaggctg
ccgtcaaccc aattgtctgc 1020ttcctgctca acaaggacct caagaagtgc ctgaggactc
acgccccctg cgcggccgca 1080cggggacgca ccccacccag cctgggtccc caagatgagt
cctgcaccac cgccagctcc 1140tccctggcca aggacacttc atcgtga
116765402PRTHomo sapiens 65Met Tyr Pro Tyr Asp Val
Pro Asp Tyr Ala Ala Ala Ala Ala Met Ala1 5
10 15Asn Thr Thr Gly Glu Pro Glu Glu Val Ser Gly Ala Leu
Ser Pro Pro20 25 30Ser Ala Ser Ala Tyr
Val Lys Leu Val Leu Leu Gly Leu Ile Met Cys35 40
45Val Ser Leu Ala Gly Asn Ala Ile Leu Ser Leu Leu Val Leu Lys
Glu50 55 60Arg Ala Leu His Lys Ala Pro
Tyr Tyr Phe Leu Leu Asp Leu Cys Leu65 70
75 80Ala Asp Gly Ile Arg Ser Ala Val Cys Phe Pro Phe
Val Leu Ala Ser85 90 95Val Arg His Gly
Ser Ser Trp Thr Phe Ser Ala Leu Ser Cys Lys Ile100 105
110Val Ala Phe Met Ala Val Leu Phe Cys Phe His Ala Ala Phe
Met Leu115 120 125Phe Cys Ile Ser Val Thr
Arg Tyr Met Ala Ile Ala His His Arg Phe130 135
140Tyr Ala Lys Arg Met Thr Leu Trp Thr Cys Ala Ala Val Ile Cys
Met145 150 155 160Ala Trp
Thr Leu Ser Val Ala Met Ala Phe Pro Pro Val Phe Asp Val165
170 175Gly Thr Tyr Lys Phe Ile Arg Glu Glu Asp Gln Cys
Ile Phe Glu His180 185 190Arg Tyr Phe Lys
Ala Asn Asp Thr Leu Gly Phe Met Leu Met Leu Ala195 200
205Val Leu Met Ala Ala Thr His Ala Val Tyr Gly Lys Leu Leu
Leu Phe210 215 220Glu Tyr Arg His Arg Lys
Met Lys Pro Val Gln Met Val Pro Ala Ile225 230
235 240Ser Gln Asn Trp Thr Phe His Gly Pro Gly Ala
Thr Gly Gln Ala Ala245 250 255Ala Asn Trp
Ile Ala Gly Phe Gly Arg Gly Pro Met Pro Pro Thr Leu260
265 270Leu Gly Ile Arg Gln Asn Gly His Ala Ala Ser Arg
Arg Leu Leu Gly275 280 285Met Asp Glu Val
Lys Gly Glu Lys Gln Leu Gly Arg Met Phe Tyr Ala290 295
300Ile Thr Leu Leu Phe Leu Leu Leu Trp Ser Pro Tyr Ile Val
Ala Cys305 310 315 320Tyr
Trp Arg Val Phe Val Lys Ala Cys Ala Val Pro His Arg Tyr Leu325
330 335Ala Thr Ala Val Trp Met Ser Phe Ala Gln Ala
Ala Val Asn Pro Ile340 345 350Val Cys Phe
Leu Leu Asn Lys Asp Leu Lys Lys Cys Leu Arg Thr His355
360 365Ala Pro Cys Ala Ala Ala Arg Gly Arg Thr Pro Pro
Ser Leu Gly Pro370 375 380Gln Asp Glu Ser
Cys Thr Thr Ala Ser Ser Ser Leu Ala Lys Asp Thr385 390
395 400Ser Ser661209DNAHomo sapiens
66atgtacccat acgacgtacc tgattacgca gcagcagcag caatggccaa cactaccgga
60gagcctgagg aggtgagcgg cgctctgtcc ccaccgtccg catcagctta tgtgaagctg
120gtactgctgg gactgattat gtgcgtgagc ctggcgggta acgccatctt gtccctgctg
180gtgctcaagg agcgtgccct gcacaaggct ccttactact tcctgctgga cctgtgcctg
240gccgatggca tacgctctgc cgtctgcttc ccctttgtgc tggcttctgt gcgccacggc
300tcttcatgga ccttcagtgc actcagctgc aagattgtgg cctttatggc cgtgctcttt
360tgcttccatg cggccttcat gctgttctgc atcagcgtca cccgctacat ggccatcgcc
420caccaccgct tctacgccaa gcgcatgaca ctctggacat gcgcggctgt catctgcatg
480gcctggaccc tgtctgtggc catggccttc ccacctgtct ttgacgtggg cacctacaag
540tttattcggg aggaggacca gtgcatcttt gagcatcgct acttcaaggc caatgacacg
600ctgggcttca tgcttatgtt ggctgtgctc atggcagcta cccatgctgt ctacggcaag
660ctgctcctct tcgagtatcg tcaccgcaag atgaagccag tgcagatggt gccagccatc
720agccagaact ggacattcca tggtcccggg gccaccggcc aggctgctgc caactggatc
780gccggctttg gccgtgggcc catgccacca accctgctgg gtatccggca gaatgggcat
840gcagccagcc ggcggctact gggcatggac gaggtcaagg gtgaaaagca gctgggccgc
900atgttctacg cgatcacact gctctttctg ctcctctggt caccctacat cgtggcctgc
960tactggcgag tgtttgtgaa agcctgtgct gtgccccacc gctacctggc cactgctgtt
1020tggatgagct tcgcccaggc tgccgtcaac ccaattgtct gcttcctgct caacaaggac
1080ctcaagaagt gcctgaggac tcacgccccc tgcgcggccg cacggggacg caccccaccc
1140agcctgggtc cccaagatga gtcctgcacc accgccagct cctccctggc caaggacact
1200tcatcgtga
120967370PRTHomo sapiens 67Met Ala Asn Tyr Ser His Ala Ala Asp Asn Ile
Leu Gln Asn Leu Ser1 5 10
15Pro Leu Thr Ala Phe Leu Lys Leu Thr Ser Leu Gly Phe Ile Ile Gly20
25 30Val Ser Val Val Gly Asn Leu Leu Ile Ser
Ile Leu Leu Val Lys Asp35 40 45Lys Thr
Leu His Arg Ala Pro Tyr Tyr Phe Leu Leu Asp Leu Cys Cys50
55 60Ser Asp Ile Leu Arg Ser Ala Ile Cys Phe Pro Phe
Val Phe Asn Ser65 70 75
80Val Lys Asn Gly Ser Thr Trp Thr Tyr Gly Thr Leu Thr Cys Lys Val85
90 95Ile Ala Phe Leu Gly Val Leu Ser Cys Phe
His Thr Ala Phe Met Leu100 105 110Phe Cys
Ile Ser Val Thr Arg Tyr Leu Ala Ile Ala His His Arg Phe115
120 125Tyr Thr Lys Arg Leu Thr Phe Trp Thr Cys Leu Ala
Val Ile Cys Met130 135 140Val Trp Thr Leu
Ser Val Ala Met Ala Phe Pro Pro Val Leu Asp Val145 150
155 160Gly Thr Tyr Ser Phe Ile Arg Glu Glu
Asp Gln Cys Thr Phe Gln His165 170 175Arg
Ser Phe Arg Ala Asn Asp Ser Leu Gly Phe Met Leu Leu Leu Ala180
185 190Leu Ile Leu Leu Ala Thr Gln Leu Val Tyr Leu
Lys Leu Ile Phe Phe195 200 205Val His Asp
Arg Arg Lys Met Lys Pro Val Gln Phe Val Ala Ala Val210
215 220Ser Gln Asn Trp Thr Phe His Gly Pro Gly Ala Ser
Gly Gln Ala Ala225 230 235
240Ala Asn Trp Leu Ala Gly Phe Gly Arg Gly Pro Thr Pro Pro Thr Leu245
250 255Leu Gly Ile Arg Gln Asn Ala Asn Thr
Thr Gly Arg Arg Arg Leu Leu260 265 270Val
Leu Asp Glu Phe Lys Met Glu Lys Arg Ile Ser Arg Met Phe Tyr275
280 285Ile Met Thr Phe Leu Phe Leu Thr Leu Trp Gly
Pro Tyr Leu Val Ala290 295 300Cys Tyr Trp
Arg Val Phe Ala Arg Gly Pro Val Val Pro Gly Gly Phe305
310 315 320Leu Thr Ala Ala Val Trp Met
Ser Phe Ala Gln Ala Gly Ile Asn Pro325 330
335Phe Val Cys Ile Phe Ser Asn Arg Glu Leu Arg Arg Cys Phe Ser Thr340
345 350Thr Leu Leu Tyr Cys Arg Lys Ser Arg
Leu Pro Arg Glu Pro Tyr Cys355 360 365Val
Ile370681113DNAHomo sapiens 68atggcgaact atagccatgc agctgacaac attttgcaaa
