Abstract:
The present invention features isolated, purified, or enriched nucleic acid encoding a SIRP polypeptide and isolated, purified, or enriched SIRP polypeptide and uses thereof.

Description:
This application claims priority to U.S. provisional application No. 60/030,964 filed Nov. 15, 1996. 
    
    
     BACKGROUND OF THE INVENTION 
     Signal transduction is a fundamental mechanism whereby external stimuli are relayed to the interior of cells. A key aspect of signal transduction involves the reversible phosphorylation of tyrosine residues on proteins. The phosphorylation state of tyrosine residues on a protein is modified through the reciprocal actions of tyrosine kinases (TKs) and tyrosine phosphatases (TPs). 
     For example, a variety of polypeptide growth factors and hormones mediate their cellular effects by interacting with cell surface receptors and soluble or cytoplasmic polypeptide containing molecules having tyrosine kinase enzymatic activity (for review, see Williams, et al.  Cell  61:203-212 (1990); Carpenter, et al.  J. Biol. Chem . 265:7709-7712 (1990)). The interaction of these ligands with their receptors induces a series of events which include receptor dimerization and stimulation of protein tyrosine kinase activity. Tyrosine autophosphorylation on multiple sites creates specific binding sites for target proteins, which bind to the activated receptor with their SH2 domains (for review, see Schlessinger and Ullrich,  Neuron  9:383-391, (1992)). 
     SH2 (src homology 2) domains are conserved sequences of about 100 amino acids found in cytoplasmic non-receptor tyrosine kinases such as pp60src, PLC-γ, GAP and v-crk (Mayer, et al.,  Nature  332:272-275 (1988); Pawson,  Oncogene  3:491-495 (1988)). While having distinct catalytic domains, all these molecules share conserved SH2 and SH3 (src homology 3) domains and the ability to associate with receptors with tyrosine kinase activity (Anderson, et al.  Science  250:979-982 (1990)). 
     Tyrosine kinase activation and receptor autophosphorylation are prerequisites for the association between growth factor receptors and SH2 domain-containing proteins (Margolis, et al.,  Mol. Cell. Biol . 10:435-441 (1990); Kumjian et al.,  Proc. Natl. Acad. Sci. USA  86:8232-8239 (1989); Kazlauskas, et al.,  Science  247:1578-1581 (1990)). In particular, the carboxy-terminal (C-terminal) fragment of the epidermal growth factor receptor (EGFR), which contains all the known autophosphorylation sites, binds specifically to the SH2 domains of GAP and PLC-γ (see below). Hence, a major site of association exists between the SH2 domain of these substrate proteins and the tyrosine phosphorylated C-terminal tail of the EGFR. 
     Target proteins which bind to activated receptors have been identified by analysis of proteins that co-immunoprecipitate with growth factor receptors, or that bind to receptors attached to immobilized matrices (Morrison, et al.,  Cell  58:649-657 (1989); Kazlauskas, et al.,  EMBO J . 9:3279-3286 (1990)). 
     Ohnishi et al.  J. Biol. Chem . 271:25569-25574 (1996), not admitted to be prior art, described that a brain specific immunoglobulin-like molecule with tyrosine-based activation motifs, BIT, is associated with protein-tyrosine phosphatase SH-PTP2, whereby two SH2 domains of SH-PTP2 simultaneously interact with two phosphotyrosines of BIT-TAM. 
     Phosphotyrosine phosphatases (PTPs) are involved with negative or positive regulation of growth factor-specific cell responses such as mitosis, differentiation, migration, survival, transformation or death. For example, SHP-2 is a phosphotyrosine phosphatase which contains a SH2 domain. SHP-2 is a positive signal transducer for a number of receptor tyrosine kinases (RTKS) and cytokine receptors. 
     SUMMARY OF THE INVENTION 
     Within the scope of this invention, applicant has identified a novel mammalian protein family of at least fifteen members designated SIgnal Regulatory Proteins (SIRPs. In particular, Applicant has cloned and sequenced the coding sequences of 4 members of SIRPs, SIRP1 and SIRP4 from human, and SIRPα1 and SIRPβ1 from mouse. In this regard, the present invention relates to SIRP polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such polypeptides or nucleic acids, antibodies to such polypeptides or nucleic acids, assays utilizing such polypeptides or nucleic acids, and methods relating to all of the foregoing. 
     SIRP family proteins play a general role in the regulation of signals that define diverse physiological and pathological processes. Thus, the present invention provides several agents and methods useful for diagnosing, treating, and preventing various diseases or conditions associated with abnormalities in these pathways as well as assay systems useful for screening for therapeutically effective agents. 
     In particular, SIRP polypeptides are involved in various signal transduction pathways such as the negative regulation of signals generated by receptor tyrosine kinases, including, but not limited to, receptors for EGF, insulin and platelet derived growth factor (PDGF). For example, acting like a tumor suppressor, SIRP4 exerts negative regulatory effects on growth factor and hormone induced cellular responses such as DNA synthesis. Oncogenesis may be associated with mutant SIRPs or not enough SIRPs. Restoring SIRPs to their normal levels such as by gene therapy could restore the cells to a normal growth pattern. Insulin receptor activity is also regulated by SIRPs. Overexpression of SIRPs may be involved in type II diabetes where sufficient insulin is present but insulin signaling is deficient. A compound that inhibits the negative regulation of insulin signaling by SIRPS, such as by interfering with the interaction between SIRP and SHP-2 may lead to enhanced insulin signaling. 
     All SIRP proteins have a receptor-like, or Immunoglubulin (Ig) like extracellular domain and a transmembrane domain. There are two subtypes of SIRPs distinguished by the presence or absence of a cytoplasmic SHP-2 binding domain. For example, SIRP4 has a cytoplasmic domain while SIRP1 doesn&#39;t. The cytoplasmic domain of SIRP4 contains two SHP-2 binding regions each having two tyrosine residues. 
     The growth inhibitory effect of SIRP4 depends on phosphorylation of tyrosines and is related to reduced MAP kinase activation. SIRP4 becomes a substrate of activated receptor tyrosine kinases (RTKs) upon EGF, insulin or PDGF stimulation. In its tyrosine phosphorylated form, SIRP4 binds a phosphotyrosine phosphatase, SHP-2, via SH2 interactions. Once SIRP4 binds SHP-2, it activates the catalytic activity of SHP-2 and becomes a substrate of SHP-2. This direct activation of SHP-2 could induce activation of Src or other Src family kinases. The above described interaction allows SIRP4 to participate in major signal transduction pathways involving SHP-2. 
     SHP-2 has two SH2 domains and is required for signaling downstream of a variety of RTKs. SHP-2 has been reported to bind directly to RTKs such as PDGF receptor, EGF receptor, and cKit in response to stimulation by their ligands. Insulin receptor substrate 1 (IRS-1) also associates with SHP-2 in response to insulin. 
     SIRP4 also binds SHP-1 and Grb2, both of which contain a SH-2 domain. Grb2 is an adapter molecule and one of its functions is to link growth factor receptors to downstream effector proteins. Grb2 is known to bind tyrosine-phosphorylated SHP-2 in response to PDGF stimulation. 
     The full length nucleic acid sequences encoding hSIRP1, hSIRP4, mSIRPα1 and mSIRPβ1 proteins are set forth respectively in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The coding regions are nt 41-1237 of SEQ ID NO:1, nt 13-1524 of SEQ ID NO:2, nt 59-1597 of SEQ ID NO:3, and nt 86-1261 of SEQ ID NO:4. 
     The full length amino acid sequences of hSIRP1, hSIRP4, mSIRPα1 and mSIRPβ1 are set forth respectively in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. The first two Ig-like domains of hSIRP1 is from aa 54-227; the third Ig-like domain is from aa 250-330; the extracellular domain next to the membrane is from aa 336-366; and the transmembrane domain is from aa 367-398. The first two Ig-like domains of hSIRP4 is from aa 1-227; the third Ig-like domain is from aa 250-336; the extracelluar domain next to the membrane, the transmembrane domain, and the cytoplasmic domain immediate next to the membrane are from aa 347-407; and the rest of the cytoplasmic domain is from aa 408-503. 
     Thus, in a first aspect the invention features an isolated, purified, enriched or recombinant nucleic acid encoding a SIRP polypeptide. Preferably such nucleic acid encodes a mammalian SIRP polypeptide, more preferably it encodes a human SIRP polypeptide. 
     By “isolated” in reference to nucleic acid is meant a polymer of 2 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA or RNA that is isolated from a natural source or that is synthesized. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but does indicate that it is the predominate sequence present (at least 10-200 more than any other nucleotide sequence) and is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it. Therefore, the term does not encompass an isolated chromosome encoding one or more SIRP polypeptides. 
     By the use of the term “enriched” in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased in a useful manner and preferably separate from a sequence library. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid. 
     It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term “purified” in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10 6 -fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. 
     By “SIRP polypeptide” is meant 9 or more contiguous amino acids set forth in the full length amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. The SIRP polypeptides can be encoded by full-length nucleic acid sequences or any portion of a full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained. Preferred functional activities include the ability to bind to a receptor tyrosine kinase or a SH-2 domain bearing protein such as SHP-2, SHP-1 or Grb-2. A non full-length SIRP polypeptide may be used to elicit an antibody against the polypeptide and the full-length polypeptide using techniques known to those skilled in the art. The present invention also encompasses deletion mutants lacking one or more isolated SIRP domains (e.g., Ig-like domain, transmembrane domain, SH2 binding domain, and tyrosine residues), and complementary sequences capable of hybridizing to full length SIRP protein under stringent hybridization conditions. 
     In preferred embodiments, isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in the full length nucleic acid sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 or at least 27, 30, 45, 60 or 90 contiguous nucleotides thereof and the SIRP polypeptide comprises, consists essentially of, or consists of at least 9, 10, 15, 20, 30, 50, 100, 200, or 300 contiguous amino acids of a SIRP polypeptide. 
     By “comprising” it is meant including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may 0 o not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements. 
     Compositions and probes of the present invention may contain human nucleic acids encoding a SIRP polypeptide but are substantially free of nucleic acid not encoding SIRP polypeptide. The human nucleic acid encoding a SIRP polypeptide is at least 18 contiguous bases of the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 and will selectively hybridize to human genomic DNA encoding a SIRP polypeptide, or is complementary to such a sequence. The nucleic acid may be isolated from a natural source by cDNA cloning or subtractive hybridization; the natural source may be blood, semen, and tissue of various organisms including eukaryotes, mammals, birds, fish, plants, gorillas, rhesus monkeys, chimpanzees and humans; and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer. In yet other preferred embodiments the nucleic acid is a conserved or unique region, for example those useful for the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, and obtaining antibodies to polypeptide regions. 
     By “conserved nucleic acid regions”, are meant regions present on two or more nucleic acids encoding a SIRP polypeptide, to which a particular nucleic acid sequence can hybridize to under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acid encoding SIRP polypeptides are provided in Abe, et al.  J. Biol. Chem ., 19:13361 (1992) (hereby incorporated by reference herein in its entirety, including any drawings). Preferably, conserved regions differ by no more than 7 out of 20 nucleotides. 
     By “unique nucleic acid region” is meant a sequence present in a full length nucleic acid coding for a SIRP polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide. Such regions preferably comprise 12 or 20 contiguous nucleotides present in the full length nucleic acid encoding a SIRP polypeptide. 
     The invention also features a nucleic acid probe for the detection of a nucleic acid encoding a SIRP polypeptide in a sample. The nucleic acid probe contains nucleic acid that will hybridize to at least one sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. 
     In preferred embodiments the nucleic acid probe hybridizes to nucleic acid encoding at least 12, 27, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. 
     By “high stringency hybridization conditions” is meant those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1 o SDS at 50° C.; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5×Denhardt&#39;s solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides. 
     Methods for using the probes include detecting the presence or amount of SIRP RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to SIRP RNA. The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a SIRP polypeptide may be used in the identification of the sequence of the nucleic acid detected (for example see, Nelson et al., in  Nonisotopic DNA Probe Techniques , p. 275 Academic Press, San Diego (Kricka, ed., 1992) hereby incorporated by reference herein in its entirety, including any drawings). Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe. 
