WO2000064928A2 - Transmembrane (7tm) receptor r35) - Google Patents

Abstract

R35 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing R35 polypeptides and polynucleotides in therapy, and diagnostic assays for such.

Description

Novel Compounds Field of the Invention

This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and in identifying compounds which may be agonists, antagonists and /or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides.

Background of the Invention

It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, Nature, 1991, 351:353-354). Herein these proteins are referred to as proteins participating in pathways with G-proteins or PPG proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B.K, et al., Proc. Natl Acad. Sci, USA, 1987, 84:46-50; Kobilka, B.K., et al., Science, 1987, 238:650-656; Bunzow, J.R., et al., Nature, 1988, 336:783-787), G-proteins themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, e.g., protein kinase A and protein kinase C (Simon, M.I., et al., Science, 1991, 252:802-8). For example, in one form of signal transduction, the effect of hormone binding is activation of the enzyme, adenylate cyclase, inside the cell. Enzyme activation by hormones is dependent on the presence of the nucleotide GTP. GTP also influences hormone binding. A G-protein connects the hormone receptor to adenylate cyclase. G-protein was shown to exchange GTP for bound GDP when activated by a hormone receptor. The GTP-carrying form then binds to activated adenylate cyclase. Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, the G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal. The membrane protein gene superfamily of G-protein coupled receptors has been characterized as having seven putative transmembrane domains. The domains are believed to represent transmembrane α-helices connected by extracellular or cytoplasmic loops. G-protein coupled receptors include a wide range of biologically active receptors, such as hormone, viral, growth factor and neuroreceptors. G-protein coupled receptors (otherwise known as 7TM receptors) have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. The G-protein family of coupled receptors includes dopamine receptors which bind to neuroleptic drugs used for treating psychotic and neurological disorders. Other examples of members of this family include, but are not limited to, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorant, and cytomegalovirus receptors. Most G-protein coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in signal transduction.

Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some G-protein coupled receptors. Most G-protein coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several G-protein coupled receptors, such as the b-adrenoreceptor, phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization. For some receptors, the ligand binding sites of G-protein coupled receptors are believed to comprise hydrophilic sockets formed by several G-protein coupled receptor transmembrane domains, said socket being surrounded by hydrophobic residues of the G-protein coupled receptors. The hydrophilic side of each G-protein coupled receptor transmembrane helix is postulated to face inward and form polar ligand binding site. TM3 has been implicated in several G-protein coupled receptors as having a ligand binding site, such as the TM3 aspartate residue. TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding. G-protein coupled receptors can be intracellularly coupled by heterotrisneric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331) Different G-protein a-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G-protein coupled receptors have been identified as an important mechanism for the regulation of G-protein coupling of some G-protein coupled receptors. G-protein coupled receptors are found in numerous sites within a mammalian host.

Over the past 15 years, nearly 350 therapeutic agents targeting 7 transmembrane (7 TM) receptors have been successfully introduced onto the market.

There is a continuing need to identify and characterise further 7TM receptor genes and their related polypeptides/proteins, as targets for drug discovery.

Summary of the Invention The present invention relates to R35, a novel 7TM receptor, in particular R35 polypeptides and R35 polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of neuropathic pain, inflammatory and chronic pain, incontinence, disorders of the gastrointestinal tract associated with gut motility and secretion (for example, irritable bowel syndrome) hereinafter referred to as "the Diseases", amongst others In a further aspect, the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with R35 imbalance with the identified compounds. In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate R35 activity or levels. In a yet further aspect the invention relates to the use of the polynucleotides and polypeptides of the invention to identify agonists or antagonists for use in therapy.

Description of the Invention In a first aspect, the present invention relates to R35 polypeptides. Such peptides include isolated polypeptides comprising an amino acid sequence which has at least 95% identity, preferably at least 97-99% identity, to that of SEQ ID NO:2, 4, 6 or 11 over the entire length of SEQ ID NO:2, 4, 6 or 1 1 respectively. Such polypeptides include those comprising the amino acid sequence of SEQ ID NO:2, 4, 6 or 11. Further peptides of the present invention include isolated polypeptides in which the amino acid sequence has at least 95% identity, preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NO:2, 4, 6 or 1 1 over the entire length of SEQ ID NO:2, 4, 6 or 11 respectively. Such polypeptides include the polypeptides of SEQ ID NO:2, 4, 6 or 1 1.

Further peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 1, 3, 5 or 10.

Polypeptides of the present invention are believed to be members of the family of polypeptides. The ligand binding and signalling properties of R35 are hereinafter referred to as "R35 activity" or "R35 polypeptide activity" or " biological activity of R35" . Also included amongst the activities of R35 are antigenic and immunogenic activities of said R35 polypeptides, in particular the antigenic and immunogenic activities of the polypeptide ofSEQ ID NO:2, 4, 6 or 1 1. Preferably, a polypeptide of the present invention exhibits at least one biological activity of R35.

The polypeptides of the present invention may be in the form of the " mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

The present invention also includes include variants of the aforementioned polypetides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Nal, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination.

Polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art. In a further aspect, the present invention relates toR35 polynucleotides. Such polynucleotides include isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide which has at least 95% identity to the amino acid sequence of SEQ ID ΝO:2, 4, 6 or 1 1 over the entire length of SEQ ID NO:2, 4, 6 or 11 respectively. In this regard, polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred. Such polynucleotides include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO:l, 3, 5or 10 encoding the polypeptides of SEQ ID NO:2, 4, 6 or 11 respectively.

Further polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence that has at least95% identity to a nucleotide sequence encoding a polypeptide of SEQ ID NO:2, 4, 6 or 11 , over the entire coding region. In this regard, polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98- 99%o identity are more highly preferred, and those with at least 99% identity are most highly preferred.

Further polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 95% identity to SEQ ID NO: l, 3, 5 or 10 over the entire length of SEQ ID NO: 1 , 3, 5 or 10 respectively. In this regard, polynucleotides which have at least 97%) identity are highly preferred, whilst those with at least 98-99% identiy are more highly preferred, and those with at least 99% identity are most highly preferred. Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO:l, 3, 5 or 10 as well as the polynucleotides of SEQ ID NO:l, 3, 5 or 10.

The invention also provides polynucleotides which are complementary to all the above described polynucleotides. The nucleotide sequences are as follows:

SEQ ID NO: 1 is a chick cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 510 to 2678) encoding a polypeptide of 723 amino acids, the polypeptide of SEQ ID NO:2. The nucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO: 1 or it may be a sequence other than the one contained in SEQ ID NO:l, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.

SEQ ID NO:3 is a mouse cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 1 to 2196) encoding a polypeptide of 732 amino acids, the polypeptide of SEQ ID NO:4. The nucleotide sequence encoding the polypeptide of SEQ ID NO:4 may be identical to the polypeptide encoding sequence contained in SEQ ID NO:3 or it may be a sequence other than the one contained in SEQ ID NO:3, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:4.