atctctcgcc tctaacagcc 60tttctgaaac tgacttcctt gggtttcata ataggagtca
gcgtggtggg caacctcctg 120atctccattt tgctagtgaa agataagacc ttgcatagag
caccttacta cttcctgttg 180gatctttgct gttcagatat cctcagatct gcaatttgtt
tcccatttgt gttcaactct 240gtcaaaaatg gctctacctg gacttatggg actctgactt
gcaaagtgat tgcctttctg 300ggggttttgt cctgtttcca cactgctttc atgctcttct
gcatcagtgt caccagatac 360ttagctatcg cccatcaccg cttctataca aagaggctga
ccttttggac gtgtctggct 420gtgatctgta tggtgtggac tctgtctgtg gccatggcat
ttcccccggt tttagacgtg 480ggcacttact cattcattag ggaggaagat caatgcacct
tccaacaccg ctccttcagg 540gctaatgatt ccttaggatt tatgctgctt cttgctctca
tcctcctagc cacacagctt 600gtctacctca agctgatatt tttcgtccac gatcgaagaa
aaatgaagcc agtccagttt 660gtagcagcag tcagccagaa ctggactttt catggtcctg
gagccagtgg ccaggcagct 720gccaattggc tagcaggatt tggaaggggt cccacaccac
ccaccttgct gggcatcagg 780caaaatgcaa acaccacagg cagaagaagg ctattggtct
tagacgagtt caaaatggag 840aaaagaatca gcagaatgtt ctatataatg acttttctgt
ttctaacctt gtggggcccc 900tacctggtgg cctgttattg gagagttttt gcaagagggc
ctgtagtacc agggggattt 960ctaacagctg ctgtctggat gagttttgcc caagcaggaa
tcaatccttt tgtctgcatt 1020ttctcaaaca gggagctgag gcgctgtttc agcacaaccc
ttctttactg cagaaaatcc 1080aggttaccaa gggaacctta ctgtgttata tga
111369384PRTHomo sapiens 69Met Tyr Pro Tyr Asp Val
Pro Asp Tyr Ala Ala Ala Ala Ala Met Ala1 5
10 15Asn Tyr Ser His Ala Ala Asp Asn Ile Leu Gln Asn Leu
Ser Pro Leu20 25 30Thr Ala Phe Leu Lys
Leu Thr Ser Leu Gly Phe Ile Ile Gly Val Ser35 40
45Val Val Gly Asn Leu Leu Ile Ser Ile Leu Leu Val Lys Asp Lys
Thr50 55 60Leu His Arg Ala Pro Tyr Tyr
Phe Leu Leu Asp Leu Cys Cys Ser Asp65 70
75 80Ile Leu Arg Ser Ala Ile Cys Phe Pro Phe Val Phe
Asn Ser Val Lys85 90 95Asn Gly Ser Thr
Trp Thr Tyr Gly Thr Leu Thr Cys Lys Val Ile Ala100 105
110Phe Leu Gly Val Leu Ser Cys Phe His Thr Ala Phe Met Leu
Phe Cys115 120 125Ile Ser Val Thr Arg Tyr
Leu Ala Ile Ala His His Arg Phe Tyr Thr130 135
140Lys Arg Leu Thr Phe Trp Thr Cys Leu Ala Val Ile Cys Met Val
Trp145 150 155 160Thr Leu
Ser Val Ala Met Ala Phe Pro Pro Val Leu Asp Val Gly Thr165
170 175Tyr Ser Phe Ile Arg Glu Glu Asp Gln Cys Thr Phe
Gln His Arg Ser180 185 190Phe Arg Ala Asn
Asp Ser Leu Gly Phe Met Leu Leu Leu Ala Leu Ile195 200
205Leu Leu Ala Thr Gln Leu Val Tyr Leu Lys Leu Ile Phe Phe
Val His210 215 220Asp Arg Arg Lys Met Lys
Pro Val Gln Phe Val Ala Ala Val Ser Gln225 230
235 240Asn Trp Thr Phe His Gly Pro Gly Ala Ser Gly
Gln Ala Ala Ala Asn245 250 255Trp Leu Ala
Gly Phe Gly Arg Gly Pro Thr Pro Pro Thr Leu Leu Gly260
265 270Ile Arg Gln Asn Ala Asn Thr Thr Gly Arg Arg Arg
Leu Leu Val Leu275 280 285Asp Glu Phe Lys
Met Glu Lys Arg Ile Ser Arg Met Phe Tyr Ile Met290 295
300Thr Phe Leu Phe Leu Thr Leu Trp Gly Pro Tyr Leu Val Ala
Cys Tyr305 310 315 320Trp
Arg Val Phe Ala Arg Gly Pro Val Val Pro Gly Gly Phe Leu Thr325
330 335Ala Ala Val Trp Met Ser Phe Ala Gln Ala Gly
Ile Asn Pro Phe Val340 345 350Cys Ile Phe
Ser Asn Arg Glu Leu Arg Arg Cys Phe Ser Thr Thr Leu355
360 365Leu Tyr Cys Arg Lys Ser Arg Leu Pro Arg Glu Pro
Tyr Cys Val Ile370 375 380701155DNAHomo
sapiens 70atgtacccat acgacgtacc tgattacgca gcagcagcag caatggcgaa
ctatagccat 60gcagctgaca acattttgca aaatctctcg cctctaacag cctttctgaa
actgacttcc 120ttgggtttca taataggagt cagcgtggtg ggcaacctcc tgatctccat
tttgctagtg 180aaagataaga ccttgcatag agcaccttac tacttcctgt tggatctttg
ctgttcagat 240atcctcagat ctgcaatttg tttcccattt gtgttcaact ctgtcaaaaa
tggctctacc 300tggacttatg ggactctgac ttgcaaagtg attgcctttc tgggggtttt
gtcctgtttc 360cacactgctt tcatgctctt ctgcatcagt gtcaccagat acttagctat
cgcccatcac 420cgcttctata caaagaggct gaccttttgg acgtgtctgg ctgtgatctg
tatggtgtgg 480actctgtctg tggccatggc atttcccccg gttttagacg tgggcactta
ctcattcatt 540agggaggaag atcaatgcac cttccaacac cgctccttca gggctaatga
ttccttagga 600tttatgctgc ttcttgctct catcctccta gccacacagc ttgtctacct
caagctgata 660tttttcgtcc acgatcgaag aaaaatgaag ccagtccagt ttgtagcagc
agtcagccag 720aactggactt ttcatggtcc tggagccagt ggccaggcag ctgccaattg
gctagcagga 780tttggaaggg gtcccacacc acccaccttg ctgggcatca ggcaaaatgc
aaacaccaca 840ggcagaagaa ggctattggt cttagacgag ttcaaaatgg agaaaagaat
cagcagaatg 900ttctatataa tgacttttct gtttctaacc ttgtggggcc cctacctggt
ggcctgttat 960tggagagttt ttgcaagagg gcctgtagta ccagggggat ttctaacagc
tgctgtctgg 1020atgagttttg cccaagcagg aatcaatcct tttgtctgca ttttctcaaa
cagggagctg 1080aggcgctgtt tcagcacaac ccttctttac tgcagaaaat ccaggttacc
aagggaacct 1140tactgtgtta tatga
115571388PRTHomo sapiens 71Met Ala Asn Tyr Ser His Ala Ala Asp
Asn Ile Leu Gln Asn Leu Ser1 5 10
15Pro Leu Thr Ala Phe Leu Lys Leu Thr Ser Leu Gly Phe Ile Ile
Gly20 25 30Val Ser Val Val Gly Asn Leu
Leu Ile Ser Ile Leu Leu Val Lys Asp35 40
45Lys Thr Leu His Arg Ala Pro Tyr Tyr Phe Leu Leu Asp Leu Cys Cys50
55 60Ser Asp Ile Leu Arg Ser Ala Ile Cys Phe
Pro Phe Val Phe Asn Ser65 70 75
80Val Lys Asn Gly Ser Thr Trp Thr Tyr Gly Thr Leu Thr Cys Lys
Val85 90 95Ile Ala Phe Leu Gly Val Leu
Ser Cys Phe His Thr Ala Phe Met Leu100 105
110Phe Cys Ile Ser Val Thr Arg Tyr Leu Ala Ile Ala His His Arg Phe115
120 125Tyr Thr Lys Arg Leu Thr Phe Trp Thr
Cys Leu Ala Val Ile Cys Met130 135 140Val
Trp Thr Leu Ser Val Ala Met Ala Phe Pro Pro Val Leu Asp Val145
150 155 160Gly Thr Tyr Ser Phe Ile
Arg Glu Glu Asp Gln Cys Thr Phe Gln His165 170
175Arg Ser Phe Arg Ala Asn Asp Ser Leu Gly Phe Met Leu Leu Leu
Ala180 185 190Leu Ile Leu Leu Ala Thr Gln