     The invention also features recombinant nucleic acid, preferably in a cell or an organism. The recombinant nucleic acid may contain a sequence (coding sequence or noncoding sequence) or a segment of sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 and a vector or a promoter effective to initiate transcription in a host cell. The recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complimentary to an RNA sequence encoding a SIRP polypeptide and a transcriptional termination region functional in a cell. 
     In another aspect the invention features an isolated, enriched or purified SIRP polypeptide. 
     By “isolated” in reference to a polypeptide is meant a polymer of 2 (preferably 7, more preferably 13, most preferably 25) or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term “isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is the predominate sequence present (at least 10-20% more than any other sequence) and is essentially free (about 90-95% pure at least) of non-amino acid material naturally associated with it. 
     By the use of the term “enriched” in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acids present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no amino acid from other sources. The other source amino acid may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to elevate the proportion of the desired amino acid. 
     It is also advantageous for some purposes that an amino acid sequence be in purified form. The term “purified” in reference to a polypeptide does not require absolute purity (such o0 as a homogeneous preparation); instead, it represents an indication.that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure. 
     In preferred embodiments SIRP polypeptides contain at least 9, 10, 15, 20, or 30 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. 
     In yet another aspect the invention features a purified antibody (e.a., a monoclonal or polyclonal antibody) having specific binding affinity to a SIRP polypeptide. The antibody contains a sequence of amino acids that is able to specifically bind to a SIRP polypeptide. 
     By “specific binding affinity” is meant that the antibody will bind to a hSIRP polypeptide at a certain detectable amount but will not bind other polypeptides to the same extent, under identical conditions. The present invention also encompasses antibodies that can distinguish hSIRP1 from hSIRP2 or hSIRP3 or can otherwise distinguish between the various SIRPs. 
     Antibodies having specific binding affinity to a SIRP polypeptide may be used in methods for detecting the presence and/or amount of a SIRP polypeptide is a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the SIRP polypeptide. Diagnostic kits for performing such methods may be constructed to include a first container means containing the antibody and a second container means having a conjugate of a binding partner of the antibody and a label. 
     In another aspect the invention features a hybridoma which produces an antibody having specific binding affinity to a SIRP polypeptide. 
     By “hybridoma” is meant an immortalized cell line which is capable of secreting an antibody, for example a SIRP antibody. 
     In preferred embodiments the SIRP antibody comprises a sequence of amino acids that is able to specifically bind a SIRP polypeptide. 
     Another aspect of the invention features a method of detecting the presence or amount of a compound capable of binding to a SIRP polypeptide. The method involves incubating the compound with a SIRP polypeptide and detecting the presence or amount of the compound bound to the SIRP polypeptide. 
     In preferred embodiments, the compound inhibits an activity of SIRP. The present invention also features compounds capable of binding and inhibiting SIRP polypeptide that are identified by methods described above. 
     In another aspect the invention features a method of screening potential agents useful for treatment of a disease or condition characterized by an abnormality in a signal transduction pathway that contains an interaction between a SIRP polypeptide and a natural binding partner (NBP). The method involves assaying potential agents for those able to promote or disrupt the interaction as an indication of a useful agent. 
     By “NBP” is meant a natural binding partner of a SIRP polypeptide that naturally associates with a SIRP polypeptide. The structure (primary, secondary, or tertiary) of the particular natural binding partner will influence the particular type of interaction between the SIRP polypeptide and the natural binding partner. For example, if the natural binding partner comprises a sequence of amino acids complementary to the SIRP polypeptide, covalent bonding may be a possible interaction. Similarly, other structural characteristics may allow for other corresponding interactions. The interaction is not limited to particular residues and specifically may involve phosphotyrosine, phosphoserine, or phosphothreonine residues. A broad range of sequences may be capable of interacting with SIRP polypeptides. One example of a natural binding partner may be SHP-2, which is described above. Other examples include, but are not limited to, SHP-1 and Grb2. Using techniques well known in the art, one may identify several natural binding partners for SIRP polypeptides such as by utilizing a two-hybrid screen. 
     By “screening” is meant investigating an organism for the presence or absence of a property. The process may include measuring or detecting various properties, including the level of signal transduction and the level of interaction between a SIRP polypeptide and a NBP. 
     By “disease or condition” is meant a state in an organism, e.g., a human, which is recognized as abnormal by members of the medicalcommunity. The disease or condition may be characterized by an abnormality in one or more signal transduction pathways in a cell wherein one of the components of the signal transduction pathway is either a SIRP polypeptide or a NBP. Specific diseases or disorders which might be treated or prevented, based upon the affected cells include cancers and diabetes. 
     In preferred embodiments, the methods described herein involve identifying a patient in need of treatment. Those skilled in the art will recognize that various techniques may be used to identify such patients. 
     By “abnormality” is meant an a level which is statistically different from the level observed in organisms not suffering from such a disease or condition and may be characterized as either an excess amount, intensity or duration of signal or a deficient amount, intensity or duration of signal. The abnormality in signal transduction may be realized as an abnormality in cell function, viability or differentiation state. The present invention is based in part on the determination that such abnormality in a pathway can be alleviated by action at the SHP-2-SIRP interaction site in the pathway. An abnormal interaction level may also either be greater or less than the normal level and may impair the normal performance or function of the organism. Thus, it is also possible to screen for agents that will be useful for treating a disease or condition, characterized by an abnormality in the signal transduction pathway, by testing compounds for their ability to affect the interaction between a SIRP polypeptide and SHP-2, since the complex formed by such interaction is part of the signal transduction pathway. However, the disease or condition may be characterized by an abnormality in the signal transduction pathway even if the level of interaction between the SIRP polypeptide and NBP is normal. 
     By “interact” is meant any physical association between polypeptides, whether covalent or non-covalent. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. Examples of non-covalent bonds include electrostatic bonds, hydrogen bonds, and Van der Waals bonds. Furthermore, the interactions between polypeptides may either be direct or indirect. Thus, the association between two given polypeptides may be achieved with an intermediary agent, or several such agents, that connects the two proteins of interest (e.g., a SIRP polypeptide and SHP-2). Another example of an indirect interaction is the independent production, stimulation, or inhibition of both a SIRP polypeptide and SHP-2 by a regulatory agent. Depending upon the type of interaction present, various methods may be used to measure the level of interaction. For example, the strengths of covalent bonds are often measured in terms of the energy required to break a certain number of bonds (i.e., kcal/mol) Non-covalent interactions are often described as above, and also in terms of the distance between the interacting molecules. Indirect interactions may be described in a number of ways, including the number of intermediary agents involved, or the degree of control exercised over the SIRP polypeptide relative to the control exercised over SHP-2 or another NBP. 
     By “disrupt” is meant that the interaction between the SIRP polypeptide and SHP-2 or a NBP is reduced either by preventing expression of the SIRP polypeptide, or by preventing expression of SHP-2 or NBP, or by specifically preventing interaction of the naturally synthesized proteins or by interfering with the interaction of the proteins. 
     By “promote” is meant that the interaction between a SIRP polypeptide and SHP-2 or NBP is increased either by increasing expression of a SIRP polypeptide, or by increasing expression of SHP-2 or a NBP, or by decreasing the dephosphorylating activity of the corresponding regulatory PTP (or other phosphatase acting on other phosphorylated signaling components) by promoting interaction of the SIRP polypeptide and SHP-2 or NBP or by prolonging the duration of the interaction. Covalent binding can be promoted either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling polypeptides, such as an antibody, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom 1984 , J. Immunol . 133:1335-2549; Jansen, F. K., et al., 1982 , Immunological Rev . 62:185-216; and Vitetta et al., supra). 
     By “signal transduction pathway” is meant the sequence of events that involves the transmission of a message from an extracellular protein to the cytoplasm through a cell membrane. The signal ultimately will cause the cell to perform a particular function, for example, to uncontrollably proliferate and therefore cause cancer. Various mechanisms for the signal transduction pathway (Fry et al., Protein Science, 2:1785-1797, 1993) provide possible methods for measuring the amount or intensity of a given signal. Depending upon the particular disease associated with the abnormality in a signal transduction pathway, various symptoms may be detected. Those skilled in the art recognize those symptoms that are associated with the various other diseases described herein. Furthermore, since some adapter molecules recruit secondary signal transducer proteins towards the membrane, one measure of signal transduction is the concentration and localization of variousproteins and complexes. In addition, conformational changes that are involved in the transmission of a signal may be observed using circular dichroism and fluorescence studies. 
     In another aspect the invention features a method of diagnosis of an organism for a disease or condition characterized by an abnormality in a signal transduction pathway that contains an interaction between a SIRP polypeptide and SHP-2 or a NBP. The method involves detecting the level of interaction as an indication of said disease or condition. 
     By “organism” is meant any living creature. The term includes mammals, and specifically humans. Preferred organisms include mice, as the ability to treat or diagnose mice is often predictive of the ability to function in other organisms such as humans. 
     By “diagnosis” is meant any method of identifying a =symptom normally associated with a given disease or condition. Thus, an initial diagnosis may be conclusively established as correct by the use of additional confirmatory evidence such as the presence of other symptoms. Current classification of various diseases and conditions is constantly changing as more is learned about the mechanisms causing the diseases or conditions. Thus, the detection of an important symptom, such as the detection of an abnormal level of interaction between SIRP polypeptides and SHP-2 or NBPs may form the basis to define and diagnose a newly named disease or condition. For example, conventional cancers are classified according to the presence of a particular set of symptoms. However, a subset of these symptoms may both be associated with an abnormality in a particular signaling pathway, such as the ras 21  pathway and in the future these diseases may be reclassified as ras 21  pathway diseases regardless of the particular symptoms observed. 
     Yet another aspect of the invention features a method for treatment of an organism having a disease or condition characterized by an abnormality in a signal transduction pathway. The signal transduction pathway contains an interaction between a SIRP polypeptide and SHP-2 or a NBP and the method involves promoting or disrupting the interaction, including methods that target the SIRP:NBP interaction directly, as well as methods that target other points along the pathway. 
     By “dominant negative mutant protein” is meant a mutant protein that interferes with the normal signal transduction pathway. The dominant negative mutant protein contains the domain of interest (e.g., an SIRP polypeptide or SHP-2 or a NBP), but has a mutation preventing proper signaling, for example by preventing binding of a second domain from the same protein. One example of a dominant negative protein is described in Millauer et al.,  Nature  Feb. 10, 1994. The agent is preferably a peptide which blocks or promotes interaction of the SIRP polypeptide and SHP-2 or another NBP. The peptide may be recombinant, purified, or placed in a pharmaceutically acceptable carrier or diluent. 