SEQ ID NO:5 is a rat cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 1 to 2190) encoding a polypeptide of 730 amino acids, the polypeptide of SEQ ID NO:6. The nucleotide sequence encoding the polypeptide of SEQ ID NO:6 may be identical to the polypeptide encoding sequence contained in SEQ ID NO:5 or it may be a sequence other than the one contained in SEQ ID NO:5, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:6.

SEQ ID NO: 10 is a partial human cDNA sequence and comprises a polypeptide encoding sequence (nucleotide 1 to 1824) encoding a polypeptide of 608 amino acids, the polypeptide of SEQ ID NO1 1 :. The nucleotide sequence encoding the polypeptide of SEQ ID NO: 1 1 may be identical to the polypeptide encoding sequence contained in SEQ ID NO: 10 or it may be a sequence other than the one contained in SEQ ID NO: 10, which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO: 1 1. The nucleotide sequence of SEQ ID NO:7 is a partial chick cDNA sequence isolated using

RNA fingerprinting methodology.

The nucleotide sequence of SEQ ID NO: 8 is a partial mouse cDNA sequence encoding a polypeptide (SEQ ID NO:9) that is homologous to chick R35. Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one R35 activity. Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NN.(1989)), from a cDΝA library derived from embryonic day 16 chick dorsal root ganglia (DRG). Polynucleotides of the invention can also be obtained from natural sources such as genomic DΝA libraries or can be synthesized using well known and commercially available techniques.

When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide, by itself; or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions. For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentzβt al, Proc Νatl Acad Sci USA (1989) 86:821-824, or is an HA tag. The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRΝA.

Further embodiments of the present invention include polynucl otides encoding polypeptide variants which comprise the amino acid sequence of SEQ ID ΝO:2, 4, 6 or 11 and in which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, amino acid residues are substituted, deleted or added, in any combination.

Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO: 1, 3, 5 or 10 may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention Typically these nucleotide sequences are greater than 95% identical to that of the referent. The probes or primers will generally comprise at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides.

A polynucleotide encoding a polypeptide of the present invention may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1, 3, 5, 10 or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to the skilled artisan. Preferred stringent hybridization conditions include overnight incubation at 42PC in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0. lx SSC at about 65°C. Thus the present invention also includes polynucleotides obtainable by screening an appropriate library under sfringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1, 3, 5, 10 or a fragment thereof.

The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA. This is a consequence of reverse transcriptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remain attached to the template during the polymerisation reaction), failing to complete a DNA copy of the mRNA template during 1st strand cDNA synthesis.

There are several methods available and well known to those skilled in the art to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al., PNAS USA 85, 8998- 9002, 1988). Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence). The products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.

Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems which comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrooket al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). Preferred such methods include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid- mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells, such asStreptococci, Staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A great variety of expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrooket al, Molecular Cloning, A Laboratory Manual (supra). Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.

If a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification. This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the polynucleotide of SEQ ID NO: 10 which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under- expression, over-expression or altered expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeledR35 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (ee, e.g., Myers et al, Science (1985) 230: 1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cottonet al, Proc Natl Acad Sci USA (1985) 85: 4397-4401). In another embodiment, an array of oligonucleotides probes comprisingR35 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al., Science, Vol 274, pp 610- 613 (1996)). The diagnostic assays offer a process for diagnosing or determining a susceptibility tothe Diseases through detection of mutation in theR35 gene by the methods described. In addition, such diseases may be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagonostic kit which comprises: (a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO: 10, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID NO: 1 1 or a fragment thereof; or

(d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO: 1 1. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or suspectability to a disease, particularly neuropathic pain, inflammatory and chronic pain, incontinence-disorders of the gastrointestinal tract associated with gut motility and secretion (for example, irritable bowel syndrome), amongst others. The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (co inheritance of physically adjacent genes). The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease. The polypeptides of the invention or their fragments or analogs thereof, or cells expressing them, can also be used as immunogens to produce antibodies immunospecific for polypeptides of the present invention. The term " immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art. Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495- 497), the trioma technique, the human B-cell hybridoma technique (Kozboret al, Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al, Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as those described in U.S. Patent No. 4,946,778, can also be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms, including other mammals, may be used to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.

Antibodies against polypeptides of the present invention may also be employed to treat the Diseases, amongst others.

In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region. In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa. Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.

Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases hereinbefore mentioned amongst others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.

A further aspect of the invention relates to an immunological/vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition comprises a polypeptide or polynucleotide of the present invention The vaccine formulation may further comprise a suitable carrier. Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

Polypeptides of the present invention are responsible for one or more biological functions, including one or more disease states, in particular the Diseases hereinbefore mentioned. It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the polypeptide of SEQ ID NO:2, 4 or 6 or a human homolog, for example a polypeptide comprising the polypeptide of SEQ ID NO: 11. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the polypeptide. In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. Such agonists, antagonists or inhibitors so- identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; or may be structural or functional mimetics thereof (see Coliganet al, Current Protocols in Immunology l(2):Chapter 5 (1991)).

The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Constitutively active polpypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring R35 activity in the mixture, and comparing the R35 activity of the mixture to a standard. Fusion proteins, such as those made from Fc portion and R35 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see D. Bennett et al, J Mol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide(also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.

The polypeptide may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^1), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide which compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.

Examples of potential polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypetide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.

Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises:

(a) a polypeptide of the present invention;

(b) a recombinant cell expressing a polypeptide of the present invention;

(c) a cell membrane expressing a polypeptide of the present invention; or

(d) antibody to a polypeptide of the present invention; which polypeptide is preferably that of SEQ ID NO:2, 4, 6 or 11.

It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.

It will be readily appreciated by the skilled artisan that a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by:

(a) determining in the first instance the three-dimensional structure of the polypeptide;

(b) deducing the three-dimensional structure for the likely reactive or binding site(s) of an agonist, antagonist or inhibitor;

(c) synthesing candidate compounds that are predicted to bind to or react with the deduced binding or reactive site; and

(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitors. It will be further appreciated that this will normally be an interative process.

In a further aspect, the present invention provides methods of treating abnormal conditions such as, for instance, neuropathic pain, inflammatory and chronic pain, incontinence, disorders of the gastrointestinal tract associated with gut motility and secretion (for example, irritable bowel syndrome), related to either an excess of, or an under-expression of, R35 polypeptide activity.

If the activity of the polypeptide is in excess, several approaches are available. One approach comprises administering to a subject in need thereof an inhibitor compound (antagonist) as hereinabove described, optionally in combination with a pharmaceutically acceptable carrier, in an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous polypeptide may be administered. Typical examples of such competitors include fragments of the R35 polypeptide.

In still another approach, expression of the gene encoding endogenous R35 polypeptide can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Alternatively, oligonucleotides which form triple helices with the gene can be supplied (see, for example, Lee et al, Nucleic Acids Res (1979) 6:3073; Cooney t α/., Science (1988) 241 :456; Dervan et al, Science (1991) 251 : 1360). These oligomers can be administered er se or the relevant oligomers can be expressed in vivo. For treating abnormal conditions related to an under-expression ofR35 and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound which activates a polypeptide of the present invention, i.e., an agonist as described above, in combination with a pharmaceutically acceptable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of R35 by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo. For an overview of gene therapy^ see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996). Another approach is to administer a therapeutic amount of a polypeptide of the present invention in combination with a suitable pharmaceutical carrier. In a further aspect, the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present invention, agonist/antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

The composition will be adapted to the route of administration, for instance by a systemic or an oral route. Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, and the like. The dosage range required depends on the choice of peptide or other compounds of the present invention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.