Leu Val Tyr Leu Lys Leu Ile Phe Phe195 200
205Val His Asp Arg Arg Lys Met Lys Pro Val Gln Phe Val Ala Ala Val210
215 220Ser Gln Asn Trp Thr Phe His Gly Pro
Gly Ala Ser Gly Gln Ala Ala225 230 235
240Ala Asn Trp Leu Ala Gly Phe Gly Arg Gly Pro Thr Pro Pro
Thr Leu245 250 255Leu Gly Ile Arg Gln Asn
Ala Asn Thr Thr Gly Arg Arg Arg Leu Leu260 265
270Val Leu Asp Glu Phe Lys Met Glu Lys Arg Ile Ser Arg Met Phe
Tyr275 280 285Ile Met Thr Phe Leu Phe Leu
Thr Leu Trp Gly Pro Tyr Leu Val Ala290 295
300Cys Tyr Trp Arg Val Phe Ala Arg Gly Pro Val Val Pro Gly Gly Phe305
310 315 320Leu Thr Ala Ala
Val Trp Met Ser Phe Ala Gln Ala Gly Ile Asn Pro325 330
335Phe Val Cys Ile Phe Ser Asn Arg Glu Leu Arg Arg Cys Phe
Ser Thr340 345 350Thr Leu Leu Tyr Cys Ala
Ala Ala Arg Gly Arg Thr Pro Pro Ser Leu355 360
365Gly Pro Gln Asp Glu Ser Cys Thr Thr Ala Ser Ser Ser Leu Ala
Lys370 375 380Asp Thr Ser
Ser385721167DNAHomo sapiens 72atggcgaact atagccatgc agctgacaac attttgcaaa
atctctcgcc tctaacagcc 60tttctgaaac tgacttcctt gggtttcata ataggagtca
gcgtggtggg caacctcctg 120atctccattt tgctagtgaa agataagacc ttgcatagag
caccttacta cttcctgttg 180gatctttgct gttcagatat cctcagatct gcaatttgtt
tcccatttgt gttcaactct 240gtcaaaaatg gctctacctg gacttatggg actctgactt
gcaaagtgat tgcctttctg 300ggggttttgt cctgtttcca cactgctttc atgctcttct
gcatcagtgt caccagatac 360ttagctatcg cccatcaccg cttctataca aagaggctga
ccttttggac gtgtctggct 420gtgatctgta tggtgtggac tctgtctgtg gccatggcat
ttcccccggt tttagacgtg 480ggcacttact cattcattag ggaggaagat caatgcacct
tccaacaccg ctccttcagg 540gctaatgatt ccttaggatt tatgctgctt cttgctctca
tcctcctagc cacacagctt 600gtctacctca agctgatatt tttcgtccac gatcgaagaa
aaatgaagcc agtccagttt 660gtagcagcag tcagccagaa ctggactttt catggtcctg
gagccagtgg ccaggcagct 720gccaattggc tagcaggatt tggaaggggt cccacaccac
ccaccttgct gggcatcagg 780caaaatgcaa acaccacagg cagaagaagg ctattggtct
tagacgagtt caaaatggag 840aaaagaatca gcagaatgtt ctatataatg acttttctgt
ttctaacctt gtggggcccc 900tacctggtgg cctgttattg gagagttttt gcaagagggc
ctgtagtacc agggggattt 960ctaacagctg ctgtctggat gagttttgcc caagcaggaa
tcaatccttt tgtctgcatt 1020ttctcaaaca gggagctgag gcgctgtttc agcacaaccc
ttctttactg cgcggccgca 1080cggggacgca ccccacccag cctgggtccc caagatgagt
cctgcaccac cgccagctcc 1140tccctggcca aggacacttc atcgtga
116773402PRTHomo sapiens 73Met Tyr Pro Tyr Asp Val
Pro Asp Tyr Ala Ala Ala Ala Ala Met Ala1 5
10 15Asn Tyr Ser His Ala Ala Asp Asn Ile Leu Gln Asn Leu
Ser Pro Leu20 25 30Thr Ala Phe Leu Lys
Leu Thr Ser Leu Gly Phe Ile Ile Gly Val Ser35 40
45Val Val Gly Asn Leu Leu Ile Ser Ile Leu Leu Val Lys Asp Lys
Thr50 55 60Leu His Arg Ala Pro Tyr Tyr
Phe Leu Leu Asp Leu Cys Cys Ser Asp65 70
75 80Ile Leu Arg Ser Ala Ile Cys Phe Pro Phe Val Phe
Asn Ser Val Lys85 90 95Asn Gly Ser Thr
Trp Thr Tyr Gly Thr Leu Thr Cys Lys Val Ile Ala100 105
110Phe Leu Gly Val Leu Ser Cys Phe His Thr Ala Phe Met Leu
Phe Cys115 120 125Ile Ser Val Thr Arg Tyr
Leu Ala Ile Ala His His Arg Phe Tyr Thr130 135
140Lys Arg Leu Thr Phe Trp Thr Cys Leu Ala Val Ile Cys Met Val
Trp145 150 155 160Thr Leu
Ser Val Ala Met Ala Phe Pro Pro Val Leu Asp Val Gly Thr165
170 175Tyr Ser Phe Ile Arg Glu Glu Asp Gln Cys Thr Phe
Gln His Arg Ser180 185 190Phe Arg Ala Asn
Asp Ser Leu Gly Phe Met Leu Leu Leu Ala Leu Ile195 200
205Leu Leu Ala Thr Gln Leu Val Tyr Leu Lys Leu Ile Phe Phe
Val His210 215 220Asp Arg Arg Lys Met Lys
Pro Val Gln Phe Val Ala Ala Val Ser Gln225 230
235 240Asn Trp Thr Phe His Gly Pro Gly Ala Ser Gly
Gln Ala Ala Ala Asn245 250 255Trp Leu Ala
Gly Phe Gly Arg Gly Pro Thr Pro Pro Thr Leu Leu Gly260
265 270Ile Arg Gln Asn Ala Asn Thr Thr Gly Arg Arg Arg
Leu Leu Val Leu275 280 285Asp Glu Phe Lys
Met Glu Lys Arg Ile Ser Arg Met Phe Tyr Ile Met290 295
300Thr Phe Leu Phe Leu Thr Leu Trp Gly Pro Tyr Leu Val Ala
Cys Tyr305 310 315 320Trp
Arg Val Phe Ala Arg Gly Pro Val Val Pro Gly Gly Phe Leu Thr325
330 335Ala Ala Val Trp Met Ser Phe Ala Gln Ala Gly
Ile Asn Pro Phe Val340 345 350Cys Ile Phe
Ser Asn Arg Glu Leu Arg Arg Cys Phe Ser Thr Thr Leu355
360 365Leu Tyr Cys Ala Ala Ala Arg Gly Arg Thr Pro Pro
Ser Leu Gly Pro370 375 380Gln Asp Glu Ser
Cys Thr Thr Ala Ser Ser Ser Leu Ala Lys Asp Thr385 390
395 400Ser Ser741209DNAHomo sapiens
74atgtacccat acgacgtacc tgattacgca gcagcagcag caatggcgaa ctatagccat
60gcagctgaca acattttgca aaatctctcg cctctaacag cctttctgaa actgacttcc
120ttgggtttca taataggagt cagcgtggtg ggcaacctcc tgatctccat tttgctagtg
180aaagataaga ccttgcatag agcaccttac tacttcctgt tggatctttg ctgttcagat
240atcctcagat ctgcaatttg tttcccattt gtgttcaact ctgtcaaaaa tggctctacc
300tggacttatg ggactctgac ttgcaaagtg attgcctttc tgggggtttt gtcctgtttc
360cacactgctt tcatgctctt ctgcatcagt gtcaccagat acttagctat cgcccatcac
420cgcttctata caaagaggct gaccttttgg acgtgtctgg ctgtgatctg tatggtgtgg
480actctgtctg tggccatggc atttcccccg gttttagacg tgggcactta ctcattcatt
540agggaggaag atcaatgcac cttccaacac cgctccttca gggctaatga ttccttagga
600tttatgctgc ttcttgctct catcctccta gccacacagc ttgtctacct caagctgata
660tttttcgtcc acgatcgaag aaaaatgaag ccagtccagt ttgtagcagc agtcagccag
720aactggactt ttcatggtcc tggagccagt ggccaggcag ctgccaattg gctagcagga
780tttggaaggg gtcccacacc acccaccttg ctgggcatca ggcaaaatgc aaacaccaca
840ggcagaagaa ggctattggt cttagacgag ttcaaaatgg agaaaagaat cagcagaatg
900ttctatataa tgacttttct gtttctaacc ttgtggggcc cctacctggt ggcctgttat
960tggagagttt ttgcaagagg gcctgtagta ccagggggat ttctaacagc tgctgtctgg