     An EC 50  or IC 50  of less than or equal to 100 μM is preferable, and even more preferably less than or equal to 50 μM, and most preferably less that or equal to 20 μM. Such lower EC 50 &#39;s or IC 50 &#39;s are advantageous since they allow lower concentrations of molecules to be used in vivo or in vitro for therapy or diagnosis. The discovery of molecules with such low EC 50 &#39;s and IC 50 &#39;s enables the design and synthesis of additional molecules having similar potency and effectiveness. In addition, the molecule may have an EC 50  or IC 50  less than or equal to 100 μM at one or more, but not all cells chosen from the group consisting of parathyroid cell, bone osteoclast, juxtaglomerular kidney cell, proximal tubule kidney cell, distal tubule kidney cell, cell of the thick ascending limb of Henle&#39;s loop and/or collecting duct, central nervous system cell, keratinocyte in the epidermis, parafollicular cell in the thyroid.(C-cell), intestinal cell, trophoblast in the placenta, platelet, vascular smooth muscle cell, cardiac atrial cell, gastrin-secreting cell, glucagon-secreting cell, kidney mesangial cell, mammary cell, beta cell, fat/adipose cell, immune cell and GI tract cell. 
     By “therapeutically effective amount” is meant an amount of a pharmaceutical composition having a therapeutically relevant effect. A therapeutically relevant effect relieves to some extent one or more symptoms of the disease or condition in the patient; or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or condition. Generally, a therapeutically effective amount is between about 1 nmole and 1 μmole of the molecule, depending on its EC 50  or IC 50  and on the age and size of the patient, and the disease associated with the patient. 
     In another aspect, the invention describes a polypeptide comprising a recombinant SIRP polypeptide or a unique fragment thereof. By “unique fragment,” is meant an amino acid sequence present in a full-length SIRP polypeptide that is not present in any other naturally occurring polypeptide. Preferably, such a sequence comprises 6 contiguous amino acids present in the full sequence. More preferably, such a sequence comprises 12 contiguous amino acids present in the full sequence. Even more preferably, such a sequence comprises 18 contiguous amino acids present in the full sequence. 
     By “recombinant SIRP polypeptide” is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g, present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature. 
     In another aspect, the invention describes a recombinant cell or tissue containing a purified nucleic acid coding for a SIRP polypeptide. In such cells, the nucleic acid may be under the control of its genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter. By “exogenous” it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the SIRP polypeptide. 
     In another aspect, the invention features a SIRP polypeptide binding agent able to bind to a SIRP polypeptide. The binding agent is preferably a purified antibody which recognizes an epitope present on a SIRP polypeptide. Other binding agents include molecules which bind to the SIRP polypeptide and analogous molecules which bind to a SIRP polypeptide. 
     By “purified” in reference to an antibody is meant that the antibody is distinct from naturally occurring antibody, such as in a purified form. Preferably, the antibody is provided as a homogeneous preparation by standard techniques. Uses of antibodies to the cloned polypeptide include those to be used as therapeutics, or as diagnostic tools. 
     In another aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having the full length amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 except that it lacks at least one of the domains selected from the group consisting of the extracellular Ig like domain, the transmembrane domain, and the SHP-2 binding domains. Such deletion mutants are useful in the design of assays for protein inhibitors. The nucleic acid molecules described above may be, for example, cDNA or genomic DNA and may be placed in a recombinant vector or expression vector. In such a vector, the nucleic acid preferably is operatively associated with the regulatory nucleotide sequence containing transcriptional and translational regulatory information that controls expression of the nucleotide sequence in a host cell. 
     Thus, the invention also provides a genetically engineered host cell containing any of the nucleotide sequences described herein and the nucleic acid preferably is operatively associated with the regulatory nucleotide sequence containing transcriptional and translational regulatory information that controls expression of the nucleotide sequence in a host cell. Such host cells may obviously be either prokaryotic or eukaryotic. 
     Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG. 1 shows the deduced amino acid sequences of SIRP4 (SEQ ID NO: 6) and SIRP 5 (SEQ ID NO: 5). Identical amino acids are boxed. The putative signal sequence and the transmembrane region are indicated by thin and thick overlines, respectively. Three Ig-like domains are indicated by stippled overlines. Potential tyrosine phosphorylation sites are shown in bold, the C-terminal proline rich region is shaded. The location of oligonucleotides flanking the Ex region is indicated by stars. 
     FIG. 2 (SEQ ID NO: 14, residues 32 to 128 of SEQ ID NO: 6, residues 31-137 of SEQ ID NO: 5, SEQ ID NOS 15-26, respectively, in order of appearance) shows the alignment of extracellular regions including the first Ig-like domain of 15 SIRP family members. Ex1 shows the amino acids encoded by the initial PCR fragment that was used for screening and GST-fusion protein construction. Ex2-11 are derived from PCR and cDNA sequences, Ex 12-13 from genomic isolates. Numbering is according to FIG.  1 . 
     FIG. 3 shows the alignment of amino acid sequences of human SIRP4 (SEQ ID NO: 6), mouse SIRP1 (SEQ ID NO: 5), human SIRPα1 (SEQ ID NO: 7) and mouse SIRPβ1 (SEQ ID NO: 8). 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The present invention relates to SIRP polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. Those skilled in the art will recognize that many of the methods described below in relation to SIRP1, SIRP4, SHP-2, SHP-1 and Grb2 could also be utilized with respect to the other members of this group. 
     Various other features and aspects of the invention are: Nucleic Acid Encoding A SIRP Polypeptide; A Nucleic Acid Probe for the Detection of SIRP; Probe Based Method And Kit For Detecting SIRP; DNA Constructs Comprising a SIRP Nucleic Acid Molecule and Cells Containing These Constructs; Purified SIRP Polypeptides; SIRP Antibody And Hybridoma; An Antibody Based Method And Kit For Detecting SIRP; Isolation of Compounds Which Interact With SIRP; Compositions; Disruption of Protein Complexes; Antibodies to Complexes; Pharmaceutical Formulations and Modes of Administration; Identification of Agents; Purification and Production of Complexes; Derivatives of Complexes; and Evaluation of Disorders. 
     All of these aspects and features are explained in detail with respect to another protein involved with signal transduction, PYK-2, in PCT publication WO 96/18738, which is incorporated herein by reference in its entirety, including any drawings. Those skilled in the art will readily appreciate that such description can be easily adapted to SIRP as well, and is equally applicable to the present invention. 
     For example, as disclosed in WO 96/18738, the nucleic acid molecules of the present invention may be cloned into a variety of vectors including those derived from plasmids, bacteriophage and viruses. Additionally, the nucleic acid molecules of the present invention may, as necessary, have restriction endonuclease recognition sites added to their 5′- end and/or 3′-ends. Examples of suitable plasmid vectors may include pBR322, pUC 118, pUC 119 and the like; suitable phage or bacteriophage vectors may include λgt10, λgt11, and the like; and suitable virus vectors may include pMAM-neo, pKRC and the like. 
     To express the nucleic acids of the present invention in a prokaryotic cell, it is necessary to operably link the nucleic acid molecules of the invention to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the β-lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pBR325, and the like. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (PL and PR), the trp, recA lacZ, LacI, and gal promoters of  E. coli , the α-amylase (Uhmanen et al., J. Bacteriol. 162:176-182′(1985)) and the ζ-28-specific promoters of  B. subtilis  (Gilman et al., Gene sequence 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)). 
     Preferred prokaryotic vectors include plasmids such as those capable of replication in  E. coli  such as, for example, pBR322, ColE1, pSC101, pACYC 184, πVX. Such plasmids are, for example, disclosed by Sambrook (cf. Molecular A cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, &amp; Maniatis, Cold Spring Harbor Laboratory, (1989)). Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include plJ101 (Kendall et al., J. Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriiophages such as φC31 (Chater et al., In: Sixth International Symposium on Actinomycetales. Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John et al., (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki (J. Bacteriol. 33:729-742 (1978)). 
     Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-micron circle, and the like, or their derivatives. Full-length cDNA sequences can be subcloned into the following mammalian expression vectors: pLSV; downstream the SV40 early promoter, PLXSN-retroviral vector; downstream the Mo-MuLV long terminal repeat; pRK5; downstream the CMV promoter. 
     Expression of the nucleic acid molecules of the present invention in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gene. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight,  Cell  31:355-365 (1982)); the SV40 early promoter (Benoist et al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)). 
     Host cells which may be used in the expression systems of the present invention are not strictly limited, provided that they are suitable for use in the expression of the SIRP peptide of interest. Suitable hosts may often include eukaryotic cells. Preferred eukaryotic hosts include for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of the fibroblastorigin such as a VERO or CHO-K1, or cells of lymphoid origin and their derivatives. Preferred mammalian host cells include PC12 cells, NIH3T3, SP2/O and J5581, as well as neuroblastoma cell lines such as IMR 332 which may provide better capacities for correct post-translational processing. 
     EXAMPLES 
     The examples below are non-limiting and are merely representative of various aspects and features of the procedures used to identify the full-length nucleic acid and amino acid sequences of a series of SIRP proteins. Experiments demonstrating SIRP expression, interaction and signaling activities are also provided. 
     Material and Methods 
     Cell Culture and Transient Expression 
     MM5/C1, Rat1-IR, A431 or human fibroblast cells were grown until confluency, starved for 18 hours in serum-free medium, and either left untreated or were POV—(1 mM sodium orthovanadate, 3 mM H 2 O 2 ), insulin—(100 nM), EGF—(1 nM), or PDGF—(100 pM) stimulated for different time intervals as indicated. SIRP4, SHP-2 (Vogel, et al .,  Science  259:1611-1614 (1994)) or SHP-2C463 A mutant (Stein-Gerlach, et al.  J. Biol. Chem . 270:24635-24637 (1995)) cDNAs were transiently cotransfected in BHK-IR, BHK-EGFR or BHK-βPDGFR cells using the calcium precipitation method (Chen, et al.  Mol, Cell. Biol . 7:2745-2752 (1987)). After stimulation, cells were lysed in buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol,. 1 mM POV, 1 mM EDTA, 1 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin. 
     Immunoprecipitation and Western Blotting 
     SHP-2 immunoprecipitations were performed with polyclonal anti-SHP-2 antibodies (Vogel, et al.,  Science  259:1611-1614 (1994)). Overexpressed SIRP4 or endogenous SIRP4-like proteins were immunoprecipitated by polyclonal anti-Ex1 antibodies raised by immunizing rabbits with a GST-fusion protein containing the Ex1 fragment (FIG.  2 ). Western blots were labeled with monoclonal anti-phosphotyrosine antibodies 5E2 (Fendly, et al.,  Cancer Res . 50:1550-1558 (1990)), and after stripping, reprobed with monoclonal anti-SHP-2 antibodies (Transduction Laboratories), or polyclonal anti-SIRP4-CT antibodies, raised against a GST-fusion protein containing the C-terminal part of SIRP4 (amino acids 336-503). For immunolabeling goat anti-mouse or -rabbit horseradish peroxidase conjugates (Bio-Rad) and the ECL detection system (Amersham) were used. 
     To obtain 293 cells stably expressing SIRP4 (293/SIRP4), cells were transfected with SIRP4 cDNA in PLXSN (Miller, et al.  Biotechniaues  7:980-988 (1989)) using the calcium precipitation method, followed by selection with G418 (1 mg/ml). SIRP4 was immunoprecipitated from quiescent or POV-stimulated (1 mM) 293/SIRP4 cells with polyclonal anti-Ex1 antibodies. Subsequently, crude lysates of [ 35 S]-methionine labeled 293 cells expressing different SH2 domain containing proteins were added to the affinity matrix and incubated for 2 h at 4° C. The immunocomplexes were washed, separated by SDS-PAGE and analyzed by autoradiography. 
     Enzymatic Deglycosylation 
     To perform in vitro deglycosylation SHP-2 immunocomplexes or the 110 kDa protein preparation were first denatured in the presence of 1% SDS at 100° C. for 5 min. Deglycosylation was done in potassium phosphate buffer (40 mM, pH 7.0), containing 20 mM EDTA, 1% b-mercaptoethanol, 1% Triton X-100 and 0.5 Unit of Endoglycosidase F/N-Glycosidase F (Boehringer Mannheim) at 37° C. for 16 hours. 
     Protein Purification 