Polynucleotide and polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology. This is most easily facilitated by storing the sequence in a computer readable medium and then using the stored data to search a sequence database using well known searching tools, such as GCG and Lasergene. Accordingly, in a further aspect, the present invention provides for a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO:l, 3, 5 or 10 and/or a polypeptide sequence encoded therefrom.

The following definitions are provided to facilitate understanding of certain terms used frequently hereinbefore.

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.

" Isolated" means altered " by the hand of man" from the natural state. If an " isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not " isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is " isolated" , as the term is employed herein.

" Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, " polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, " polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. " Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

" Polypeptide" refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. " Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. " Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Post-translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al, "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al, " Protein Synthesis: Post-translational Modifications and Aging", Ann NY Acad Sci (1992) 663:48-62).

" Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.

"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: 1) Algorithm: Needleman and Wunsch, j. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA.

89: 10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = +10, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3 Available as: The "gap" program from Genetics Computer Group, Madison WI. These are the default parameters for nucleic acid comparisons.

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: 1 , that is be 100% identical, or it may include up to a certain integer number of nucleotide alterations as compared to the reference sequence. Such alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the numerical percent of the respective percent identity(divided by 100) and subtracting that product from said total number of nucleotides in SEQ ID NO: 1, or: n_n < x_n - (x_n • y), wherein n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO: l, and y is, for instance, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc, and wherein any non-integer product of x_n and y is rounded down to the nearest integer prior to subtracting it from x_n. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations. Similarly, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO:2, that is be 100%) identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%). Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity(divided by 100) and then subtracting that product from said total number of amino acids in SEQ ID NO:2, or: n_a<x_a - (x_a • y), wherein n_a is the number of amino acid alterations, x_a is the total number of amino acids in SEQ ID NO:2, and y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integer product of x_a and y is rounded down to the nearest integer prior to subtracting it from x_a.

"Homolog" is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a subject sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the sequences being compared as hereinbefore described. Falling within this generic term are the terms "ortholog", meaning a polynucleotide or polypeptide that is the functional equivalent of a polynucleotide or polypeptide in another species, and "paralog" meaning a functionally similar sequence when considered within the same species. "Fusion protein" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0 464 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e.g., EP-A 0232 262]. On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth. Examples

Example 1 - Cloning of partial chick R35 cDNA by RNA fingerprinting The initial R35 fingerprint fragment was identified in an RNA-fingerprinting approach, comparing RNA from NGF and NT3 treated DRG explants, as described (Friedel, R.H., Schnurch, H., Stubbusch, J., and Barde, Y.-A., Proc. Natl. Acad. Sci. USA, 1992; 94: 12670- 12675).

Experimental design DRG Explant Cultures

Lumbosacral ganglia 4, 5, and 6 of El 0 chicken embryos were carefully dissected, washed in PBS, and transferred to 48-well tissue culture plates (Falcon) coated with polyornithine (Sigma) and mouse laminin (Life Technologies) according to Ernsberger, U. & Rohrer, H. (1988) Dev. Biol. 126, 420-432.

Each well contained 0.5 ml of culture medium consisting of Ham's F-14 (Life Technologies) supplemented with 0.1 mg/ml transferrin, 16 ug/ml putrescine, 6 ng/ml progesterone, 8 ng/ml sodium selenite, 100 ug/ml penicillin G, 60 ug ml streptomycin sulfate (all reagents from Sigma), and 2 ng ml NGF or NT3. After 4 days of incubation at 37°C in a 3% C02 atmosphere at 95% relative humidity, ganglia were detached from the wells with a small spatula and total RNA was isolated by using an RNeasy kit (Qiagen).

RNA Fingerprinting. cDNAs were synthesized from total RNA with oligo(dT) primers and Moloney murine leukemia virus reverse transcriptase (Superscript II; Life Technologies) at 45°C for 1 hour. Reaction products were purified from excess oligonucleotides by using a QIAquick kit from Qiagen. cDNA derived from 60 ng of total RNA was used as template for a PCR. Amplification was performed in a volume of 25 ul containing lx PCR buffer (Perkin-Elmer), 20 uM dNTPs (Pharmacia), 50 nM each primer, and 5 uCi (1 uCi = 37 kBq) of [α-33P]dATP (Amersham). For each reaction, two 18-mer oligonucleotides of arbitrary sequence and a G+C content of 61% were used (see list below). All PCRs were run on thermal cyclers 2400/9600 from Perkin-Elmer. After 3 minutes of denaturation at 95°C, 1.5 units of AmpliTaq DNA polymerase (Perkin-Elmer) was added (manual "hot start"). After 2 minutes at 95°C the profile was started with two low- stringency cycles (95°C for 30 seconds, 50°C for 2 minutes, and 72°C for 2 minutes) followed by 25-30 high-stringency cycles (95°C for 30 seconds, 65°C for 1 minute, and 72°C for 1 minute), and a final 5-minute extension at 72°C. Reaction products were separated by electrophoresis on nondenaturing 4% polyacrylamide gels for 3 hours at 40 W. Gels were vacuum dried on chromatography paper (3MM; Whatman), fixed in a cassette, and overlaid with x-ray film. Exposure times varied from overnight to 3 days. Bands of interest were marked on the gel by piercing through the developed film. Gel pieces were excised with a scalpel and DNA was eluted by boiling in 50 ul of 10 mM TrisHCl, 1 mM EDTA buffer, pH 7.5, for 5 minutes. One microliter of the eluted DNA was used as template in a second PCR (reamplification). The reaction volume was 50 ul containing lx PCR buffer, 0.2 mM dNTPs, and 0.1 uM primers (the same as used for first PCR). Then 1.5 units of AmpliTaq were added by manual "hot start." The temperature profile consisted of 30 cycles (95°C for 15 seconds, 65°C for 30 seconds, and 72°C for 30 seconds) and a final 5-minute extension at 72°C. A lOul aliquot of the reaction was separated on an ethidium bromide-stained 1% agarose gel. Occasionally, reamplification products contained by-products of lower molecular weight. These were excluded by cutting out only the band of the highest molecular weight. The DNA was purified with a QIAquick kit and cloned in the pCRII vector of the TA-Cloning kit (Invitrogen). DNA sequencing was done with an automated sequencer (ABI 373 A; Applied Biosystems). Primers used for RNA fingerprinting: PI 5' AGTCGCAGCACAGGTGAG; P2 5' CGTCCGTAGGTCCAGGT; P3 5' CCCAGGTCGTCGTGTTCA; P4 5' GTCAGGGATACCGAGAGC; P5 5' CCAGCCGCCAGAGTTTTG; P6 5' GAGAGTTAGCCGCAGCG; P7 5' GTTACAGCAACCGCCAGG; P8 5' GGGGTCCTTCAATGGGAG;

P9 5' TCCGAGGGAGATTCCGTC; and

P10 5' GCCACAGGGCAGAGTCTT.