1020atgagttttg cccaagcagg aatcaatcct tttgtctgca ttttctcaaa cagggagctg
1080aggcgctgtt tcagcacaac ccttctttac tgcgcggccg cacggggacg caccccaccc
1140agcctgggtc cccaagatga gtcctgcacc accgccagct cctccctggc caaggacact
1200tcatcgtga
120975333PRTHomo sapiensMOD_RES(305)Variable amino acid 75Met Gln Ala Ala
Gly His Pro Glu Pro Leu Asp Ser Arg Gly Ser Phe1 5
10 15Ser Leu Pro Thr Met Gly Ala Asn Val Ser Gln
Asp Asn Gly Thr Gly20 25 30His Asn Ala
Thr Phe Ser Glu Pro Leu Pro Phe Leu Tyr Val Leu Leu35 40
45Pro Ala Val Tyr Ser Gly Ile Cys Ala Val Gly Leu Thr
Gly Asn Thr50 55 60Ala Val Ile Leu Val
Ile Leu Arg Ala Pro Lys Met Lys Thr Val Thr65 70
75 80Asn Val Phe Ile Leu Asn Leu Ala Val Ala
Asp Gly Leu Phe Thr Leu85 90 95Val Leu
Pro Val Asn Ile Ala Glu His Leu Leu Gln Tyr Trp Pro Phe100
105 110Gly Glu Leu Leu Cys Lys Leu Val Leu Ala Val Asp
His Tyr Asn Ile115 120 125Phe Ser Ser Ile
Tyr Phe Leu Ala Val Met Ser Val Asp Arg Tyr Leu130 135
140Val Val Leu Ala Thr Val Arg Ser Arg His Met Pro Trp Arg
Thr Tyr145 150 155 160Arg
Gly Ala Lys Val Ala Ser Leu Cys Val Trp Leu Gly Val Thr Val165
170 175Leu Val Leu Pro Phe Phe Ser Phe Ala Gly Val
Tyr Ser Asn Glu Leu180 185 190Gln Val Pro
Ser Cys Gly Leu Ser Phe Pro Trp Pro Glu Arg Val Trp195
200 205Phe Lys Ala Ser Arg Val Tyr Thr Leu Val Leu Gly
Phe Val Leu Pro210 215 220Val Cys Thr Ile
Cys Val Leu Tyr Thr Asp Leu Leu Arg Arg Leu Arg225 230
235 240Ala Val Arg Leu Arg Ser Gly Ala Lys
Ala Leu Gly Lys Ala Arg Arg245 250 255Lys
Val Thr Val Leu Val Leu Val Val Leu Ala Val Cys Leu Leu Cys260
265 270Trp Thr Pro Phe His Leu Ala Ser Val Val Ala
Leu Thr Thr Asp Leu275 280 285Pro Gln Thr
Pro Leu Val Ile Ser Met Ser Tyr Val Ile Thr Ser Leu290
295 300Xaa Tyr Ala Asn Ser Cys Leu Asn Pro Phe Leu Tyr
Ala Phe Leu Asp305 310 315
320Asp Asn Phe Arg Lys Asn Phe Arg Ser Ile Leu Arg Cys325
330761002DNAHomo sapiens 76atgcaggccg ctgggcaccc agagcccctt gacagcaggg
gctccttctc cctccccacg 60atgggtgcca acgtctctca ggacaatggc actggccaca
atgccacctt ctccgagcca 120ctgccgttcc tctatgtgct cctgcccgcc gtgtactccg
ggatctgtgc tgtggggctg 180actggcaaca cggccgtcat ccttgtaatc ctaagggcgc
ccaagatgaa gacggtgacc 240aacgtgttca tcctgaacct ggccgtcgcc gacgggctct
tcacgctggt actgcccgtc 300aacatcgcgg agcacctgct gcagtactgg cccttcgggg
agctgctctg caagctggtg 360ctggccgtcg accactacaa catcttctcc agcatctact
tcctagccgt gatgagcgtg 420gaccgatacc tggtggtgct ggccaccgtg aggtcccgcc
acatgccctg gcgcacctac 480cggggggcga aggtcgccag cctgtgtgtc tggctgggcg
tcacggtcct ggttctgccc 540ttcttctctt tcgctggcgt ctacagcaac gagctgcagg
tcccaagctg tgggctgagc 600ttcccgtggc ccgagcgggt ctggttcaag gccagccgtg
tctacacttt ggtcctgggc 660ttcgtgctgc ccgtgtgcac catctgtgtg ctctacacag
acctcctgcg caggctgcgg 720gccgtgcggc tccgctctgg agccaaggct ctaggcaagg
ccaggcggaa ggtgaccgtc 780ctggtcctcg tcgtgctggc cgtgtgcctc ctctgctgga
cgcccttcca cctggcctct 840gtcgtggccc tgaccacgga cctgccccag accccactgg
tcatcagtat gtcctacgtc 900atcaccagcc tcasstacgc caactcgtgc ctgaacccct
tcctctacgc ctttctagat 960gacaacttcc ggaagaactt ccgcagcata ttgcggtgct
ga 100277347PRTHomo sapiensMOD_RES(319)Variable
amino acid 77Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ala Ala Ala Ala Met
Gln1 5 10 15Ala Ala Gly
His Pro Glu Pro Leu Asp Ser Arg Gly Ser Phe Ser Leu20 25
30Pro Thr Met Gly Ala Asn Val Ser Gln Asp Asn Gly Thr
Gly His Asn35 40 45Ala Thr Phe Ser Glu
Pro Leu Pro Phe Leu Tyr Val Leu Leu Pro Ala50 55
60Val Tyr Ser Gly Ile Cys Ala Val Gly Leu Thr Gly Asn Thr Ala
Val65 70 75 80Ile Leu
Val Ile Leu Arg Ala Pro Lys Met Lys Thr Val Thr Asn Val85
90 95Phe Ile Leu Asn Leu Ala Val Ala Asp Gly Leu Phe
Thr Leu Val Leu100 105 110Pro Val Asn Ile
Ala Glu His Leu Leu Gln Tyr Trp Pro Phe Gly Glu115 120
125Leu Leu Cys Lys Leu Val Leu Ala Val Asp His Tyr Asn Ile
Phe Ser130 135 140Ser Ile Tyr Phe Leu Ala
Val Met Ser Val Asp Arg Tyr Leu Val Val145 150
155 160Leu Ala Thr Val Arg Ser Arg His Met Pro Trp
Arg Thr Tyr Arg Gly165 170 175Ala Lys Val
Ala Ser Leu Cys Val Trp Leu Gly Val Thr Val Leu Val180
185 190Leu Pro Phe Phe Ser Phe Ala Gly Val Tyr Ser Asn
Glu Leu Gln Val195 200 205Pro Ser Cys Gly
Leu Ser Phe Pro Trp Pro Glu Arg Val Trp Phe Lys210 215
220Ala Ser Arg Val Tyr Thr Leu Val Leu Gly Phe Val Leu Pro
Val Cys225 230 235 240Thr
Ile Cys Val Leu Tyr Thr Asp Leu Leu Arg Arg Leu Arg Ala Val245
250 255Arg Leu Arg Ser Gly Ala Lys Ala Leu Gly Lys
Ala Arg Arg Lys Val260 265 270Thr Val Leu
Val Leu Val Val Leu Ala Val Cys Leu Leu Cys Trp Thr275
280 285Pro Phe His Leu Ala Ser Val Val Ala Leu Thr Thr
Asp Leu Pro Gln290 295 300Thr Pro Leu Val
Ile Ser Met Ser Tyr Val Ile Thr Ser Leu Xaa Tyr305 310
315 320Ala Asn Ser Cys Leu Asn Pro Phe Leu
Tyr Ala Phe Leu Asp Asp Asn325 330 335Phe
Arg Lys Asn Phe Arg Ser Ile Leu Arg Cys340
345781044DNAHomo sapiens 78atgtacccat acgacgtacc tgattacgca gcagcagcag
caatgcaggc cgctgggcac 60ccagagcccc ttgacagcag gggctccttc tccctcccca
cgatgggtgc caacgtctct 120caggacaatg gcactggcca caatgccacc ttctccgagc
cactgccgtt cctctatgtg 180ctcctgcccg ccgtgtactc cgggatctgt gctgtggggc
tgactggcaa cacggccgtc 240atccttgtaa tcctaagggc gcccaagatg aagacggtga
ccaacgtgtt catcctgaac 300ctggccgtcg ccgacgggct cttcacgctg gtactgcccg
tcaacatcgc ggagcacctg 360ctgcagtact ggcccttcgg ggagctgctc tgcaagctgg
tgctggccgt cgaccactac 420aacatcttct ccagcatcta cttcctagcc gtgatgagcg
tggaccgata cctggtggtg 480ctggccaccg tgaggtcccg ccacatgccc tggcgcacct