     Approximately 10 10  Rat1-IR cells were used to purify the 110 kDa protein. Starved Rat1-IR cells were insulin-stimulated (100 nM) for 10 min, washed briefly with ice-cold hypotonic buffer containing 20 mM HEPES, pH 7.5, 1 mM POV, 1 mM EDTA, 1 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin, scraped into the same buffer and homogenized. Obtained cell extracts were pelleted at 1000 rpm for 15 min, and supeinatants were spun at 48.000 g for 1 hour. Membranes were solubilized in lysis buffer as described above. hIR was depleted from membrane extracts using an affinity column with monoclonal anti-hIR antibody 83-14 (Redemann et al.,  Mol. Cell. Biol . 12:491-498 (1992)), covalently coupled to Protein A-Sepharose beads (Pharmacia). Depleted extracts were applied onto a WGA-agarose 6 MB column (Sigma), and glycoproteins were eluted with 0.3 M N-acetyl-glucosamine in HNTG (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 1 mM POV). After concentration protein extracts were applied onto an anti, phosphotyrosine antibody column (Sigma). Bound proteins were eluted with 20 mM phosphotyrosine in HNTG. The eluate was subjected to SDS-PAGE, proteins were transferred to a PVDF membrane (Millipore) and stained with Coomassie blue. The protein of 10 kD apparent molecular weight was microsequenced. The following five tryptic peptides were obtained: PIYSFIGGEHFPR (SEQ ID NO: 9), IVEPDTEIK (SEQ ID NO: 10), YGFSPR (SEQ ID NO: 11), IKEVAHVNLEVR (SEQ ID NO: 12), VAAGDSAT (SEQ ID NO: 13). 
     Biological Assays 
     To produce retroviruses expressing pLXSN, wild type SIRP4 and mutated SIRP4 constructs, BOSC 23 cells were transiently transfected by expression plasmids as described (Pear, et al.  Proc. Natl. Acad. Sci . 90:8392-8396 (1993)). To obtain NIH3T3 cells stably expressing wild type SIRP4, SIRP4-4Y or SIRP4-DCT mutants subconfluent NIH3T3 cells (10 5  cells per 6 cm dish) were incubated with supernatants of transfected BOSC 23 cells for 4 h in the presence of Polybrene (4 mg/ml), followed by selection with G418 (1 mg/ml). 
     To perform focus formation assays cell lines 3T3/pLXSN, 3T3/SIRP4, 3T3/SIRP4-4Y or 3T3/SIRP4-DCT were superinfected for 4 hours with equal volumes of v-fms-virus supernatant (10 5  cells/6 cm dish). Cells were cultivated for 14 days in 4% FCS with medium change every second day. Cell foci were stained with Crystal violet (0.1% crystal violet, 30% methanol). 
     Example 1 
     Identification and Cloning of Signal Regulatory Proteins 
     Western blot of mammalian cells with anti-phosphotyrosine antibodies and anti-SHP-2 antibodies was used to identify tyrosine phosphorylated SHP-2 associated proteins. 
     Western blots containing anti-SHP-2 immunoprecipitates from starved or POV-treated mouse MM5/C1 mammary carcinoma, rat fibroblast Rat1-IR or human epidermal carcinoma A431 cells were incubated with anti-phosphotyrosine antibodies or anti-SHP-2 antibodies. Samples were deglycosylated with or treated without Endoglycosidase F/N-Glycosidase F (Endo.F/F). As a control, insulin-stimulated Rat1-IR cell lysates were immunoprecipitated with preimmune rabbit serum (aNS). 
     Samples from each purification step (i.e., solubilized crude membrane extract, hIR-depleted extracts, concentrated eluate from WGA-agarose beads, and eluate from anti-phosphotyrosine antibody column) were analyzed by 10% SDS-PAGE and visualized by silver staining and in Western blots using monoclonal anti-phosphotyrosine antibodies. 
     A major tyrosine phosphorylated protein was revealed in analysis of anti-SHP-2 immunoprecipitates from both pervanadate (POV) and growth factor stimulated cells. This phosphoprotein migrated at 120 kDa, 110 kDa and 90 kDa positions in mouse mammary tumor (MM5/C1) cells, Rat1 cells overexpressing the human insulin receptor (Rat1-IR), and human epidermoid carcinoma (A431) cells, respectively. 
     Upon in vitro deglycosylation, this glycoprotein was reduced to 65 kDa apparent molecular weight (MW) in all cases. This indicated that the same SHP-2 binding protein of 65 kDa was differentially glycosylated in a species specific manner. 
     In some cell lines such as A431, other tyrosine phosphorylated proteins in the 90-120 kDa range remained unaffected by the deglycosylation treatment. These proteins may represent Gab1 and/or the human homologue of the Drosophila DOS protein. 
     Insulin treated Rat1-IR were used to purify the 110 kDa SHP-2 binding glycoprotein using standard chromatography procedures. Approximately 4 mg of the glycoprotein that copurified with SHP-2 were obtained and subject to microsequence analysis. This yielded five peptide sequences: PIYSFIGGEHFPR (SEQ ID NO: 9), IVEPDTEIK (SEQ ID NO: 10), YGFSPR (SEQ ID NO: 11), IKEVAHVNLEVR (SEQ ID NO: 12), VAAGDSAT (SEQ ID NO: 13). Computer aided search in the EST database led to the identification of a 305 bp rat sequence (accession Nr.: H31804) and subsequent human cDNA fragment of 2 kb (EMBL databank, accession Nr.: U6701) containing matching and homologous sequences, respectively. 
     Specific primers flanking the very 5′ portion of this sequence were used to amplify a 360 bp human DNA fragment (encoding Ex1 in FIG. 2) which was used to screen a human placenta cDNA library. 
     Several positive clones were isolated. One clone of 2.4 kb encoded a polypeptide of 503 amino acids designated SIRP4 (for signal Regulating Protein 4) with a calculated mass of 57,000. The deduced sequence identifies SIRP4 as a transmembrane protein with three Ig-like domains and a cytoplasmic portion containing four potential tyrosine phosphorylation sites and one proline-rich region. 
     A second cDNA clone, SIRP1, is also identified. This protein is highly homologous to SIRP4 within the Ig-like domains (Ig-1: 83%; Ig-2: 88%; Ig-3: 83%), but displays striking sequence divergence at the amino terminus and upstream of the transmembrane domain which gives rise to a shorter protein that still contains a transmembrane-like region but lacks the cytoplasmic C-terminal portion. 
     SIRP4 and SIRP1 are members of a novel protein family. This protein family has a variety of distinct sequence isoforms as evidenced by comparison of fifteen cDNA and genomic sequences within the first Ig-like domain (FIG.  2 ). Two major classes exist in SIRP family distinguished by the presence or absence of a cytoplasmic SHP-2 binding domain. 
     Example 2 
     Analyzing the Functions of SIRP4 
     SIRP4 binds to SHP-2 and serves as a substrate for SHP-2, IR, EGFR, and βPDGFR 
     The identity of SIRP4 as SHP-2 binding protein and substrate was confirmed by expression of the SIRP4 cDNA either alone or in combination with SHP-2 or an enzymatically inactive mutant SHP-2C463 A in BHK cells. BHK cells stably express human EGF-, insulin- or βPDGF receptors. 
     Immunoprecipitations were performed with a polyclonal antibody raised against a GST-fusion protein containing the extracellular Ex1 region (FIG.  2 ). 
     Western blots containing anti-SIRP4 immunoprecipitations from quiescent or ligand-stimulated BHK-IR, BHK-EGFR or BHK-5 PDGF cells were labeled with anti-phosphotyrosine, anti-SHP-2 and anti-SIRP4 antibodies, respectively. 
     Anti-SIRP4 immunoprecipitation revealed a tyrosine phosphorylated protein of 85-90 kDa upon ligand stimulation which associated with SHP-2. 
     The results suggested SIRP4 to be a direct substrate of SHP-2 since expression of the SHP-2 mutant SHP-2C463 A led to a significant increase in its phosphotyrosine content (even in starved cells) while coexpression of wt SHP-2 resulted in dephosphorylation. The MW of overexpressed SIRP4 matches that of the endogenous protein detected in SHP-2 immunoprecipitates from A431 cells. 
     Endogenous SIRP4-like proteins were immunoprecipitated from untreated or EGF-stimulated A431 cells, from quiescent or PDGF-treated human fibroblasts, or from starved or insulin-stimulated HBL-100 cells. As a control, ligand-stimulated cell lysates were immunoprecipitated with preimmune rabbit serum (aNS). Immunoblots were probed with monoclonal anti-phosphotyrosine and monoclonal anti-SHP-2 antibodies. 
     Polyclonal anti-Ex1 antibodies immunoprecipitate a protein of 85-90 kDa apparent MW from A431, HBL-100 tumor cells and human fibroblasts. This protein was tyrosine phosphorylated upon EGF, insulin or PDGF stimulation, respectively, and coprecipitated with SHP-2 in a ligand dependent manner. 
     These data indicate the existence of SIRP4 in several human cell lines where SIRP4 serves as a substrate for insulin-, EGF- and βPDGF receptors, binds SHP-2 in its tyrosine phosphorylated form and serves as a substrate for the phosphatase activity of SHP-2. The interaction of SHP-2 with SIRP4 likely involves one or both SH2 domains of SHP-2 as suggested by the requirement of phosphotyrosine residues and the abrogation of detectable association by mutation of critical residues in SHP-2 SH2 domains. 
     In vitro binding assays were performed to determine whether SIRP4 is able to interact with other SH2 domain-containing proteins. SIRP4-associated [ 35 S]-Methionine labeled proteins were resolved on SDS-PAGE and detected by autoradiography. The result shows that SIRP4 associates with both SHP-1 and Grb2 but not p85, Shc, Grb7, PLC-g, c-src, Nck, Vav, GAP, or ISGF-3. 
     A catalytically inactive SHP-1 mutant has recently been shown to bind an as yet unidentified tyrosine phosphorylated protein of 90-95 kDa in human 293 cells. This tyrosine phosphorylated protein is likely to be SIRP4 or one of its family members. 
     Effects of SIRP4 on Cell Growth and Transformation 
     To investigate the biological function of SIRP4, three stable transfectants of NIH3T3 cells were constructed to express wild type SIRP4 or SIRP4 mutants carrying either point mutations of the putative SHP-2 tyrosine binding sites (SIRP4-4Y) or a deletion of most of the cytoplasmic region (SIRP4-DCT). 
     Ligand-stimulated [ 3 H]-thymidine incorporation of NIH3T3 cells expressing empty vector (3T3/pLXSN), wild type SIRP4 (3T3/SIRP4), SIRP4-4Y (3T3/SIRP4-4Y) or SIRP4-DCT (3T3/SIRP4-DCT, amino acids 402-503 are deleted) mutants. Cells were grown to confluence in 24-well dishes (Nunc), starved for 24 h in DMEM/0.5% FCS, stimulated with different concentrations of insulin or EGF for 18 h, then incubated with 0.5 mCi [ 3 H]-thymidine per well for 4 h. Incorporation into DNA was determined as described (Redemann, et al.  Mol. Cell. Biol . 12:491-498 (1992)). 
     Upon stimulation of cells with insulin, EGF and PDGF, control cells showed growth factor-induced DNA synthesis as measured by [ 3 H]-thymidine incorporation. Overexpression of SIRP4 led to a decrease of [ 3 H]-thymidine incorporation. In contrast, both SIRP4 mutants had nearly no effect on DNA synthesis. The observed inhibitory effect on DNA synthesis must be connected to SIRP4 tyrosine phosphorylation and/or its association with SHP-2 since wt SIRP4 became tyrosine phosphorylated and bound to SHP-2 upon ligand stimulation, and SIRP4 mutants did not. 
     SIRP4 effected growth inhibition upon insulin or EGF stimulation is correlated with reduced MAP kinase activation in 3T3/SIRP4 cells. 3T3/pLXSN, 3T3/SIRP4 or 3T3/SIRP4-4Y cells were starved for 18 hours in DMEM/0.50 FCS and stimulated with insulin or EGF for the time indicated. MAP kinase was detected in Western blots by using polyclonal erk1 and erk2 antibodies (Santa Cruz). In contrast, expression of SIRP4 mutants defective in SHP-2 binding had no effect on MAP kinase activation. Similar observations were made upon stimulation of the cells with PDGF. 
     These data strongly indicate that SIRP4 represents a novel regulatory element in the pathway that leads to MAP kinase activation. 
     We next determined the consequence of SIRP4 overexpression on oncogene mediated transformation of NIH3T3 cells. To examine the ability of SIRP4 to influence the formation of cell foci, subconfluent 3T3/pLXSN, 3T3/SIRP4, 3T3/SIRP4-4Y or 3T3/SIRP4-DCT cells were infected with v-fms virus supernatants. 
     As measured by focus formation, transformation by a v-fms retrovirus was significantly suppressed in cells overexpressing wt SIRP4 but not in cells expressing mutant SIRP4. 
     Previous reports have described certain SHP-2 binding proteins of 110-130 kDa apparent MW in mouse, rat or hamster cells. Tyrosine hyperphosphorylation of these proteins was observed when an enzymatically inactive SHP-2 mutant was overexpressed. In addition, disruption of SHP-2 function induced a variety of negative effects on growth factor-induced cellular signals. Our experiments strongly indicate that these proteins belong to the SIRP family and that the biological effects previously observed are due to the function of these SIRP proteins. 
     Without being bound by any theory, applicant proposes that tyrosine docking sites on SIRP proteins for either SHP-2 and/or other SH2 proteins such as SHP-1 or Grb2 play a significant role since the inhibitory effect of SIRP4 on NIH3T3 cell proliferation and transformation depends on phosphorylation of tyrosines. 
     One or both of the SHP phosphatases may tightly regulate the SIRP4 phosphorylation state. 
     SIRP4 may also act in its phosphorylated state as a “trapping” protein that sequesters SHP-2 from activated RTKs. The sequestion makes SHP-2 unavailable for other positive regulatory functions such as an adapter which recruits the Grb2-SOS complex to activated receptors. Such a function is supported by the observation that SHP-2 has higher affinity to the tyrosine phosphorylated form of SIRP4 than to autophosphorylated insulin and EGF receptors (Yamauchi, et al.,  J. Biol. Chem . 270:17716-17722, Yamauchi, et al.  J. Biol. Chem . 270:14871-14874 (1995)). 
     A third possibility is based on the membrane-spanning structural features of the SIRP4 variant. The high degree of sequence diversity within the Ig-domains is reminiscent of immunoglobulin variable regions and suggests a role of extracellular determinants in the SIRP related signal transduction. Structurally defined interaction of SIRP with specific receptors, soluble ligands, extracellular matrix components or other factors may result in specific regulatory consequences for intracellular signaling events. 
     All publications referenced are incorporated by reference herein, including the nucleotide sequences, amino acid sequences, drawings and tables in each publication. All the compounds disclosed and referred to in the publications mentioned above are incorporated by reference herein, including those compounds disclosed and referred to in articles cited by the publications mentioned above. 
     Other embodiments of this invention are disclosed in the following claims. As will be obvious to those skilled in the art, may variations and modifications may be made without departing from the spirit and scope of the invention. 
     