The initial 248 nucleotide cDNA fragment of chick R35 (SEQ ID NO:7) was cloned by this means from the RNA of NGF-treated DRG explants of E10 chicken embryos with primer pair P3 and P7 (NB: the primer sequences are not included in the sequence of SEQ ID NO:7; P3 is the 5' primer and P7 is the 3' primer).

Example 2: Cloning of full length (ie. including entire coding region) chick R35 cDNA The fingerprint fragment isolated from Example 1 comprising the 248 nucleotide chick R35 cDNA sequence (SEQ ID NO: 7) served as a probe to screen a lambda gtlO cDNA-library from chicken El 6 DRGs. Subsequently 3 cDNA clones were obtained, that overlap and cover 4.5 kb of the R35 cDNA. Two of these lambda clones (called R351ambda-13 and R351ambda-12) cover the entire open reading frame. The screening of the lambda library was essentially done as described (Sambrook. et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The full-length chick R35 cDNA sequence is given in SEQ ID NO: 1.

Example 3: Cloning of partial mouse R35 cDNA

The 431 nucleotide mouse partial cDNA fragment (SEQ ID NO: 8) was cloned by PCR, using mouse genomic DNA as template. Non-degenerate primers were deduced from conserved regions in the alignment of the full length chicken R35 cDNA seqeunce with the sequences of other species we have cloned (i.e. goose, nuthatch, ostrich, snake and turtle). The primers used for the PCR were:

SK-R35-S2; 5' TTTGCCTCTCTGTGGTTGGG;

N-R35-A3; 5' GAGCCAAAGAATGACTTTGGTCAG.

PCR conditions:

300 ng mouse genomic DNA was used as template. The reaction volume was 50 ul containing lx PCR buffer(Perkin Elmer) with 2.5mM Mg2+, 0.2 mM dNTPs, and 0.2 uM of each primer SK-R35-S2 and N-R35-A3. 1.5 units of AmpliTaq were added by manual "hot start." The temperature profile consisted of 40 cycles (95°C for 30 seconds, 63°C for 30 seconds, and 72°C for 1 minute).

Example 4: Cloning of full-length (ie. the entire coding region) mouse R35 cDNA

Full length mouse R35 cDNA was cloned by PCR extension of the initial fragment from example 3 with using a lamda gtlO cDNA library from mouse P0 brain (constructed by H. Schnuerch) as template. For this purpose R35 specific primers from the fragment obtained in example 3 were designed and used in combination with lambda gtlO specific primers which flanked the cDNA insertion site of the lambda DNA.

Each cDNA-PCR extension consisted of two rounds of PCR: a first PCR with an aliquot of the lamda gtlO cDNA library as template, and a R35 specific primer and a lambda specific primer. The second PCR was done with an aliquot of the first PCR as template and nested primers (again one R35 specific and one lambda specific). PCR conditions were cycles of 95°C 15s, 65°C 30s, and 72°C 1 min.

PCR products were seperated by gel electrophoresis in an agarose gel, the band with the highest molecular weight was cut out and cloned in the pCRII vector (Invitrogen).

Primers for the mouse R35 5' extension:

1. PCR: LI and MM-R35-EU1

2. PCR: L2 and MM-R35-EU2

The resulting PCR fragment was designated "EU"

Primers for the R35 3' extension:

1. PCR: LI and MM-R35-ED1

2. PCR: L2 and MM-R35-ED2

The resulting PCR fragment was designated "ED"

The full-length mouse R35 cDNA sequence was compiled from EU + fragment obtained in example 3 + ED.

The sequence was confirmed by RT-PCR with mouse R35 specific primers from an independent cDNA of P3 mouse brain.

Primer sequences:

The lambda specific primers:

LI: AAG AGC TGA CGC AAG TTC TGG TA (for first PCR)

L2: TTG AGC AAG TTC AGC CTG GTT AAG (nested, for second PCR)

The mouse R35 specific primers for 5' extension:

MM-R35-EU1 : TATACCGCGAAGAGGAGCAGCA (for first PCR) MM-R35-EU2: AAGGCGTGCGCACGAAGACA (nested, for second PCR)

The mouse R35 specific primers for 3' extension: MM-R35-ED1 : GCACCAACAGCTTCCCCTTG (for first PCR)

MM-R35-ED2: GAGCTTGGCACAGAAGCGCT (nested, for second PCR).

Example 5: Cloning of rat R35 cDNA The rat R35 cDNA was cloned by RT-PCR with a rat P2 cDNA library, using mouse primers under low stringency conditions: cycles of 95°C 15s, 56°C 30s, and 72°C 1 min. The mouse primers used to amplify the rat cDNA: MM-R35-S6: TCAGACTCGTTGAAGAGGGA (upstream primer) MM-R35-A15: CAAAAACTCATGCACGAGAAC (downstream primer)

The sequences of MM-R35-S6 and -A15 are not included in the rat R35 cDNA sequence given in SEQ ID N05; they are located in the 5' and 3' untranslated regions respectively.

Example 6: Cloning of human HS-R35 cDNA

An initial human R35 cDNA fragment of 223 nt was cloned by RT-PCR with primers from the murine R35 sequnce, using cDNA of adult human brain as template.

Mouse R35 primers used to clone the initial human fragment: MM-R35-S2 : TGGGAATGGTGCAGGAGTAA MM-R35-A3: ACACTGGTGTCCGCAAACGT

PCR conditions were standard: cycles of 95°C 15s, 65°C 30s, and 72°C 1 min The 5' extension of the initial cDNA was done by RT-PCR using a adult human brain cDNA library, cloned in the expression vector pcDNA3.1 (Invitrogen), as template. Primers were human specific (HS-R35-A1) and vector specific (PCT7).

Primer sequences:

HS-R35-A 1 : AC AC AAGGGAATGGC AAGGG PCT7: TAATACGACTCACTATAGGGAGACC

PCR conditions were standard: cycles of 95°C 15s, 65°C 30s, and 72°C 1 min.

The sequence obtained was a large fragment of the human sequence, encompassing the 5' coding region, encoding the N-terminal 608 amino acid residues. The polynucleotide sequence of this fragment is shown in SEQ ID NO: 10 and the polypeptide encoded thereby is shown in SEQ ID NO: l l. The 3' human cDNA sequence, which together with the sequence disclosed in SEQ ID NO: 10 will provide the complete full-length human sequence, may be cloned according to methods well known in the art. For example a 3' RACE reaction may be performed using a PCR primer sequence specific to the human sequence in combination with a vector specific primer from a suitable library. From this information a single full-length clone may be obtained, for example by PCR, or the 5' and 3' fragments may be joined using methods well known in the art.