accggggggc gaaggtcgcc 540agcctgtgtg tctggctggg cgtcacggtc ctggttctgc
ccttcttctc tttcgctggc 600gtctacagca acgagctgca ggtcccaagc tgtgggctga
gcttcccgtg gcccgagcgg 660gtctggttca aggccagccg tgtctacact ttggtcctgg
gcttcgtgct gcccgtgtgc 720accatctgtg tgctctacac agacctcctg cgcaggctgc
gggccgtgcg gctccgctct 780ggagccaagg ctctaggcaa ggccaggcgg aaggtgaccg
tcctggtcct cgtcgtgctg 840gccgtgtgcc tcctctgctg gacgcccttc cacctggcct
ctgtcgtggc cctgaccacg 900gacctgcccc agaccccact ggtcatcagt atgtcctacg
tcatcaccag cctcasstac 960gccaactcgt gcctgaaccc cttcctctac gcctttctag
atgacaactt ccggaagaac 1020ttccgcagca tattgcggtg ctga
104479364PRTHomo sapiens 79Met Gln Ala Ala Gly His
Pro Glu Pro Leu Asp Ser Arg Gly Ser Phe1 5
10 15Ser Leu Pro Thr Met Gly Ala Asn Val Ser Gln Asp Asn
Gly Thr Gly20 25 30His Asn Ala Thr Phe
Ser Glu Pro Leu Pro Phe Leu Tyr Val Leu Leu35 40
45Pro Ala Val Tyr Ser Gly Ile Cys Ala Val Gly Leu Thr Gly Asn
Thr50 55 60Ala Val Ile Leu Val Ile Leu
Arg Ala Pro Lys Met Lys Thr Val Thr65 70
75 80Asn Val Phe Ile Leu Asn Leu Ala Val Ala Asp Gly
Leu Phe Thr Leu85 90 95Val Leu Pro Val
Asn Ile Ala Glu His Leu Leu Gln Tyr Trp Pro Phe100 105
110Gly Glu Leu Leu Cys Lys Leu Val Leu Ala Val Asp His Tyr
Asn Ile115 120 125Phe Ser Ser Ile Tyr Phe
Leu Ala Val Met Ser Val Asp Arg Tyr Leu130 135
140Val Val Leu Ala Thr Val Arg Ser Arg His Met Pro Trp Arg Thr
Tyr145 150 155 160Arg Gly
Ala Lys Val Ala Ser Leu Cys Val Trp Leu Gly Val Thr Val165
170 175Leu Val Leu Pro Phe Phe Ser Phe Ala Gly Val Tyr
Ser Asn Glu Leu180 185 190Gln Val Pro Ser
Cys Gly Leu Ser Phe Pro Trp Pro Glu Arg Val Trp195 200
205Phe Lys Ala Ser Arg Val Tyr Thr Leu Val Leu Gly Phe Val
Leu Pro210 215 220Val Cys Thr Ile Cys Val
Leu Tyr Thr Asp Leu Leu Arg Arg Leu Arg225 230
235 240Ala Val Arg Leu Arg Ser Gly Ala Lys Ala Leu
Gly Lys Ala Arg Arg245 250 255Lys Val Thr
Val Leu Val Leu Val Val Leu Ala Val Cys Leu Leu Cys260
265 270Trp Thr Pro Phe His Leu Ala Ser Val Val Ala Leu
Thr Thr Asp Leu275 280 285Pro Gln Thr Pro
Leu Val Ile Ser Met Ser Tyr Val Ile Thr Ser Leu290 295
300Ser Tyr Ala Asn Ser Cys Leu Asn Pro Phe Leu Tyr Ala Phe
Leu Asp305 310 315 320Asp
Asn Phe Arg Lys Asn Phe Arg Ser Ile Leu Arg Cys Ala Ala Ala325
330 335Arg Gly Arg Thr Pro Pro Ser Leu Gly Pro Gln
Asp Glu Ser Cys Thr340 345 350Thr Ala Ser
Ser Ser Leu Ala Lys Asp Thr Ser Ser355 360801095DNAHomo
sapiens 80atgcaggccg ctgggcaccc agagcccctt gacagcaggg gctccttctc
cctccccacg 60atgggtgcca acgtctctca ggacaatggc actggccaca atgccacctt
ctccgagcca 120ctgccgttcc tctatgtgct cctgcccgcc gtgtactccg ggatctgtgc
tgtggggctg 180actggcaaca cggccgtcat ccttgtaatc ctaagggcgc ccaagatgaa
gacggtgacc 240aacgtgttca tcctgaacct ggccgtcgcc gacgggctct tcacgctggt
actgcccgtc 300aacatcgcgg agcacctgct gcagtactgg cccttcgggg agctgctctg
caagctggtg 360ctggccgtcg accactacaa catcttctcc agcatctact tcctagccgt
gatgagcgtg 420gaccgatacc tggtggtgct ggccaccgtg aggtcccgcc acatgccctg
gcgcacctac 480cggggggcga aggtcgccag cctgtgtgtc tggctgggcg tcacggtcct
ggttctgccc 540ttcttctctt tcgctggcgt ctacagcaac gagctgcagg tcccaagctg
tgggctgagc 600ttcccgtggc ccgagcgggt ctggttcaag gccagccgtg tctacacttt
ggtcctgggc 660ttcgtgctgc ccgtgtgcac catctgtgtg ctctacacag acctcctgcg
caggctgcgg 720gccgtgcggc tccgctctgg agccaaggct ctaggcaagg ccaggcggaa
ggtgaccgtc 780ctggtcctcg tcgtgctggc cgtgtgcctc ctctgctgga cgcccttcca
cctggcctct 840gtcgtggccc tgaccacgga cctgccccag accccactgg tcatcagtat
gtcctacgtc 900atcaccagcc tcagctacgc caactcgtgc ctgaacccct tcctctacgc
ctttctagat 960gacaacttcc ggaagaactt ccgcagcata ttgcggtgcg cggccgcacg
gggacgcacc 1020ccacccagcc tgggtcccca agatgagtcc tgcaccaccg ccagctcctc
cctggccaag 1080gacacttcat cgtga
109581378PRTHomo sapiens 81Met Tyr Pro Tyr Asp Val Pro Asp Tyr
Ala Ala Ala Ala Ala Met Gln1 5 10
15Ala Ala Gly His Pro Glu Pro Leu Asp Ser Arg Gly Ser Phe Ser
Leu20 25 30Pro Thr Met Gly Ala Asn Val
Ser Gln Asp Asn Gly Thr Gly His Asn35 40
45Ala Thr Phe Ser Glu Pro Leu Pro Phe Leu Tyr Val Leu Leu Pro Ala50
55 60Val Tyr Ser Gly Ile Cys Ala Val Gly Leu
Thr Gly Asn Thr Ala Val65 70 75
80Ile Leu Val Ile Leu Arg Ala Pro Lys Met Lys Thr Val Thr Asn
Val85 90 95Phe Ile Leu Asn Leu Ala Val
Ala Asp Gly Leu Phe Thr Leu Val Leu100 105
110Pro Val Asn Ile Ala Glu His Leu Leu Gln Tyr Trp Pro Phe Gly Glu115
120 125Leu Leu Cys Lys Leu Val Leu Ala Val
Asp His Tyr Asn Ile Phe Ser130 135 140Ser
Ile Tyr Phe Leu Ala Val Met Ser Val Asp Arg Tyr Leu Val Val145
150 155 160Leu Ala Thr Val Arg Ser
Arg His Met Pro Trp Arg Thr Tyr Arg Gly165 170
175Ala Lys Val Ala Ser Leu Cys Val Trp Leu Gly Val Thr Val Leu
Val180 185 190Leu Pro Phe Phe Ser Phe Ala
Gly Val Tyr Ser Asn Glu Leu Gln Val195 200
205Pro Ser Cys Gly Leu Ser Phe Pro Trp Pro Glu Arg Val Trp Phe Lys210
215 220Ala Ser Arg Val Tyr Thr Leu Val Leu
Gly Phe Val Leu Pro Val Cys225 230 235
240Thr Ile Cys Val Leu Tyr Thr Asp Leu Leu Arg Arg Leu Arg
Ala Val245 250 255Arg Leu Arg Ser Gly Ala
Lys Ala Leu Gly Lys Ala Arg Arg Lys Val260 265
270Thr Val Leu Val Leu Val Val Leu Ala Val Cys Leu Leu Cys Trp
Thr275 280 285Pro Phe His Leu Ala Ser Val
Val Ala Leu Thr Thr Asp Leu Pro Gln290 295
300Thr Pro Leu Val Ile Ser Met Ser Tyr Val Ile Thr Ser Leu Ser Tyr305
310 315 320Ala Asn Ser Cys
Leu Asn Pro Phe Leu Tyr Ala Phe Leu Asp Asp Asn325 330
335Phe Arg Lys Asn Phe Arg Ser Ile Leu Arg Cys Ala Ala Ala
Arg Gly340 345 350Arg Thr Pro Pro Ser Leu
Gly Pro Gln Asp Glu Ser Cys Thr Thr Ala355 360