       
         
           
             26 
           
           
             1 
             3804 
             DNA 
             Homo sapiens 
           
            1
cacagacgtt tggacagagc aggctcctaa ggtctccaga atgcccgtgc cagcctcctg     60
gccccacctt cctagtcctt tcctgctgat gacgctactg ctggggagac tcacaggagt    120
ggcaggtgag gacgagctac aggtgattca gcctgaaaag tccgtatcag ttgcagctgg    180
agagtcggcc actctgcgct gtgctatgac gtccctgatc cctgtggggc ccatcatgtg    240
gtttagagga gctggagcag gccgggaatt aatctacaat cagaaagaag gccacttccc    300
acgggtaaca actgtttcag aactcacaaa gagaaacaac ctgaactttt ccatcagcat    360
cagtaacatc accccagcag acgccggcac ctactactgt gtgaagttcc ggaaagggag    420
ccctgacgac gtggagttta agtctggagc aggcactgag ctgtctgtgc gcgccaaacc    480
ctctgccccc gtggtatcgg gccctgcggt gagggccaca cctgagcaca cagtgagctt    540
cacctgcgag tcccatggct tctctcccag agacatcacc ctgaaatggt tcaaaaatgg    600
gaatgagctc tcagacttcc agaccaacgt ggaccccgca ggagacagtg tgtcctacag    660
catccacagc acagccaggg tggtgctgac ccgtggggac gttcactctc aagtcatctg    720
cgagatggcc cacatcacct tgcaggggga ccctcttcgt gggactgcca acttgtctga    780
ggccatccga gttccaccca ccttggaggt tactcaacag cccatgaggg cagagaacca    840
ggcaaacgtc acctgccagg tgagcaattt ctacccccgg ggactacagc tgacctggtt    900
ggagaatgga aatgtgtccc ggacagaaac agcttcgacc ctcatagaga acaaggatgg    960
cacctacaac tggatgagct ggctcctggt gaacacctgt gcccacaggg acgatgtggt   1020
gctcacctgt caggtggagc atgatgggca gcaagcagtc agcaaaagct atgccctgga   1080
gatctcagca caccagaagg agcacggctc agatatcacc catgaaccag cgctggctcc   1140
tactgctcca ctcctcgtag ctctcctcct gggccccaag ctgctactgg tggttggtgt   1200
ctctgccatc tacatctgct ggaaacagaa ggcctgactg accctcagtc tctgctgcct   1260
cctcctttct tgagaagctc agcctgagag aaggagctgg cgagaacctt ccccacactc   1320
agctccaaac gcctcctctc ccaggtcatc tgcctgccca cacgctcctg ttccaccttc   1380
acaagaccat gatgccccaa agcagtgtct ctattcacgg tcctgagcag gggccatggg   1440
attgggctct gggcactgac tcatggcacc tccctagaag gtgagaaaca ctccaaatct   1500
aaacacacca ggacttctcc catccgtcgc cttgggactg gccataaacc acagactctc   1560
tccaggctct caagagttat cctgtcttct ggattcctgc ctaccccaac tcccccagcc   1620
ttgttgaggt tctctactgc ctcctgaata cacatgaacc cctataccaa ttttaagaaa   1680
aaaatgattc tctttcctct ttgtccaagc atcctatccc tcaaacccaa aaagaaagaa   1740
gctctccctt ctctctctgt gatggagaca gtatttcttc tagtatcctg cagccttccc   1800
agtcctgctg cttgtggtag aaattgctgc cacagcccaa cattgaggag ccctcgatga   1860
ctgcccttta caactcatat tcagttctgc ctccaaaatg catgtgtcca cttacatgag   1920
atggtaaatg tttaacaatg gactttctga aagggaaaaa ccaaaagctg ttttgcagtg   1980
cttgccaatt tctctagtgt aataactccc aacctgacca atttcagcac tgccaacagt   2040
taaacaacca gattcgaaga ttcctgaaat ttaacaattg gttttcaggg cccagtccaa   2100
gcctgctgct ggaaacctca gagttaaatc cctattctcc acacctctca cctccaccac   2160
ccctccctgt cccagccagc atcatctctt tggggaccac tcctctggct ttcatttttc   2220
agccacagtg attctttgga aaagtcaaat catatcactt ctctgcttct tccccaacac   2280
agctgcatgg tcccgctctc cctccttcaa gtctctgctc aatgtcactt cattaaaggc   2340
ggccttctat aaactacctt gtataaaata ttatttattt tctctatccc ggcattctaa   2400
tttctcttat cctaattaat ttttctttag cccttatttt gatgagtatt atgccgaata   2460
caggcagccc tcacttttca tggccagtgc aagattgcaa aaagactgtg caacctgaaa   2520
cccaggaaag cagtctccat agtcaatcag aaaaacaatg atcattctgt gacctttacc   2580
attttttgtc aaaatattag aaactctcac actctcagtt acaaatgtag aggacaatga   2640
aaatataatg aaataaatat ttatttgtgc actacaattc aaagcattag aaacattgaa   2700
gtcaatggcg tttcttgtaa atgtatccag atgaggttgg aagagtgctt gacctttttg   2760
tatatttcta atatggagtg atatagtttg gctctgtgtc tccatccaaa tctcatctta   2820
aattgtaatc tgcatgtgtt gtgggaatgg gacctaggta ggaggtgact gaatacatgg   2880
gggcggactt cccccttgct gttcttgtga tagtgagttc tcataagatc tcagtgagtt   2940
ctcatgagat ctggtttttt gaaagtgtgt ggcaagtccc ccttcgctct ctctctctct   3000
ctccctcctg ccaccatgtg aagaaggtgc ctgcttcctt ttctccttcc accatggttg   3060
taagtttcct gaggcctccc agtcatgctt cctgttaagc ctgtggaact gtgagtccaa   3120
ttaaacctct tttattcata aaatatccag tttctggtag ttctttatag cagtgtgaga   3180
atgggctaat acacggagca agcatcgttc tttcattttt atttatttta ttttttgaga   3240
tggagtttca ccttattccc aggctggagt gcaatgtcgt gatcttggct cactgcaacc   3300
cccgcctcca gggttcaagt gattctcctg cctcagcctc ctgagtagct gggattacag   3360
gcatgtacca ccacacccag ctaattttgt atttttagta gagatggggt ttctccatgt   3420
tgatcagact agtcttgaac tcccgacctc aggtgatcca cctgtcttgg cctcccaaag   3480
tgctgggatt acaggcatga gccaccatgc ctagccagca agcatcattt ctattatacc   3540
ttggtgtttg cctctttcta agtttggact agcttccaac atcttatccc ttgaattttc   3600
aatattgtgg aatcactcca gaagatcctt tcatgtgaag ttttttgctg gcatttcaac   3660
ctttgggaca tcttcagccc ttttattacc actcctctcc catttgtggc agtttgcgtt   3720
tactacctcc ctctggctgc ctatctgaag ttcctgcatc agggtctaca ttgccacagt   3780
caactatttg tacttctaga attc                                          3804
 