Example 7: Expression profile of (chick) R35 mRNA by in situ hybridisation

Preparation of tissue sections and hybridisation with single stranded RNA-probes were performed as described (Schnurch, H. and Risau, W., 1991; Development 1 11 : 1 143-1154). The probe, R35-S1-A1, used for in situ hybridisation was synthesised from a 478 nucleotide cDNA fragment of the R35 open reading frame (nucleotides 1123 to 1600 in SEQ ID NO:l). Results:

Probing transverse sections of chick embryonic dorsal root ganglia (DRGs) at days 6, 8, 10, 12, 14 and 16 of gestation revealed R35 mRNA to be restricted to a specific subpopulation of DRG neurons. At all developmental stages examined, this R35-positive population was concentrated in the medial part of the DRG. The medial neurons (also called mediodorsal neurons) are those that express the NGF receptor trkA and are equivalent to the "small" NGF- dependent neurons of rodents. This subpopulation is generally assumed to be responsible for noci- and thermoception. R35 may therefore have a role in noci- or thermoception. No R35 mRNA was detectable in the lateral part of the DRG.

Transverse sections of chick embryonic spinal cord at 6 and 8 days gestation revealed robust R35 expression detectable in the entire spinal cord, including the meninges. In 10 day gestation embryos, a relatively weak R35-specific hybridization signal was detected throughout the spinal cord, but expression remained high in the motoneuron pool. No R35 signal was detectable in the meninges at this stage. At 12, 14 and 16 days gestation, R35 expression was restricted to the motoneuron pool with no signal detectable in other regions of spinal cord, or in the meninges. The marked decrease in R35 expression from E8 to E10 in the spinal cord is concommitant with the end of proliferation and the onset of terminal differentiation in this region. This fact would suggest a potential role of R35 in the regulation of proliferation/differentiation of spinal cord neurons.

In the developing brain at 10 days gestation, R35 expression was detected in the tectum where it was restricted to two layers of neurons, which lie close to the ventricle. Other regions of the tectum showed no detectable R35 expression. At this stage of development R35 mRNA was also detected in retina, being mainly restricted to the retinal ganglion cell layer. Other layers in the retina showed only very weak, or no, signal. At 14 days gestation a strong R35- specific hybridisation signal was detected in the external granule cell layer of the cerebellum.. No other regions of the cerebellum show R35 signal at this stage. The external granule cell layer consists mainly of proliferating granule cells, that subsequently will move inside the cerebellum to form the mature granule cell layer.

R35 mRNA was also detected in neurons of the developing gut. At 12 days gestation the R35 hybridization signal in the gut was restricted to two concentric layers of neurons, which presumably correspond to the plexus myentericus (outer ring) and the plexus submucosus (inner ring). Within developing gonads, R35 hybridization signal was evenly distributed in the gonad anlage at 6 days gestation and expression remained detectable in cells of the gonads at 12 days gestation.

Conclusions: these data validate findings from initial RNA fingerprinting and confirm R35 as a novel seven transmembrane receptor. Restricted expression of R35 in peripheral, central and autonomic nervous systems implicate functions in molecular processes and signalling pathways that regulate these neuronal tissues. As such, modulation of R35 function may be of value for therapeutic intervention in relation to neuropathic pain, inflammatory and chronic pain, incontinence, disorders of the gastrointestinal tract associated with gut motility and secretion (for example, irritable bowel syndrome).