365Ser Ser Ser Leu Ala Lys Asp Thr Ser Ser370
375821137DNAHomo sapiens 82atgtacccat acgacgtacc tgattacgca gcagcagcag
caatgcaggc cgctgggcac 60ccagagcccc ttgacagcag gggctccttc tccctcccca
cgatgggtgc caacgtctct 120caggacaatg gcactggcca caatgccacc ttctccgagc
cactgccgtt cctctatgtg 180ctcctgcccg ccgtgtactc cgggatctgt gctgtggggc
tgactggcaa cacggccgtc 240atccttgtaa tcctaagggc gcccaagatg aagacggtga
ccaacgtgtt catcctgaac 300ctggccgtcg ccgacgggct cttcacgctg gtactgcccg
tcaacatcgc ggagcacctg 360ctgcagtact ggcccttcgg ggagctgctc tgcaagctgg
tgctggccgt cgaccactac 420aacatcttct ccagcatcta cttcctagcc gtgatgagcg
tggaccgata cctggtggtg 480ctggccaccg tgaggtcccg ccacatgccc tggcgcacct
accggggggc gaaggtcgcc 540agcctgtgtg tctggctggg cgtcacggtc ctggttctgc
ccttcttctc tttcgctggc 600gtctacagca acgagctgca ggtcccaagc tgtgggctga
gcttcccgtg gcccgagcgg 660gtctggttca aggccagccg tgtctacact ttggtcctgg
gcttcgtgct gcccgtgtgc 720accatctgtg tgctctacac agacctcctg cgcaggctgc
gggccgtgcg gctccgctct 780ggagccaagg ctctaggcaa ggccaggcgg aaggtgaccg
tcctggtcct cgtcgtgctg 840gccgtgtgcc tcctctgctg gacgcccttc cacctggcct
ctgtcgtggc cctgaccacg 900gacctgcccc agaccccact ggtcatcagt atgtcctacg
tcatcaccag cctcagctac 960gccaactcgt gcctgaaccc cttcctctac gcctttctag
atgacaactt ccggaagaac 1020ttccgcagca tattgcggtg cgcggccgca cggggacgca
ccccacccag cctgggtccc 1080caagatgagt cctgcaccac cgccagctcc tccctggcca
aggacacttc atcgtga 113783433PRTHomo sapiens 83Met Cys Phe Ser Pro
Ile Leu Glu Ile Asn Met Gln Ser Glu Ser Asn1 5
10 15Ile Thr Val Arg Asp Asp Ile Asp Asp Ile Asn Thr
Asn Met Tyr Gln20 25 30Pro Leu Ser Tyr
Pro Leu Ser Phe Gln Val Ser Leu Thr Gly Phe Leu35 40
45Met Leu Glu Ile Val Leu Gly Leu Gly Ser Asn Leu Thr Val
Leu Val50 55 60Leu Tyr Cys Met Lys Ser
Asn Leu Ile Asn Ser Val Ser Asn Ile Ile65 70
75 80Thr Met Asn Leu His Val Leu Asp Val Ile Ile
Cys Val Gly Cys Ile85 90 95Pro Leu Thr
Ile Val Ile Leu Leu Leu Ser Leu Glu Ser Asn Thr Ala100
105 110Leu Ile Cys Cys Phe His Glu Ala Cys Val Ser Phe
Ala Ser Val Ser115 120 125Thr Ala Ile Asn
Val Phe Ala Ile Thr Leu Asp Arg Tyr Asp Ile Ser130 135
140Val Lys Pro Ala Asn Arg Ile Leu Thr Met Gly Arg Ala Val
Met Leu145 150 155 160Met
Ile Ser Ile Trp Ile Phe Ser Phe Phe Ser Phe Leu Ile Pro Phe165
170 175Ile Glu Val Asn Phe Phe Ser Leu Gln Ser Gly
Asn Thr Trp Glu Asn180 185 190Lys Thr Leu
Leu Cys Val Ser Thr Asn Glu Tyr Tyr Thr Glu Leu Gly195
200 205Met Tyr Tyr His Leu Leu Val Gln Ile Pro Ile Phe
Phe Phe Thr Val210 215 220Val Val Met Leu
Ile Thr Tyr Thr Lys Ile Leu Gln Ala Leu Asn Ile225 230
235 240Arg Ile Gly Thr Arg Phe Ser Thr Gly
Gln Lys Lys Lys Ala Arg Lys245 250 255Lys
Lys Thr Ile Ser Leu Thr Thr Gln His Glu Ala Thr Asp Met Ser260
265 270Gln Ser Ser Gly Gly Arg Asn Val Val Phe Gly
Val Arg Thr Ser Val275 280 285Ser Val Ile
Ile Ala Leu Arg Arg Ala Val Lys Arg His Arg Glu Arg290
295 300Arg Glu Arg Gln Lys Arg Val Phe Arg Met Ser Leu
Leu Ile Ile Ser305 310 315
320Thr Phe Leu Leu Cys Trp Thr Pro Ile Ser Val Leu Asn Thr Thr Ile325
330 335Leu Cys Leu Gly Pro Ser Asp Leu Leu
Val Lys Leu Arg Leu Cys Phe340 345 350Leu
Val Met Ala Tyr Gly Thr Thr Ile Phe His Pro Leu Leu Tyr Ala355
360 365Phe Thr Arg Gln Lys Phe Gln Lys Val Leu Lys
Ser Lys Met Lys Lys370 375 380Arg Val Val
Ser Ile Val Glu Ala Asp Pro Leu Pro Asn Asn Ala Val385
390 395 400Ile His Asn Ser Trp Ile Asp
Pro Lys Arg Asn Lys Lys Ile Thr Phe405 410
415Glu Asp Ser Glu Ile Arg Glu Lys Cys Leu Val Pro Gln Val Val Thr420
425 430Asp841302DNAHomo sapiens 84atgtgttttt
ctcccattct ggaaatcaac atgcagtctg aatctaacat tacagtgcga 60gatgacattg
atgacatcaa caccaatatg taccaaccac tatcatatcc gttaagcttt 120caagtgtctc
tcaccggatt tcttatgtta gaaattgtgt tgggacttgg cagcaacctc 180actgtattgg
tactttactg catgaaatcc aacttaatca actctgtcag taacattatt 240acaatgaatc
ttcatgtact tgatgtaata atttgtgtgg gatgtattcc tctaactata 300gttatccttc
tgctttcact ggagagtaac actgctctca tttgctgttt ccatgaggct 360tgtgtatctt
ttgcaagtgt ctcaacagca atcaacgttt ttgctatcac tttggacaga 420tatgacatct
ctgtaaaacc tgcaaaccga attctgacaa tgggcagagc tgtaatgtta 480atgatatcca
tttggatttt ttcttttttc tctttcctga ttccttttat tgaggtaaat 540tttttcagtc
ttcaaagtgg aaatacctgg gaaaacaaga cacttttatg tgtcagtaca 600aatgaatact
acactgaact gggaatgtat tatcacctgt tagtacagat cccaatattc 660tttttcactg
ttgtagtaat gttaatcaca tacaccaaaa tacttcaggc tcttaatatt 720cgaataggca
caagattttc aacagggcag aagaagaaag caagaaagaa aaagacaatt 780tctctaacca
cacaacatga ggctacagac atgtcacaaa gcagtggtgg gagaaatgta 840gtctttggtg
taagaacttc agtttctgta ataattgccc tccggcgagc tgtgaaacga 900caccgtgaac
gacgagaaag acaaaagaga gtcttcagga tgtctttatt gattatttct 960acatttcttc
tctgctggac accaatttct gttttaaata ccaccatttt atgtttaggc 1020ccaagtgacc
ttttagtaaa attaagattg tgttttttag tcatggctta tggaacaact 1080atatttcacc
ctctattata tgcattcact agacaaaaat ttcaaaaggt cttgaaaagt 1140aaaatgaaaa
agcgagttgt ttctatagta gaagctgatc ccctgcctaa taatgctgta 1200atacacaact
cttggataga tcctaaaaga aacaaaaaaa ttacctttga agatagtgaa 1260ataagagaaa
aatgtttagt gcctcaggtt gtcacagact ag 130285447PRTHomo
sapiens 85Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ala Ala Ala Ala Met
Cys1 5 10 15Phe Ser Pro
Ile Leu Glu Ile Asn Met Gln Ser Glu Ser Asn Ile Thr20 25
30Val Arg Asp Asp Ile Asp Asp Ile Asn Thr Asn Met Tyr
Gln Pro Leu35 40 45Ser Tyr Pro Leu Ser
Phe Gln Val Ser Leu Thr Gly Phe Leu Met Leu50 55
60Glu Ile Val Leu Gly Leu Gly Ser Asn Leu Thr Val Leu Val Leu