           
             2 
             2433 
             DNA 
             Homo sapiens 
           
            2
cagccgcggc ccatggagcc cgccggcccg gcccccggcc gcctcgggcc gctgctctgc     60
ctgctgctcg ccgcgtcctg cgcctggtca ggagtggcgg gtgaggagga gctgcaggtg    120
attcagcctg acaagtccgt atcagttgca gctggagagt cggccattct gcactgcact    180
gtgacctccc tgatccctgt ggggcccatc cagtggttca gaggagctgg accagcccgg    240
gaattaatct acaatcaaaa agaaggccac ttcccccggg taacaactgt ttcagagtcc    300
acaaagagag aaaacatgga cttttccatc agcatcagta acatcacccc agcagatgcc    360
ggcacctact actgtgtgaa gttccggaaa gggagccctg acacggagtt taagtctgga    420
gcaggcactg agctgtctgt gcgtgccaaa ccctctgccc ccgtggtatc gggccctgcg    480
gcgagggcca cacctcagca cacagtgagc ttcacctgcg agtcccacgg cttctcaccc    540
agagacatca ccctgaaatg gttcaaaaat gggaatgagc tctcagactt ccagaccaac    600
gtggaccccg taggagagag cgtgtcctac agcatccaca gcacagccaa ggtggtgctg    660
acccgcgagg acgttcactc tcaagtcatc tgcgaggtgg cccacgtcac cttgcagggg    720
gaccctcttc gtgggactgc caacttgtct gagaccatcc gagttccacc caccttggag    780
gttactcaac agcccgtgag ggcagagaac caggtgaatg tcacctgcca ggtgaggaag    840
ttctaccccc agagactaca gctgacctgg ttggagaatg gaaacgtgtc ccggacagaa    900
acggcctcaa ccgttacaga gaacaaggat ggtacctaca actggatgag ctggctcctg    960
gtgaatgtat ctgcccacag ggatgatgtg aagctcacct gccaggtgga gcatgacggg   1020
cagccagcgg tcagcaaaag ccatgacctg aaggtctcag cccacccgaa ggagcagggc   1080
tcaaataccg ccgctgagaa cactggatct aatgaacgga acatctatat tgtggtgggt   1140
gtggtgtgca ccttgctggt ggccctactg atggcggccc tctacctcgt ccgaatcaga   1200
cagaagaaag cccagggctc cacttcttct acaaggttgc atgagcccga gaagaatgcc   1260
agagaaataa cacaggacac aaatgatatc acatatgcag acctgaacct gcccaagggg   1320
aagaagcctg ctccccaggc tgcggagccc aacaaccaca cggagtatgc cagcattcag   1380
accagcccgc agcccgcgtc ggaggacacc ctcacctatg ctgacctgga catggtccac   1440
ctcaaccgga cccccaagca gccggccccc aagcctgagc cgtccttctc agagtacgcc   1500
agcgtccagg tcccgaggaa gtgaatggga ccgtggtttg ctctagcacc catctctacg   1560
cgctttcttg tcccacaggg agccgccgtg atgagcacag ccaacccagt tcccggaggg   1620
ctggggcggt gcaggctctg ggacccaggg gccagggtgg ctcttctctc cccacccctc   1680
cttggctctc cagcacttcc tgggcagcca cggccccctc ccccaacatt gccacacacc   1740
tggaggctga cgttgccaaa ccagccaggg aaccaacctg ggaagtggcc agaactgcct   1800
ggggtccaag aactcttgtg cctccgtcca tcaccatgtg ggttttgaag accctcgact   1860
gcctccccga tgctccgaag cctgatcttc cagggtgggg aggagaaaat cccacctccc   1920
ctgacctcca ccacctccac caccaccacc accaccacca ccaccactac caccaccacc   1980
caactggggc tagagtgggg aagatttccc ctttagatca aactgcccct tccatggaaa   2040
agctggaaaa aaactctgga acccatatcc aggcttggtg aggttgctgc caacagtcct   2100
ggcctccccc atccctaggc aaagagccat gagtcctgga ggaggagagg acccctccca   2160
aaggactgga agcaaaaccc tctgcttcct tgggtccctc caagactccc tggggcccaa   2220
ctgtgttgct ccacccggac ccatctctcc cttctagacc tgagcttgcc cctccagcta   2280
gcactaagca acatctcgct gtaagcgcct gtaaattact gtgaaatgtg aaacgtgcaa   2340
tcttgaaact gaggtgttag aaaacttgat ctgtggtgtt ttgttttgtt ttttttctta   2400
aaacaacagc aacgtgaaaa aaaaaaaaaa aaa                                2433
 
           
             3 
             3645 
             DNA 
             Mus sp. 
           
            3
gcccgcctgc cgagcgcgct caccgccgct ctccctcctt gctctgcagc cgcggcccat     60
ggagcccgcc ggcgcccctg gccgcctagg gccgctgctg ctctgcctgc tgctctccgc    120
gtcctgtttc tgtacaggag tcacggggaa agaactgaag gtgactcagc ctgagaaatc    180
agtgtctgtt gctgctgggg attcgaccgt tctgaactgc actttgacct ccttgttgcc    240
ggtgggaccc attaagtggt acagaggagt aggcaaagcc ggctgtttga tctacagttt    300
cacaggagaa cactttcctc gagttacaaa tgtttcagat gctactaaga gaaacaatat    360
ggacttttcc atccgtatca gtaatgtcac cccagaagat gccggtacct actactgtgt    420
gaagttccag aaaggaccat cagagcctga cacagaaata caatctggag ggggaacaga    480
ggtctatgta ctcgccaaac cttctccacc ggaggatccc cccaggagac aggggcatac    540
tgaccagaaa gtgaacttca cctgcaagtc tcatggcttc tctccccgga atatcaccct    600
gaagtggttc aaagatgggc aagaactcca ccccttggag accaccgtga accctagtgg    660
aaagaatgtc tcctacaaca tctccagcac agtcagggtg gtactaaact ccatggatgt    720
tcattctaag gtcatctgcg aggtagccca catcaccttg gatagaagcc ctcttcgtgg    780
gattgctaac ctgtctaact tcatccgagt ttcacccacc gtgaaggtca cccaacagtc    840
cccgacgtca atgaaccagg tgaacctcac ctgccgggat gagaggttct accccgagga    900
tctccagctg atctggctgg agaatggaaa cgtatcacgg aatgacacgc ccaagaatct    960
cacaaagaac acggatggga cctataatta cacaagcttg ttcctggtga actcatctgc   1020
tcatagagag gacgtggtgt tcacgtgcca ggtgaagcac gaccaacagc cagcgatcac   1080
ccgaaaccat accgtgctgg gacttgccca ctcgagtgat caagggagca tgcaaacctt   1140
ccctggtaat aatgctaccc acaactggaa tgtcttcatc ggtgtgggcg tggcgtgtgc   1200
tttgctcgta gtcctgctga tggctgctct ctacctcctc cggatcaaac agaagaaagc   1260
caaggggtca acatcttcca cacggttgca cgagcccgag aagaacgcca gggaaataac   1320
ccaggtacag tctttgatcc aggacacaaa tgacatcaac gacatcacat acgcagacct   1380
gaatctccca aagagaagga agcccgcacc cggctccctt gagttcctta acaaccacac   1440
agaatatgca agcattgaga caggcaaagt gcctaggcca gaggataccc tcacctatgc   1500
tgacctggac atggtccacc tcagccgggc acagccagcc cccaagcctg agccatcttt   1560
ctcagagtat gctagtgtcc aggtccagag gaagtgaatg gggctgtggt ctgtactagg   1620
ccccatcccc acaagttttc ttgtcctaca tggagtggcc atgacgagga catccagcca   1680
gccaatcctg tccccagaag gccaggtggc acgggtccta ggaccagggg taagggtggc   1740
ctttgtcttc cctccgtggc tcttcaacac ctcttgggca ccacgtcccc ttcttccgga   1800
ggctgggtct tgcagaacca gagggcgaac tggagaaatc tgcctggaat ccaagaagtg   1860
ttgtgcctcg gcccatcact cgtgggctcg gatcctggtc ttggcaaccc caggttgcgt   1920
ccttgatgtt ccagagcttg gtcttctgtg tggagaagag ctcaccatct ctacccaact   1980
tgagctttgg gaccagactc cctttagatc aaaccgcccc atctgtggaa gaactacacc   2040
agaagtcgac aagttttcag ccaacagtgt ctggcctccc cacctcccag gctgactagc   2100
ctggggagaa ggaaccctct cctcctagac cagcagagac tccctgggca tgttcagtgt   2160
ggccccacct cccttccagt cccagcttgc ttcctccagc tagcactaac tcagcagcat   2220
cgctctgtgg acgcctgtaa attattgaga aatgtgaact gtgcagtctt aaagctaagg   2280
tgttagaaaa tttgatttat gctgtttagt tgttgttggg tttcttttct ttttaatttc   2340
tttttctttt ttgatttttt ttctttccct taaaacaaca gcagccagca tcttggctct   2400
ttgtcatgtg ttgaatggtt gggtcttgtg aagtctgagg tctaacagtt tattgtcctg   2460
gaaggatttt cttacagcag aaacagattt ttttcaaatt cccagaatcc tgaggaccaa   2520
gaaggatccc tcagctgcta cttccagcac gcagcgtcac tgggacgaac caggccctgt   2580
tcttacaagg ccacatggcg ggcctttgcc tccatggcta ctgtggtaag tgcagccttg   2640
tctgacccaa tgctgaccta atgttggcca ttccacattg aggggacaag gtcagtgatg   2700
ccccccttgg ctcacaagca cttcagaggc atgcagagag aagggacact cgtccagctc   2760
tctgaggtaa tcagtgcaag gaggagtccg ttttttgcca gcaaacctca gcaggatcac   2820
actggaacag aacctggtca tacctgtgac aacacagctg tgagccaggg caaaccaccc   2880
actgtcactg gctcgagagt ctgggagagc tctgacccga caccctttaa actggatgcc   2940
ggggcctggc tgggcaatgc caagtggtta tggcaaccct gactatctgg tcttaacatg   3000
tagctcagga agtggaggcg ctaatgtccc caatccctgg ggattcctga ttccagctat   3060
tcatgtaagc agagccaacc tgcctatttc tgtagggtgc gactgggatg ttaggagcac   3120
agcaaggacc cagctctgta gggctggtga cctgatacct tctcataatg gcatctagaa   3180
gttaggctga gttgcctcac tggcccagca aaccagaact tgtctttggc cgggccatgt   3240
tcttgggctg tcttctaatt ccaaagggtt ggttggtaaa gctccacccc cttctcctct   3300
gcctaaagac ataacatgtg tatacacaca cgggtgtata gatgagttaa aagaatgtcc   3360
tcgctggcat cctaattttg tcttaagttt ttttggaggg agaaaggaac aaggcaaggg   3420
aagatgtgta gctttggctt taaccaggca gcctgggggc tcccaagcct atggaaccct   3480
ggtacaaaga agagaacaga agcgccctgt gaggagtggg atttgttttt ctgtagacca   3540
gatgagaagg aaacaggccc tgttttgtac atagttgcaa cttaaaattt ttggcttgca   3600
aaatattttt gtaataaaga tttctgggta acaataaaaa aaaaa                   3645
 
           
             4 
             2020 
             DNA 
             Mus sp. 
           