SEQUENCE INFORMATION SEQ ID NO: 1

CTAAGAGGCAGCTCTGTCTGGGAAGATACTCACTCCCCATTTCTCTGCTATCCTGCTTGC TTCTGACTCCTCCCTCCTGCAGCTCAGATCAGCACAGGGTGAATCCAACAGCCTCCCATT TCTACCGTGTTTCTCCCTGAGCTTGAGCATACCTGGTACTCTGCCTTCGTTTCAGCTGAA CTCGTGAGTAAGGCACATTGCCTGATATCTGTCAAAAGTGAATGGCTTGTTATTACCTGA AGAGACTGAATTAACTATCTTCTGGTACTGAAAGGGTAATGAAGAACTGTATTTTTTCCA GCACCTTTTGTACTTCTTTGTTGCCTGATTTTTCAAGAGCGTGAGTTATTCTGGTTATCA AGGGAGCTTAAACCTGAGTATTTCTTCACCGGATGCTACAAAAGAAGAAAGGAAAACAAA CTTAAGATGTGGAGCTAGCAGTTATGGTTTCAGCTTGTGAAAACTTGCAGAGTCAGTGAG AAGGCAAGGAACAACATTTGCCAAGGACCATGTCGGTAATGCCTAGTAATTTGTCCCTCA ATGGGACAAGCTTCTTCGCTGAAAATCATAGCATTATGGATAAACCTAATGAGCAGAGAA CTTTGAATGTCTTTCTATTTTGCTCGACTTTTATCATTGCATTCACAGTGCTTCTGGGCA GCATTTATTCACTAGTTTCCCTGCTGAAACTGCAGAACAAATCCACAATTTCAATGATTG TGACCTCTTTGTCAATAGATGATTTGATCAGCATTGTGCCAGTGATTATTTTCATGCTCA CCCAGTGGTCAAGTGATGCCCTTCCTCAGCCTCTGTGCACCACCTCAGCACTTATATATT TATTCCAGGGCATTTCTAGCAATCTGAAAGGGTCTCTTATAGTTTCCTACAACTTCTATA GCATCAACAAAACTGAGACAATGAACTGCAGTGCTTCCAAACGACGAGTGAGCATGGTAT GGGCAATTCTTAGCATTTGGATCGTCAGTTTGCTGATATGCATTTTGCCTCTCTGTGGCT GGGGCAAATACATCCCCACCACCTGGGGTTGCTTCACAGACCATGCAAGTTCCTACATCT TGTTCTTATTTATTGTCTACTCATTGTGCTTTTGCCTCCTCACAGTGCTGTCTGTCCCTC TAACTTACCAGCTGTTGTGCTCAGATGAGCAACAGCTATTACACGTCAACTACCAGGAAA TCTCTCGAGGCTACATCACCCCTGGGACACCTGCAGGCTGCAGTACTGCCACCCCATGTC TGTCACCAGTGGACCCTGTGGATAAAACCCTGAAGCATTTCCAAAATGCATGTCTAAGCT CAAAGGCAGTTTTTAGGAAAGGTGTGGCTGAGAGCAGTGGACTCGAGCCCCGATGCATGA ACAACGCAAAGAGCAGGAGCTTCACAGTGGGCTTTGCCCAGAAACGGTTCTCACTGATTC TTGCATTGACAAAAGTCATTCTCTGGCTCCCAATGATGATACAAATGGTTGTCCAGCACA TCACTGGCTATCAAAGCTTTTCATTTGAGACACTTAGCTTCCTGCTGACATTGCTAGCTG CCACCGTCACCCCAGTATTTGTCCTGTCAGAGCACTGGATCCACCTGCCGTGTGGCTGCA TCATAAACTGCAGGAGGAATTCATATGCTGTGTCTTCAGAAGAACTCAAAACCAAGCATA GAGGTTTCGAATTCAACCTTTCCTTCCAGCATGGTTATGGAATCTACCGAATATCCCCCG AGAGCCACCACCACGATGGCGATGGTAAATCCACATCGTGCCACAACCTGCTGGTTTGCG AGAAACCGTACGAGCCGCCGAGGGGAGGTGGCGGCGGTGCGGCGCTGGCGGAACTCAGCA CCACGGATAGCGCCCGGCCAGGCCCGGCGGGGCTCAGCGCCACGGACAGCGCCAGGCCCG GTCCCGCGGGGTTCAGCACCACAGACAGCGCCAGGCCCAGCGCCGCGGGGGTGCGCGGGG AGGCGGCCTCGGGCCTCGGCAGGACCGTGGAGGGACCCGAGAGGAGGCTGTCGCACGAAG AGGGCCATAAGCCAGAGCTCACAGACTGGGAGTGGTGCAGGAGTAAATCAGAGAGGACCC CTCGACAGCGGTCAGGTGGCGCCTTAGCTATTCCTCTGTGTGCATTTCAAGGAACCGTGT CACTTCAGGCACCCACAGGAAAAACTCTCTCCTTGTCTACATACGAGGTAAGCACTGAAG GACAGAAAATAACACCCACATCAAAGAAAATTGAAGTTTATCGATCTAAAAGTGTTGGTC ATGACCCCAACCCAGAGGAGTGTCCTAATACGTTTGCTGACACGAGTGTTAAAATTCATT TAGAGGTTCTGGAAATTTGTGACAATGAAGAGGCTCTGGACACTGTTTCAATCATCAGTA ATATCAGCCAGTCCTCCACACAGGTAAGATCTCCATCCTTACGCTACTCACGGAAGGAGA ACAGATTTGTCTCATGTGATTTGGGGGAAACTGCTTCCTACTCACTCTTCATACCATCCA ACAATCCCGACAGCGATATTAACATAACAATTCCAGACACTGTGGAAGCTCACCGGCAGA ATAGTAAAAAGCAACACATGGAAAGAGGTGGTTATCAGGAAGAAATACAGATGTTGAATA AAGCGTACAGAAAACGGGAAGAAGATGGCAACAGCAATTAAAAGACATTTAATCTAAACA TTTAAACTGATTCAGCAGACATTGCTCCTCTTGACTCAAGTTACCCTAACAAATACAATG CCAACCGGTTTGTTTTTTTTTTTTTCCTAGCATATAAATGATTTTTATTTGGGTACACTG TTAAATAGTTAACATTTACAAATGGGAAAACAAACGCTTCAGTGCTTTGCAGCTTTGTGC AATGAAATCCAAGTTATTTACCAATCTCTTTATTTGAGGAAGACTTGCATCTGAAAGGAA TCCTAAATGAACTCTTAAAAGCCACCCTTGCTTGCTTGTTGGCACTGGGTGGTCTGTGGG AAGCCAGAAAATGAGAGAGCAGCTCTACACCCCTGAAGATGTAACAGGTTTCAATGACTG TGGCAGTTCTCCTACTGATGTGCCAGACTTGAAACATTTACTGAAAATATGTAGCACAAG CCTAAGACTAAGCCATGCATTTGGTTAGGGATTGTAAGTTACCTACGTGGTTTAATTTTC TACTGCTCCATACGTAATTGATACGTAATGTCTCTGTAAATGGCCATTGTATAGACCTTT GAATGTGGGTGTCTGAAATTTAAGCTTGCACATTTTATATGTCCAGCACTGTGAAAATGT GTTCTCATGGCTGGACATTTCTTTCATCGAGTGTGTCAAATGTTGGTTGATCTTATGCTG GTTACTCTAAAGTGTGCCATTTTCTGCCTCCTTGATCTCTTAGGAGAGGGGACAGGGCTG CTCAAGGAGGCCAAACTTAGAGACCCAGCTTAGCTGTTGACCTCAGCTTGAGGCTGAGTG CCCAGATCAGCTTCCCTGCCCTGGAGCACCTCTGGAGGAGCCTGTTCTTATGCTGCCTTA CAGGAAGCGTAGAGCCAAGTCTGAAATCAAACTATAACCCACCAATAGACTGCTTCTTGT CAAATTCAAACAGACTGAGGGTAAAACAGCAAATGAAACACTTAAGTCTAATGCTTTTTG TCTGAGAGCTCATTTTCTGAGATGTTGTCATACATTTCAGCCAGATAGAAGTAGACTTCA GCAGGACTTTAAGAGGTTTGACTAAAAGTTACACCACTTCTTTAATTAATAGATTTAATT TTCAGTGAGTGACAACATAATAATTCAGGTGTCTGCAGTCACCTTGTTATCCATTCAGTG GGAACATGCACAGGAAAGTAAGCACAGCCCTATTTAGGATTCCTTTATGGATTGTGAACA GGTAGAGCAAAAGGGCCAACTAGCTCAGCTTGTGTTCAGTATGGAGGCATGGAAAGAACA GGTGATTTGCATTCTCACCTCACCAACATAAATCAGGCCTGCCACCATGATCAGACGGGC TATGCTGCTGTGACAGTGATGGAGAGCTGAAGAGAATCAGGGTCAGAGACTGCAGGCCGC TGTGATGGAGATGGATCACTCTGTGCAAGATGTTGCTTTTCACGTGCTGC CTTCCAAGG TTGAAAACAAAGTTCCTCCATAAGATTGCCTCAAGGCTCCTTAGTACCTGGTGATGCTGC GTTACACTTCAGATTTATCAAGAAACTATAGTAGACAGAGCAATCAATGGTAAGTGTATT AGTAGGTTATATCCATGTGCATGCAGCAGGGAGACACGTTCTGCTGTCTCTGTATTACTT TGCTTTGTGTTTGTAGGATTAGCTCAGAAAGTTGCTTTATAGCAACAGATGTGCTCAATC CAGAAAGACGTTGTGAGGGAGGATGCAGTGCATCACTGCCTCAGTTTGGTCACGTTCTCT GATGGGTGCATTTAGATTGTGTTCAGGTTTGGAGGCATTAAAATTCTGCATAGCTCTGAC CCTGAGCTTGACCTAATTGTAGAATGTCGCCTTGCTGGTGAAAAAGGAAAGACATTTTCT ATAATGAATCTGTAAATCTGATCATGTACGAATGTCTGACTGTTTTATATTTTATTTTAG TGACTCACATCATTAAACATTTTAAAGTGTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAA

SEQ ID NO:2

MSVMPSNLSLNGTSFFAENHSIMDKPNEQRTLNVFLFCSTFIIAFTVLLGSIYSLVSLLKLQNKSTISMIVT SLSIDD ISIVPVIIFMLTQ SSDALPQPLCTTSALIYLFQGISSNLKGSLIVSYNFYSINKTETMNCSASK RRVSMV AILSI IVSLLICILPLCG GKYIPTT GCFTDHASSYILF FIVYSLCFCLLTVLSVPLTYQLL CSDEQQLLHVNYQEISRGYITPGTPAGCSTATPCLSPVDPVDKTLKHFQNACLSSKAVFRKGVAESSGLEPR CMNNAKSRSFTVGFAQKRFSLILALTKVILWLPMMIQMVVQHITGYQSFSFETLSFLLTLLAATVTPVFVLS EH IHLPCGCIINCRRNSYAVSSEELKTKHRGFEFNLSFQHGYGIYRISPESHHHDGDGKSTSCHN LVCEK PYEPPRGGGGGAA AELSTTDSARPGPAG SATDSARPGPAGFSTTDSARPSAAGVRGEAASGLGRTVEGPE RRLSHEEGHKPELTD E CRSKSERTPRQRSGGALAIPLCAFQGTVSLQAPTGKTLSLSTYEVSTEGQKITP TSKKIEVYRSKSVGHDPNPEECPNTFADTSVKIHLEVLEICDNEEALDTVSIISNISQSSTQVRSPSLRYSR KENRFVSCDLGETASYSLFIPSNNPDSDINITIPDTVEAHRQNSKKQHMERGGYQEEIQMLNKAYRKREEDG NSN