Tyr65 70 75 80Cys Met
Lys Ser Asn Leu Ile Asn Ser Val Ser Asn Ile Ile Thr Met85
90 95Asn Leu His Val Leu Asp Val Ile Ile Cys Val Gly
Cys Ile Pro Leu100 105 110Thr Ile Val Ile
Leu Leu Leu Ser Leu Glu Ser Asn Thr Ala Leu Ile115 120
125Cys Cys Phe His Glu Ala Cys Val Ser Phe Ala Ser Val Ser
Thr Ala130 135 140Ile Asn Val Phe Ala Ile
Thr Leu Asp Arg Tyr Asp Ile Ser Val Lys145 150
155 160Pro Ala Asn Arg Ile Leu Thr Met Gly Arg Ala
Val Met Leu Met Ile165 170 175Ser Ile Trp
Ile Phe Ser Phe Phe Ser Phe Leu Ile Pro Phe Ile Glu180
185 190Val Asn Phe Phe Ser Leu Gln Ser Gly Asn Thr Trp
Glu Asn Lys Thr195 200 205Leu Leu Cys Val
Ser Thr Asn Glu Tyr Tyr Thr Glu Leu Gly Met Tyr210 215
220Tyr His Leu Leu Val Gln Ile Pro Ile Phe Phe Phe Thr Val
Val Val225 230 235 240Met
Leu Ile Thr Tyr Thr Lys Ile Leu Gln Ala Leu Asn Ile Arg Ile245
250 255Gly Thr Arg Phe Ser Thr Gly Gln Lys Lys Lys
Ala Arg Lys Lys Lys260 265 270Thr Ile Ser
Leu Thr Thr Gln His Glu Ala Thr Asp Met Ser Gln Ser275
280 285Ser Gly Gly Arg Asn Val Val Phe Gly Val Arg Thr
Ser Val Ser Val290 295 300Ile Ile Ala Leu
Arg Arg Ala Val Lys Arg His Arg Glu Arg Arg Glu305 310
315 320Arg Gln Lys Arg Val Phe Arg Met Ser
Leu Leu Ile Ile Ser Thr Phe325 330 335Leu
Leu Cys Trp Thr Pro Ile Ser Val Leu Asn Thr Thr Ile Leu Cys340
345 350Leu Gly Pro Ser Asp Leu Leu Val Lys Leu Arg
Leu Cys Phe Leu Val355 360 365Met Ala Tyr
Gly Thr Thr Ile Phe His Pro Leu Leu Tyr Ala Phe Thr370
375 380Arg Gln Lys Phe Gln Lys Val Leu Lys Ser Lys Met
Lys Lys Arg Val385 390 395
400Val Ser Ile Val Glu Ala Asp Pro Leu Pro Asn Asn Ala Val Ile His405
410 415Asn Ser Trp Ile Asp Pro Lys Arg Asn
Lys Lys Ile Thr Phe Glu Asp420 425 430Ser
Glu Ile Arg Glu Lys Cys Leu Val Pro Gln Val Val Thr Asp435
440 445861344DNAHomo sapiens 86atgtacccat acgacgtacc
tgattacgca gcagcagcag caatgtgttt ttctcccatt 60ctggaaatca acatgcagtc
tgaatctaac attacagtgc gagatgacat tgatgacatc 120aacaccaata tgtaccaacc
actatcatat ccgttaagct ttcaagtgtc tctcaccgga 180tttcttatgt tagaaattgt
gttgggactt ggcagcaacc tcactgtatt ggtactttac 240tgcatgaaat ccaacttaat
caactctgtc agtaacatta ttacaatgaa tcttcatgta 300cttgatgtaa taatttgtgt
gggatgtatt cctctaacta tagttatcct tctgctttca 360ctggagagta acactgctct
catttgctgt ttccatgagg cttgtgtatc ttttgcaagt 420gtctcaacag caatcaacgt
ttttgctatc actttggaca gatatgacat ctctgtaaaa 480cctgcaaacc gaattctgac
aatgggcaga gctgtaatgt taatgatatc catttggatt 540ttttcttttt tctctttcct
gattcctttt attgaggtaa attttttcag tcttcaaagt 600ggaaatacct gggaaaacaa
gacactttta tgtgtcagta caaatgaata ctacactgaa 660ctgggaatgt attatcacct
gttagtacag atcccaatat tctttttcac tgttgtagta 720atgttaatca catacaccaa
aatacttcag gctcttaata ttcgaatagg cacaagattt 780tcaacagggc agaagaagaa
agcaagaaag aaaaagacaa tttctctaac cacacaacat 840gaggctacag acatgtcaca
aagcagtggt gggagaaatg tagtctttgg tgtaagaact 900tcagtttctg taataattgc
cctccggcga gctgtgaaac gacaccgtga acgacgagaa 960agacaaaaga gagtcttcag
gatgtcttta ttgattattt ctacatttct tctctgctgg 1020acaccaattt ctgttttaaa
taccaccatt ttatgtttag gcccaagtga ccttttagta 1080aaattaagat tgtgtttttt
agtcatggct tatggaacaa ctatatttca ccctctatta 1140tatgcattca ctagacaaaa
atttcaaaag gtcttgaaaa gtaaaatgaa aaagcgagtt 1200gtttctatag tagaagctga
tcccctgcct aataatgctg taatacacaa ctcttggata 1260gatcctaaaa gaaacaaaaa
aattaccttt gaagatagtg aaataagaga aaaatgttta 1320gtgcctcagg ttgtcacaga
ctag 134487419PRTHomo sapiens
87Met Cys Phe Ser Pro Ile Leu Glu Ile Asn Met Gln Ser Glu Ser Asn1
5 10 15Ile Thr Val Arg Asp Asp
Ile Asp Asp Ile Asn Thr Asn Met Tyr Gln20 25
30Pro Leu Ser Tyr Pro Leu Ser Phe Gln Val Ser Leu Thr Gly Phe Leu35
40 45Met Leu Glu Ile Val Leu Gly Leu Gly
Ser Asn Leu Thr Val Leu Val50 55 60Leu
Tyr Cys Met Lys Ser Asn Leu Ile Asn Ser Val Ser Asn Ile Ile65
70 75 80Thr Met Asn Leu His Val
Leu Asp Val Ile Ile Cys Val Gly Cys Ile85 90
95Pro Leu Thr Ile Val Ile Leu Leu Leu Ser Leu Glu Ser Asn Thr Ala100
105 110Leu Ile Cys Cys Phe His Glu Ala
Cys Val Ser Phe Ala Ser Val Ser115 120
125Thr Ala Ile Asn Val Phe Ala Ile Thr Leu Asp Arg Tyr Asp Ile Ser130
135 140Val Lys Pro Ala Asn Arg Ile Leu Thr
Met Gly Arg Ala Val Met Leu145 150 155
160Met Ile Ser Ile Trp Ile Phe Ser Phe Phe Ser Phe Leu Ile
Pro Phe165 170 175Ile Glu Val Asn Phe Phe
Ser Leu Gln Ser Gly Asn Thr Trp Glu Asn180 185
190Lys Thr Leu Leu Cys Val Ser Thr Asn Glu Tyr Tyr Thr Glu Leu
Gly195 200 205Met Tyr Tyr His Leu Leu Val
Gln Ile Pro Ile Phe Phe Phe Thr Val210 215
220Val Val Met Leu Ile Thr Tyr Thr Lys Ile Leu Gln Ala Leu Asn Ile225
230 235 240Arg Ile Gly Thr
Arg Phe Ser Thr Gly Gln Lys Lys Lys Ala Arg Lys245 250
255Lys Lys Thr Ile Ser Leu Thr Thr Gln His Glu Ala Thr Asp
Met Ser260 265 270Gln Ser Ser Gly Gly Arg
Asn Val Val Phe Gly Val Arg Thr Ser Val275 280
285Ser Val Ile Ile Ala Leu Arg Arg Ala Val Lys Arg His Arg Glu
Arg290 295 300Arg Glu Arg Gln Lys Arg Val
Phe Arg Met Ser Leu Leu Ile Ile Ser305 310
315 320Thr Phe Leu Leu Cys Trp Thr Pro Ile Ser Val Leu
Asn Thr Thr Ile325 330 335Leu Cys Leu Gly
Pro Ser Asp Leu Leu Val Lys Leu Arg Leu Cys Phe340 345
350Leu Val Met Ala Tyr Gly Thr Thr Ile Phe His Pro Leu Leu
Tyr Ala355 360 365Phe Thr Arg Gln Lys Phe
Gln Lys Val Leu Lys Ser Lys Met Lys Lys370 375
380Arg Val Val Cys Ala Ala Ala Arg Gly Arg Thr Pro Pro Ser Leu
Gly385 390 395 400Pro Gln
Asp Glu Ser Cys Thr Thr Ala Ser Ser Ser Leu Ala Lys Asp405
410 415Thr Ser Ser881260DNAHomo sapiens 88atgtgttttt