            4
ccctcactaa agggaacaaa agctggagct ccaccgcggt ggcggccgct ctagaactag     60
tggatccccc gggctgcagg caaccatgct tctcctagat gcctggaccc acattcctca    120
ctgtgtcctg ctgttgatcc tgcttctggg acttaaagga gcagctatga gagagctgaa    180
ggtgatccaa cctgttaaat cattttttgt tggtgctgga gggtcagcca ctctgaactg    240
cacagtgaca tctctcctcc ctgtggggcc catgaggtgg tacaggggta taggacaaag    300
tcgactcttg atatactcgt tcacaggaga aggcttcccc agaataacaa atacttcaga    360
tactacaaag agaaacaaca tggacttttc catccgtatc agtaatgtca ctcctgctga    420
ttcgggtacc tactactgtg tgaagttcca gagaggacca tcagactttt acactgagat    480
tcagtctgga ggtggcactg agttgtcagt acttgctaaa ccatcttcac ctatggtctc    540
cggtcctgca gccagagctg tccctcagca gacagtgacc tttacatgca gatcccatgg    600
attctttccg cggaacctca cgctgaagtg gttcaagaat ggagatgaga tctctcactt    660
ggaaacttct gtggaaccgg aagaaacaag tgtctcctat agagtttcca gcacagtcca    720
ggtggtgttg gaacctaggg atgtccgctc tcagatcatc tgtgaagtgg atcatgtcac    780
tttagatcga gcccctctca gagggattgc tcacatctct gagttcattc aagttccacc    840
caccctggag atccgccagc agccaacaat ggtttggaat gtgataaatg ttacctgcca    900
aatacagaag ttctatcctc caagttttca gttgacctgg ttagagaatg gaaatatatc    960
ccggagagaa gtacctttta cacttacagt aaacaaggat ggaacttaca actggatcag   1020
ctgtctcttg gtgaacatat ctgcccttga ggagaacatg gtagtgacat gccaggttga   1080
gcatgatgga caagcagaag tcattgaaac ccatactgtg gtggtcactg aacatcagag   1140
agtgaaaggt actgctacca agtctggtga ggtcttcacc ccacccttat gtctaaatgt   1200
aaattgggct ttatttttta tgtataaggt aacattcttg attattgtag cattatcctg   1260
acaactacaa agtaaaatgt taacgtcata tttcattccc aacttctcac acgtctcaca   1320
tatctttcca ctaatagatt aaatagttaa gaatggaagg tatcatcaaa ttccagtatc   1380
ttgccccttc cctgttttac ctaacatttg tgaacatcct tatgctcatg tgtttccttt   1440
accatatctt tactgactcc attacatttt agatatttcc taaatatagt gtcctaatgg   1500
agtgaaattt caacgggtca cctgacaacc tgtttgtaca cacacacaca cacacacaca   1560
cacacacaca cacacagcat atgatctgga ctaatgaaat aaaggaaaat caaatgtcca   1620
ttggagcact gctatcacta aggtataagg aaaacttgct agcaaagtat ttcttttcaa   1680
cttgttacga tgctagcagt tagtttgcat tagattggac ccatttatgt gaatatcttt   1740
ttccttctct taaaacaaca aaaaagatcc tcaactccag tgacttttga aaaactcatg   1800
ttccttggca tccctccttt gctgtgagtt cattggctgg ataaacactg ggtcgcctaa   1860
ttatctataa atatgccagt taaaaatgtc aaggttagaa agcatcagtc catacagtgc   1920
aaatatagtc cacagtgggt gctcaggtaa atcatgatat tttcatttaa aatatacatt   1980
caataaaatt aactgtagtt caaaaaaaaa aaaaaaaaaa                         2020
 
           
             5 
             398 
             PRT 
             Homo sapiens 
           
            5
Met Pro Val Pro Ala Ser Trp Pro His Leu Pro Ser Pro Phe Leu Leu
  1               5                  10                  15
Met Thr Leu Leu Leu Gly Arg Leu Thr Gly Val Ala Gly Glu Asp Glu
             20                  25                  30
Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala Gly Glu
         35                  40                  45
Ser Ala Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val Gly Pro
     50                  55                  60
Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile Tyr Asn
 65                  70                  75                  80
Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Leu Thr
                 85                  90                  95
Lys Arg Asn Asn Leu Asn Phe Ser Ile Ser Ile Ser Asn Ile Thr Pro
            100                 105                 110
Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser Pro
        115                 120                 125
Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg
    130                 135                 140
Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Val Arg Ala Thr
145                 150                 155                 160
Pro Glu His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro
                165                 170                 175
Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp
            180                 185                 190
Phe Gln Thr Asn Val Asp Pro Ala Gly Asp Ser Val Ser Tyr Ser Ile
        195                 200                 205
His Ser Thr Ala Arg Val Val Leu Thr Arg Gly Asp Val His Ser Gln
    210                 215                 220
Val Ile Cys Glu Met Ala His Ile Thr Leu Gln Gly Asp Pro Leu Arg
225                 230                 235                 240
Gly Thr Ala Asn Leu Ser Glu Ala Ile Arg Val Pro Pro Thr Leu Glu
                245                 250                 255
Val Thr Gln Gln Pro Met Arg Ala Glu Asn Gln Ala Asn Val Thr Cys
            260                 265                 270
Gln Val Ser Asn Phe Tyr Pro Arg Gly Leu Gln Leu Thr Trp Leu Glu
        275                 280                 285
Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Leu Ile Glu Asn
    290                 295                 300
Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Thr Cys
305                 310                 315                 320
Ala His Arg Asp Asp Val Val Leu Thr Cys Gln Val Glu His Asp Gly
                325                 330                 335
Gln Gln Ala Val Ser Lys Ser Tyr Ala Leu Glu Ile Ser Ala His Gln
            340                 345                 350
Lys Glu His Gly Ser Asp Ile Thr His Glu Pro Ala Leu Ala Pro Thr
        355                 360                 365
Ala Pro Leu Leu Val Ala Leu Leu Leu Gly Pro Lys Leu Leu Leu Val
    370                 375                 380
Val Gly Val Ser Ala Ile Tyr Ile Cys Trp Lys Gln Lys Ala
385                 390                 395
 
           
             6 
             503 
             PRT 
             Homo sapiens 
           
            6
Met Glu Pro Ala Gly Pro Ala Pro Gly Arg Leu Gly Pro Leu Leu Cys
  1               5                  10                  15
Leu Leu Leu Ala Ala Ser Cys Ala Trp Ser Gly Val Ala Gly Glu Glu
             20                  25                  30
Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala Gly
         35                  40                  45
Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val Gly
     50                  55                  60
Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile Tyr
 65                  70                  75                  80
Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu Ser
                 85                  90                  95
Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile Thr
            100                 105                 110
Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser
        115                 120                 125
Pro Asp Thr Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val Arg
    130                 135                 140
Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala Thr
145                 150                 155                 160
Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser Pro
                165                 170                 175
Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser Asp
            180                 185                 190
Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser Ile
        195                 200                 205
His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser Gln
    210                 215                 220
Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu Arg
225                 230                 235                 240
Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu Glu
                245                 250                 255
Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr Cys
            260                 265                 270
Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu Glu
        275                 280                 285
Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu Asn
    290                 295                 300
Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val Ser
305                 310                 315                 320
Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp Gly
                325                 330                 335
Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His Pro
            340                 345                 350
Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn Glu
        355                 360                 365
Arg Asn Ile Tyr Ile Val Val Gly Val Val Cys Thr Leu Leu Val Ala
    370                 375                 380
Leu Leu Met Ala Ala Leu Tyr Leu Val Arg Ile Arg Gln Lys Lys Ala
385                 390                 395                 400
Gln Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn Ala
                405                 410                 415
Arg Glu Ile Thr Gln Asp Thr Asn Asp Ile Thr Tyr Ala Asp Leu Asn
            420                 425                 430
Leu Pro Lys Gly Lys Lys Pro Ala Pro Gln Ala Ala Glu Pro Asn Asn
        435                 440                 445
His Thr Glu Tyr Ala Ser Ile Gln Thr Ser Pro Gln Pro Ala Ser Glu
    450                 455                 460
Asp Thr Leu Thr Tyr Ala Asp Leu Asp Met Val His Leu Asn Arg Thr
465                 470                 475                 480
Pro Lys Gln Pro Ala Pro Lys Pro Glu Pro Ser Phe Ser Glu Tyr Ala
                485                 490                 495
Ser Val Gln Val Pro Arg Lys
            500
 
           
             7 
             512 
             PRT 
             Mus sp. 
           
            7
Met Glu Pro Ala Gly Ala Pro Gly Arg Leu Gly Pro Leu Leu Leu Cys
  1               5                  10                  15
Leu Leu Leu Ser Ala Ser Cys Phe Cys Thr Gly Val Thr Gly Lys Glu
             20                  25                  30
Leu Lys Val Thr Gln Pro Glu Lys Ser Val Ser Val Ala Ala Gly Asp
         35                  40                  45
Ser Thr Val Leu Asn Cys Thr Leu Thr Ser Leu Leu Pro Val Gly Pro
     50                  55                  60
Ile Lys Trp Tyr Arg Gly Val Gly Lys Ala Gly Cys Leu Ile Tyr Ser
 65                  70                  75                  80
Phe Thr Gly Glu His Phe Pro Arg Val Thr Asn Val Ser Asp Ala Thr
                 85                  90                  95
Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn Val Thr Pro
            100                 105                 110
Glu Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Gln Lys Gly Pro Ser
        115                 120                 125
Glu Pro Asp Thr Glu Ile Gln Ser Gly Gly Gly Thr Glu Val Tyr Val
    130                 135                 140
Leu Ala Lys Pro Ser Pro Pro Glu Asp Pro Pro Arg Arg Gln Gly His
145                 150                 155                 160
Thr Asp Gln Lys Val Asn Phe Thr Cys Lys Ser His Gly Phe Ser Pro
                165                 170                 175
Arg Asn Ile Thr Leu Lys Trp Phe Lys Asp Gly Gln Glu Leu His Pro
            180                 185                 190
Leu Glu Thr Thr Val Asn Pro Ser Gly Lys Asn Val Ser Tyr Asn Ile
        195                 200                 205
Ser Ser Thr Val Arg Val Val Leu Asn Ser Met Asp Val His Ser Lys
    210                 215                 220
Val Ile Cys Glu Val Ala His Ile Thr Leu Asp Arg Ser Pro Leu Arg
225                 230                 235                 240
Gly Ile Ala Asn Leu Ser Asn Phe Ile Arg Val Ser Pro Thr Val Lys
                245                 250                 255
Val Thr Gln Gln Ser Pro Thr Ser Met Asn Gln Val Asn Leu Thr Cys
            260                 265                 270
Arg Asp Glu Arg Phe Tyr Pro Glu Asp Leu Gln Leu Ile Trp Leu Glu
        275                 280                 285
Asn Gly Asn Val Ser Arg Asn Asp Thr Pro Lys Asn Leu Thr Lys Asn
    290                 295                 300
Thr Asp Gly Thr Tyr Asn Tyr Thr Ser Leu Phe Leu Val Asn Ser Ser
305                 310                 315                 320
Ala His Arg Glu Asp Val Val Phe Thr Cys Gln Val Lys His Asp Gln
                325                 330                 335
Gln Pro Ala Ile Thr Arg Asn His Thr Val Leu Gly Leu Ala His Ser
            340                 345                 350
Ser Asp Gln Gly Ser Met Gln Thr Phe Pro Gly Asn Asn Ala Thr His
        355                 360                 365
Asn Trp Asn Val Phe Ile Gly Val Gly Val Ala Cys Ala Leu Leu Val
    370                 375                 380
Val Leu Leu Met Ala Ala Leu Tyr Leu Leu Arg Ile Lys Gln Lys Lys
385                 390                 395                 400
Ala Lys Gly Ser Thr Ser Ser Thr Arg Leu His Glu Pro Glu Lys Asn
                405                 410                 415
Ala Arg Glu Ile Thr Gln Val Gln Ser Leu Ile Gln Asp Thr Asn Asp
            420                 425                 430
Ile Asn Asp Ile Thr Tyr Ala Asp Leu Asn Leu Pro Lys Arg Arg Lys
        435                 440                 445
Pro Ala Pro Gly Ser Leu Glu Phe Leu Asn Asn His Thr Glu Tyr Ala
    450                 455                 460
Ser Ile Glu Thr Gly Lys Val Pro Arg Pro Glu Asp Thr Leu Thr Tyr
465                 470                 475                 480
Ala Asp Leu Asp Met Val His Leu Ser Arg Ala Gln Pro Ala Pro Lys
                485                 490                 495
Pro Glu Pro Ser Phe Ser Glu Tyr Ala Ser Val Gln Val Gln Arg Lys
            500                 505                 510
 
           
             8 
             391 
             PRT 
             Mus sp. 
           