SEQIDNO:3

ATGTCTTTCTTTCTCAGCAACTTAACAAATGACTCTAACCTGTGGAAAGCAAGTCATAAT TCTACGGAGACGGACCTTATGAATTCACCAGCAACTCTGACTCTTTCTCTCTTTTGCCTG ATCTGTATAATGACTCTCGCAGCTCTGGTGGGCAGCATTTTCTCACTGGTTTCGCTGCTG ACCATGCAATACAGAACTGTTTTGTCCATACTTGTGACATCTTGGTCTGTGGATGATCTC TTGAGCGTCTTGTCCGTGGCCATCTTCATGGTTTTACAGTGGCCAAAAGAGGCACAGGGC TACTTCCAGTCTCTGTGCACCACCTCTGCCCTACTGTATATGTGCCAGGGCCTCTCCAGC AACTTGAAGGCGACTCTCATAGTCTGCTACAACTTTTATACGATGAACAGGACGGTGGAG AGTCAGTCAAGCTCCTGGCGATTAGGACAGGTACTTGGTGTGACTTTAACCGTTTGGGCA GTCAGCCTGCTGCTGGCTTCGCTCCCGCTCTGTGGCTGGGGTGTCTTCGTGCGCACGCCT TGGGGCTGCCTGACCGACTGCTCCAGCCCATACGTGCTGCTCCTCTTCGCGGTATACGCT TCCGCTTTCGGGCTCCTGGCGGTTCTCTCTGTCCCTCTCACTCACCAGCTGCTGTGTTCG GAGGAACCGCTGAGACTCCATGCCAACTACCAGGAAATTTCTCGTGGCGCCTCTACTCCT GGGACCCCTGCTGCTGCGGGAAGAGTGCTCTGTCTGCCGCCAGAGGATGTTGAGATCCCT GCTCTGCGATGCACTGGGGGATGCTCCCCGAGCTCCGACGTGGTGTTTGCCCCGGGTCAG CCGGCTGCCTCCGGCGCTGGAGCCGGCAGGCGAGAGAATCCCGGGACTCCCCAGGGCACC AACAGCTTCCCCTTGAGCTTGGCACAGAAGCGCTTTTCTTTGATTCTAGCGCTGACGAAA GTCATCGTTTGGCTACCCATGATGATCCACATGGTGGTAAAACACGTGGTGGGGTTTCAG AGCCTTCCGGTCGATATGCTCAGCTTTCTACTAAGCCTGCTGGCCAGCAGTGTAACACCA GTGTTTGTCTTATCCAAACGTTGGGCCCACTTGCCTTGTGGCTGCATCATCAACTGCCAG CCAGACACCTACTCCGTTGCCTTCGATGGGAAAAAGTCGAAGAGGAAAGGCTTTGAATTC AATCTATCATTCCAACAAAGTTATGGACTCTATAAAATACCACATGCAGATTGCTATGAT GATGATGAAAATTCCATATCCTATCACAACCCAAAGAATTATGAGTGTGAAGCTACAAAG GAGCCTTGGGGAGACAACCGCAGTGTCTTCAATACCATCACAGTAGAAATCAGCACCACG CCACCGCTGGACAGCGCCACACTGACGGGTGTCCACAAGTGTACCAATACTGACATCCCA GAATCCAAACAGGCCATGAAAGAGGAAAAGGGTGCTTTTTCTGTTAAAACAGAAAGCGAT ATTAACTATGGAGAAACCACCTCTTTTGAAGGTCCAGAGAGACGACTGTCCCATGAGGAG AATCAGAAACCAGATCTTTCAGACTGGGAATGGTGCAGGAGTAAGTCTGAACGAACGCCT CGCCAGCGTTCCGGTGGTGGGCTGGCTATTCCCATATGCGCGTTCCAGGGGACTGTGTCT CTCCAAGCACCTACTGGGAAAACGCTCTCTCTTTCTACTTATGAGGTCAGCGCAGAAGGG CAAAAGATAACCCCAGCCTCTAAGAAAATAGAAGTCTATCGATCTAAAAGTGTTGGCCAT GAACCGAACTCAGAGGAGTCTCCCTCCACGTTTGCGGACACCAGTGTAAAAATACACTTG GAAGTTCTTGAAATTTGTGACAACGATGAGGCTCTAGACACTGTGTCAATTATCAGCAAC ATCAGCCAATCCTCCACAAAAGTCAGGTCTCCTTCTCTGCGCTACTCACGGAAGGAAAAC AGATTCGTCTCCTGCGACTTAGGAGAAACTGCCTCATACTCGCTCTTTTTACCCACAAGT GATCCTGATGGTGATATTAACATCTCTATCCCTGACACGGTGGAGGCCCACAGACAGAAC AGCAGGCGGCAGCATGAAGAGAGGGATGGCTACCAGGAAGAGATCCAGTTGTTAAACAAA GCCTACAGGAAAAGAGAGGCAGAAAGCAAGGGTGATTAG SEQ ED NO:4

MSFFLSNLTNDSNL KASHNSTETDLMNSPATLTLSLFCLICI TLAALVGSIFSLVSLL

TMQYRTVLSILVTS SVDDLLSVLSVAIF VLQWPKEAQGYFQSLCTTSALLYMCQGLSS

NLKATLIVCYNFYTMNRTVESQSSS RLGQVLGVTLTV AVSLLLASLPLCG GVFVRTP WGCLTDCSSPYVLLLFAVYASAFGLLAVLSVPLTHQLLCSEEPLRLHANYQEISRGASTP

GTPAAAGRVLCLPPEDVEIPALRCTGGCSPSSDWFAPGQPAASGAGAGRRENPGTPQGT

NSFPLSLAQKRFSLIl-JUTKVIL LPMMIHi-VVKHVVGFQSLPVDMLSFLLSLLASSVTP

VFVLSKR AHLPCGCIINCQPDTYSVAFDGKKSKRKGFEFNLSFQQSYGLYKIPHADCYD

DDENSISYHNPKNYECEATKEP GDNRSVFNTITVEISTTPPLDSATLTGVHKCTNTDIP ESKQAMKEEKGAFSVKTESDINYGETTSFEGPERRLSHEENQKPDLSDWEWCRSKSERTP