ctcccattct ggaaatcaac atgcagtctg aatctaacat tacagtgcga 60gatgacattg
atgacatcaa caccaatatg taccaaccac tatcatatcc gttaagcttt 120caagtgtctc
tcaccggatt tcttatgtta gaaattgtgt tgggacttgg cagcaacctc 180actgtattgg
tactttactg catgaaatcc aacttaatca actctgtcag taacattatt 240acaatgaatc
ttcatgtact tgatgtaata atttgtgtgg gatgtattcc tctaactata 300gttatccttc
tgctttcact ggagagtaac actgctctca tttgctgttt ccatgaggct 360tgtgtatctt
ttgcaagtgt ctcaacagca atcaacgttt ttgctatcac tttggacaga 420tatgacatct
ctgtaaaacc tgcaaaccga attctgacaa tgggcagagc tgtaatgtta 480atgatatcca
tttggatttt ttcttttttc tctttcctga ttccttttat tgaggtaaat 540tttttcagtc
ttcaaagtgg aaatacctgg gaaaacaaga cacttttatg tgtcagtaca 600aatgaatact
acactgaact gggaatgtat tatcacctgt tagtacagat cccaatattc 660tttttcactg
ttgtagtaat gttaatcaca tacaccaaaa tacttcaggc tcttaatatt 720cgaataggca
caagattttc aacagggcag aagaagaaag caagaaagaa aaagacaatt 780tctctaacca
cacaacatga ggctacagac atgtcacaaa gcagtggtgg gagaaatgta 840gtctttggtg
taagaacttc agtttctgta ataattgccc tccggcgagc tgtgaaacga 900caccgtgaac
gacgagaaag acaaaagaga gtcttcagga tgtctttatt gattatttct 960acatttcttc
tctgctggac accaatttct gttttaaata ccaccatttt atgtttaggc 1020ccaagtgacc
ttttagtaaa attaagattg tgttttttag tcatggctta tggaacaact 1080atatttcacc
ctctattata tgcattcact agacaaaaat ttcaaaaggt cttgaaaagt 1140aaaatgaaaa
agcgagttgt ttgtgcggcc gcacggggac gcaccccacc cagcctgggt 1200ccccaagatg
agtcctgcac caccgccagc tcctccctgg ccaaggacac ttcatcgtga 126089433PRTHomo
sapiens 89Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ala Ala Ala Ala Met
Cys1 5 10 15Phe Ser Pro
Ile Leu Glu Ile Asn Met Gln Ser Glu Ser Asn Ile Thr20 25
30Val Arg Asp Asp Ile Asp Asp Ile Asn Thr Asn Met Tyr
Gln Pro Leu35 40 45Ser Tyr Pro Leu Ser
Phe Gln Val Ser Leu Thr Gly Phe Leu Met Leu50 55
60Glu Ile Val Leu Gly Leu Gly Ser Asn Leu Thr Val Leu Val Leu
Tyr65 70 75 80Cys Met
Lys Ser Asn Leu Ile Asn Ser Val Ser Asn Ile Ile Thr Met85
90 95Asn Leu His Val Leu Asp Val Ile Ile Cys Val Gly
Cys Ile Pro Leu100 105 110Thr Ile Val Ile
Leu Leu Leu Ser Leu Glu Ser Asn Thr Ala Leu Ile115 120
125Cys Cys Phe His Glu Ala Cys Val Ser Phe Ala Ser Val Ser
Thr Ala130 135 140Ile Asn Val Phe Ala Ile
Thr Leu Asp Arg Tyr Asp Ile Ser Val Lys145 150
155 160Pro Ala Asn Arg Ile Leu Thr Met Gly Arg Ala
Val Met Leu Met Ile165 170 175Ser Ile Trp
Ile Phe Ser Phe Phe Ser Phe Leu Ile Pro Phe Ile Glu180
185 190Val Asn Phe Phe Ser Leu Gln Ser Gly Asn Thr Trp
Glu Asn Lys Thr195 200 205Leu Leu Cys Val
Ser Thr Asn Glu Tyr Tyr Thr Glu Leu Gly Met Tyr210 215
220Tyr His Leu Leu Val Gln Ile Pro Ile Phe Phe Phe Thr Val
Val Val225 230 235 240Met
Leu Ile Thr Tyr Thr Lys Ile Leu Gln Ala Leu Asn Ile Arg Ile245
250 255Gly Thr Arg Phe Ser Thr Gly Gln Lys Lys Lys
Ala Arg Lys Lys Lys260 265 270Thr Ile Ser
Leu Thr Thr Gln His Glu Ala Thr Asp Met Ser Gln Ser275
280 285Ser Gly Gly Arg Asn Val Val Phe Gly Val Arg Thr
Ser Val Ser Val290 295 300Ile Ile Ala Leu
Arg Arg Ala Val Lys Arg His Arg Glu Arg Arg Glu305 310
315 320Arg Gln Lys Arg Val Phe Arg Met Ser
Leu Leu Ile Ile Ser Thr Phe325 330 335Leu
Leu Cys Trp Thr Pro Ile Ser Val Leu Asn Thr Thr Ile Leu Cys340
345 350Leu Gly Pro Ser Asp Leu Leu Val Lys Leu Arg
Leu Cys Phe Leu Val355 360 365Met Ala Tyr
Gly Thr Thr Ile Phe His Pro Leu Leu Tyr Ala Phe Thr370
375 380Arg Gln Lys Phe Gln Lys Val Leu Lys Ser Lys Met
Lys Lys Arg Val385 390 395
400Val Cys Ala Ala Ala Arg Gly Arg Thr Pro Pro Ser Leu Gly Pro Gln405
410 415Asp Glu Ser Cys Thr Thr Ala Ser Ser
Ser Leu Ala Lys Asp Thr Ser420 425
430Ser901302DNAHomo sapiens 90atgtacccat acgacgtacc tgattacgca gcagcagcag
caatgtgttt ttctcccatt 60ctggaaatca acatgcagtc tgaatctaac attacagtgc
gagatgacat tgatgacatc 120aacaccaata tgtaccaacc actatcatat ccgttaagct
ttcaagtgtc tctcaccgga 180tttcttatgt tagaaattgt gttgggactt ggcagcaacc
tcactgtatt ggtactttac 240tgcatgaaat ccaacttaat caactctgtc agtaacatta
ttacaatgaa tcttcatgta 300cttgatgtaa taatttgtgt gggatgtatt cctctaacta
tagttatcct tctgctttca 360ctggagagta acactgctct catttgctgt ttccatgagg
cttgtgtatc ttttgcaagt 420gtctcaacag caatcaacgt ttttgctatc actttggaca
gatatgacat ctctgtaaaa 480cctgcaaacc gaattctgac aatgggcaga gctgtaatgt
taatgatatc catttggatt 540ttttcttttt tctctttcct gattcctttt attgaggtaa
attttttcag tcttcaaagt 600ggaaatacct gggaaaacaa gacactttta tgtgtcagta
caaatgaata ctacactgaa 660ctgggaatgt attatcacct gttagtacag atcccaatat
tctttttcac tgttgtagta 720atgttaatca catacaccaa aatacttcag gctcttaata
ttcgaatagg cacaagattt 780tcaacagggc agaagaagaa agcaagaaag aaaaagacaa
tttctctaac cacacaacat 840gaggctacag acatgtcaca aagcagtggt gggagaaatg
tagtctttgg tgtaagaact 900tcagtttctg taataattgc cctccggcga gctgtgaaac
gacaccgtga acgacgagaa 960agacaaaaga gagtcttcag gatgtcttta ttgattattt
ctacatttct tctctgctgg 1020acaccaattt ctgttttaaa taccaccatt ttatgtttag
gcccaagtga ccttttagta 1080aaattaagat tgtgtttttt agtcatggct tatggaacaa
ctatatttca ccctctatta 1140tatgcattca ctagacaaaa atttcaaaag gtcttgaaaa
gtaaaatgaa aaagcgagtt 1200gtttgtgcgg ccgcacgggg acgcacccca cccagcctgg
gtccccaaga tgagtcctgc 1260accaccgcca gctcctccct ggccaaggac acttcatcgt
ga 1302915PRTHomo sapiens 91Ser Ser Leu Ser Thr1
5924PRTHomo sapiens 92Ser Thr Leu Ser1935PRTHomo sapiens 93Thr
Thr Ile Ser Thr1 5944PRTHomo sapiens 94Cys Val Leu
Leu1954PRTHomo sapiensMISC_FEATURE(2)..(3)The Xaa at positions 2-3 are
aliphatic amino acid 95Cys Xaa Xaa Xaa1
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