            8
Met Leu Leu Leu Asp Ala Trp Thr His Ile Pro His Cys Val Leu Leu
  1               5                  10                  15
Leu Ile Leu Leu Leu Gly Leu Lys Gly Ala Ala Met Arg Glu Leu Lys
             20                  25                  30
Val Ile Gln Pro Val Lys Ser Phe Phe Val Gly Ala Gly Gly Ser Ala
         35                  40                  45
Thr Leu Asn Cys Thr Val Thr Ser Leu Leu Pro Val Gly Pro Met Arg
     50                  55                  60
Trp Tyr Arg Gly Ile Gly Gln Ser Arg Leu Leu Ile Tyr Ser Phe Thr
 65                  70                  75                  80
Gly Glu Gly Phe Pro Arg Ile Thr Asn Thr Ser Asp Thr Thr Lys Arg
                 85                  90                  95
Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Asn Val Thr Pro Ala Asp
            100                 105                 110
Ser Gly Thr Tyr Tyr Cys Val Lys Phe Gln Arg Gly Pro Ser Asp Phe
        115                 120                 125
Tyr Thr Glu Ile Gln Ser Gly Gly Gly Thr Glu Leu Ser Val Leu Ala
    130                 135                 140
Lys Pro Ser Ser Pro Met Val Ser Gly Pro Ala Ala Arg Ala Val Pro
145                 150                 155                 160
Gln Gln Thr Val Thr Phe Thr Cys Arg Ser His Gly Phe Phe Pro Arg
                165                 170                 175
Asn Leu Thr Leu Lys Trp Phe Lys Asn Gly Asp Glu Ile Ser His Leu
            180                 185                 190
Glu Thr Ser Val Glu Pro Glu Glu Thr Ser Val Ser Tyr Arg Val Ser
        195                 200                 205
Ser Thr Val Gln Val Val Leu Glu Pro Arg Asp Val Arg Ser Gln Ile
    210                 215                 220
Ile Cys Glu Val Asp His Val Thr Leu Asp Arg Ala Pro Leu Arg Gly
225                 230                 235                 240
Ile Ala His Ile Ser Glu Phe Ile Gln Val Pro Pro Thr Leu Glu Ile
                245                 250                 255
Arg Gln Gln Pro Thr Met Val Trp Asn Val Ile Asn Val Thr Cys Gln
            260                 265                 270
Ile Gln Lys Phe Tyr Pro Pro Ser Phe Gln Leu Thr Trp Leu Glu Asn
        275                 280                 285
Gly Asn Ile Ser Arg Arg Glu Val Pro Phe Thr Leu Thr Val Asn Lys
    290                 295                 300
Asp Gly Thr Tyr Asn Trp Ile Ser Cys Leu Leu Val Asn Ile Ser Ala
305                 310                 315                 320
Leu Glu Glu Asn Met Val Val Thr Cys Gln Val Glu His Asp Gly Gln
                325                 330                 335
Ala Glu Val Ile Glu Thr His Thr Val Val Val Thr Glu His Gln Arg
            340                 345                 350
Val Lys Gly Thr Ala Thr Lys Ser Gly Glu Val Phe Thr Pro Pro Leu
        355                 360                 365
Cys Leu Asn Val Asn Trp Ala Leu Phe Phe Met Tyr Lys Val Thr Phe
    370                 375                 380
Leu Ile Ile Val Ala Leu Ser
385                 390
 
           
             9 
             13 
             PRT 
             Rattus sp. 
           
            9
Pro Ile Tyr Ser Phe Ile Gly Gly Glu His Phe Pro Arg
  1               5                  10
 
           
             10 
             9 
             PRT 
             Rattus sp. 
           
            10
Ile Val Glu Pro Asp Thr Glu Ile Lys
  1               5
 
           
             11 
             6 
             PRT 
             Rattus sp. 
           
            11
Tyr Gly Phe Ser Pro Arg
  1               5
 
           
             12 
             12 
             PRT 
             Rattus sp. 
           
            12
Ile Lys Glu Val Ala His Val Asn Leu Glu Val Arg
  1               5                  10
 
           
             13 
             8 
             PRT 
             Rattus sp. 
           
            13
Val Ala Ala Gly Asp Ser Ala Thr
  1               5
 
           
             14 
             107 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            14
Asp Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala
  1               5                  10                  15
Gly Glu Ser Ala Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val
             20                  25                  30
Gly Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile
         35                  40                  45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
     50                  55                  60
Leu Thr Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Glu Gly
                 85                  90                  95
Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
            100                 105
 
           
             15 
             107 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            15
Asp Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala
  1               5                  10                  15
Gly Glu Ser Ala Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val
             20                  25                  30
Gly Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile
         35                  40                  45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
     50                  55                  60
Leu Thr Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
                 85                  90                  95
Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
            100                 105
 
           
             16 
             107 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            16
Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Leu Val Ala Ala
  1               5                  10                  15
Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val
             20                  25                  30
Gly Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile
         35                  40                  45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp
     50                  55                  60
Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
                 85                  90                  95
Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
            100                 105
 
           
             17 
             107 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            17
Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Pro
  1               5                  10                  15
Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val
             20                  25                  30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile
         35                  40                  45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
     50                  55                  60
Ser Thr Lys Arg Glu Asn Met Asn Phe Ser Ile Ser Ile Ser Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
                 85                  90                  95
Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
            100                 105
 
           
             18 
             107 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            18
Asp Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Ala
  1               5                  10                  15
Gly Glu Ser Ala Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val
             20                  25                  30
Gly Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile
         35                  40                  45
Ser Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
     50                  55                  60
Ser Thr Lys Arg Glu Asn Met Asp Phe Ser Ile Ser Ile Ser Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
                 85                  90                  95
Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
            100                 105
 
           
             19 
             107 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            19
Asp Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Pro
  1               5                  10                  15
Gly Glu Ser Ala Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val
             20                  25                  30
Gly Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile
         35                  40                  45
Ser Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
     50                  55                  60
Leu Thr Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
                 85                  90                  95
Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
            100                 105
 
           
             20 
             106 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            20
Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Pro
  1               5                  10                  15
Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val
             20                  25                  30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile
         35                  40                  45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp
     50                  55                  60
Leu Thr Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
                 85                  90                  95
Ser Pro Asp Val Glu Phe Lys Ser Gly Ala
            100                 105
 
           
             21 
             106 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            21
Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Ser Val Ala Pro
  1               5                  10                  15
Gly Glu Ser Ala Ile Leu His Cys Thr Val Thr Ser Leu Ile Pro Val
             20                  25                  30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Ala Arg Glu Leu Ile
         35                  40                  45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
     50                  55                  60
Ser Thr Lys Arg Glu Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
                 85                  90                  95
Ser Pro Asp Val Glu Phe Lys Ser Gly Ala
            100                 105
 
           
             22 
             107 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            22
Asp Glu Leu Gln Val Ile Gln Ser Glu Lys Ser Val Ser Val Ala Ala
  1               5                  10                  15
Gly Glu Ser Ala Ala Leu His Cys Ala Met Thr Ser Leu Ile Pro Val
             20                  25                  30
Gly Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile
         35                  40                  45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
     50                  55                  60
Leu Thr Lys Arg Asn Asn Leu Asp Phe Ser Ile Ser Ile Ser Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
                 85                  90                  95
Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
            100                 105
 
           
             23 
             107 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            23
Asp Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala
  1               5                  10                  15
Gly Glu Ser Ala Thr Leu Arg Cys Ala Met Thr Ser Leu Ile Pro Val
             20                  25                  30
Gly Pro Ile Met Trp Phe Arg Gly Ala Gly Ala Gly Arg Glu Leu Ile
         35                  40                  45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Glu
     50                  55                  60
Leu Thr Lys Arg Asn Asn Leu Asp Phe Ser Ile Arg Ile Ser Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
                 85                  90                  95
Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala
            100                 105
 
           
             24 
             107 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            24
Asp Glu Leu Gln Val Ile Gln Pro Glu Ala Phe Val Ser Val Ala Ala
  1               5                  10                  15
Gly Glu Met Ala Thr Leu Asn Cys Thr Val Thr Ser Leu Leu Pro Val
             20                  25                  30
Gly Pro Ile Gln Trp Phe Arg Gly Ala Cys Pro Gly Gln Lys Leu Ile
         35                  40                  45
Tyr Ser Pro Lys Arg Cys His Ser Pro Arg Val Thr Thr Ile Ser Asp
     50                  55                  60
Gln Arg Lys Arg Asn Ser Thr Asp Tyr Ser Ile Arg Ile Ser Ser Ile
 65                  70                  75                  80
Thr Leu Glu Asp Ala Gly Thr Tyr Tyr Cys Met Lys Leu Arg Arg Ala
                 85                  90                  95
Ile Pro Ala Asn Val Glu Ile Lys Ser Gly Thr
            100                 105
 
           
             25 
             107 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            25
Glu Glu Leu Gln Met Ile Gln Pro Glu Lys Leu Leu Leu Val Thr Val
  1               5                  10                  15
Gly Lys Thr Ala Thr Leu His Cys Thr Val Thr Ser Leu Leu Pro Val
             20                  25                  30
Gly Pro Val Leu Trp Phe Arg Gly Val Gly Pro Gly Arg Glu Leu Ile
         35                  40                  45
Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Arg Val Ser Asp
     50                  55                  60
Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Ser Ser Ile
 65                  70                  75                  80
Thr Pro Ala Val Val Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly
                 85                  90                  95
Ser Pro Glu Asn Val Glu Phe Lys Ser Gly Pro
            100                 105
 
           
             26 
             106 
             PRT 
             Unknown Organism 
             
               Description of Unknown Organism  Mus sp. or
      Homo sapiens 
             
           
            26
Glu Glu Leu Gln Val Ile Gln Pro Glu Lys Ser Val Ser Val Ala Ala
  1               5                  10                  15
Gly Glu Ser Ala Ala Leu Gln Cys Thr Val Thr Ser Leu Asn Pro Val
             20                  25                  30
Gly Pro Ile Gln Arg Phe Arg Gly Ala Gly Pro Gly Arg Lys Leu Ile
         35                  40                  45
Tyr His Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp
     50                  55                  60
Leu Thr Lys Arg Thr Asn Met Asp Phe Ser Ile Cys Ile Ser Asn Ile
 65                  70                  75                  80
Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Gln Lys Gly
                 85                  90                  95
Ser Pro Asp Val Glu Leu Lys Ser Gly Ala
            100                 105