RQRSGGGLAIPICAFQGTVSLQAPTGKTLSLSTYEVSAEGQKITPASKKIEVYRSKSVGH

EPNSEESPSTFADTSVKIHLEVLEICDNDEALDTVSIISNISQSSTKVRSPSLRYSRKEN

RFVSCDLGETASYSLFLPTSDPDGDINISIPDTVEAHRQNSRRQHEERDGYQEEIQLLNK AYRKREAESKGD

SEQ ED NO:5

ATGTCGTTCTTCCTCAGCAACTTAACAAATGACTCTAGACTGTGGAAAGTAAGTCATAAT TCTACGGACCTTATGAATTCGCCAGAAACTCTGACTCTTTCTCTCTTTTGCCTGATCTGT TTAATGACTCTCGTAGCTCTGGTAGGCAGCATTTTCTCACTGGTTTCTCTGCTCACCATG CAGTACAGAACTGTTGTGTCTATGCTTGTGACCTCTTGGTCTGTGGATGATCTCTTGAGC GTCTTGTCAGTGGCCATCTTCATGGTTTTGCAGTGGCCAAGAGAGGCACCGGGCTACTTC CAGTCTCTGTGTACCACCTCTGCCTTACTGTATATGTGCCAGGGCCTCTCCAGCAACTTG AAGGCGACTCTTATAGTCTTCTACAACTTTTATACGATGCACAGGACAGTGGTGAGTCAG TCAAGCTCCTGGCGATCAGGACAGGTACTCGGTGTGGCTTTAACCGTGTGGGCAGTCAGC CTGCTGCTGGCCTCGCTCCCGCTGTGCGGCTGGGGTGTCTTCGTGCGCACGCCTTGGGGC TGCCTGACCGACTGCTCCAGCCCATACGTGCTGCTCCTCTTCGCGGTGTACGCTTCCGCT TTCGGGCTCCTGGCGGTTCTTTCTGTCCCTCTCACTCACCAGCTGCTGTGTTCAGAGGAG CCGCCAAGACTCCATGCCAACTACCAGGAAATTTCTCGTGGCGCCTCTACTCCTGGGACC CCTGCTGCTGGGGGAAGAGTGCTCTGTCTGTTGCCAGAGGATGTTGAAATCCCTGCTCTG CCAGGCACTGGGAGCTCCCTGAGCTCCGACATGGTGTTTGCACCGGGTCAGCCTGCTGCC TCTAGCGCTGGAGCCGGCAAGCGAGAGAATCTCTGGACTCCCCGTGGCTCGAGCAGCTTC CCCGTGAGCCTGGCACAGAAGCGCTTTGCTTTGATCCTAGCGCTGACTAAAGTCATCCTC TGGCTGCCTATGATGATCCACATGGTGGTAAAACACGTGGTGGGGTTTCAGAGCCTTCCG GTCGATATGCTCAGCTTTCTACTAACCCTGCTGGCCAGCACTGTAACACCAGTGTTTGTC TTGTCTAAACGATGGGCCCACTTGCCTTGTGGCTGCATCATCAACTGCCAGCCTGACACC TACTCCGTTGCATTCGATGGGAAAAAGTCAAAGAGGAAAGGCTTTGAATTCAATCTATCG TTCCAACAAAGTTATGGACTCTATAAAATGACACATGCAGATTACTATGACGATGATGAT GAAAATCCCATATCCTACCACAACCCAAAGAAATATGAGTGTGAAGCTACAAAGGAGCCT CGGGAAGACAACCACGGTGTGTTCAATACCATCACAGTAGAAATCAGCACCACGCCACCG CTGGACAGCGCCACACTGACCGGTGTCAACAAGTGCACCAACACGGATATCCCTGAGCCC AAACAGGCCGTGAGCGAAGAAAAGGGTGCCTTTTCTATTAAAACAGAATGTGCTATTAAC TATGGAGAAGCCACCTCGTTTGAAGGTCCAGAGAGACGACTGTCCCATGAGGAGACTCAG AAACCAGATCTTTCAGACTGGGAGTGGTGCAGGAGTAAGTCAGAACGAACACCTCGCCAG CGTTCTGGTGGTGGGCTGGCTATTCCCATTTGTGCGTTCCAAGGGACTGTGTCTCTCCAA GCACCAACTGGGAAAACCCTCTCTCTTTCTACTTATGAGGTCAGTGCAGAAGGGCAAAAG ATAACCCCGCCCTCTAAGAAAATAGAAGTCTATCGATCTAAAAGTGTTGGCCATGAACCG AACTCAGAAGAGTCTCCCTCCACGTTTGCAGACACCAACGTAAAAATACACTTGGAAGTT CTTGAAATTTGTGACAACGATGAGGCTTTAGACACTGTGTCAATCATCAGCAACATCAGC CAGTCCTCTACAAAAGTTCGGTCTCCTTCTCTGCGCTACTCACGGAAGGAAAACAGGTTT GTCTCATGCGACTTAGGAGAAACTGCCTCCTACTCCCTCTTCTTACCCACAAGTGATCCT GACGGAGATATCAACATCTCTATCCCAGACACAGTGGAGGCACACAGACAGAACAGCAGG CGGCAGCATCAAGACAGGGATGGCTACCAGGAAGAGATCCAGTTGTTAAACAAAGCCTAC AGGAAAAGAGAAGCAGAAAGCAAGGGTAACTAG

SEQ ID NO:6

MSFFLSNLTNDSRL KVSHNSTDLMNSPETLTLSLFCLICLMTLVALVGSIFSLVSLLTM QYRTWSMLVTSWSVDDLLSVLSVAIFMVLQ PREAPGYFQSLCTTSALLYMCQGLSSNL

KATLIVFYNFYTMHRTWSQSSSWRSGQV GVALTV AVSLLLASLPLCG GVFVRTP G

CLTDCSSPYVLLLFAVYASAFGLLAVLSVPLTHQLLCSEEPPRLHANYQEISRGASTPGT

PAAGGRVLCLLPEDVEIPALPGTGSSLSSD VFAPGQPAASSAGAGKRENL TPRGSSSF

PVSLAQKRFALILALTKVIL LPM IHMVVKHVVGFQSLPVDMLSFLLTLLASTVTPVFV LSKR AHLPCGCIINCQPDTYSVAFDGKKSKRKGFEFNLSFQQSYGLYKMTHADYYDDDD

ENPISYH PKKYECEATKEPREDNHGVFNTITVEISTTPPLDSATLTGVNKCTNTDIPEP

KQAVSEEKGAFSIKTECAINYGEATSFEGPERRLSHEETQKPDLSD EWCRSKSERTPRQ

RSGGGLAIPICAFQGTVSLQAPTGKTLSLSTYEVSAEGQ ITPPSKKIEVYRSKSVGHEP

NSEESPSTFADTNVKIHLEVLEICDNDEALDTVSIISNISQSSTKVRSPSLRYSRKENRF VSCDLGETASYSLFLPTSDPDGDINISIPDTVEAHRQNSRRQHQDRDGYQEEIQLLNKAY RKREAESKGN

SEQ ID NO:7

GTATGGAGGCATGGAAAGAACAGGTGATTTGCATTCTCACCTCACCAACATAAATCAGGCCTGCCACCATGA TCAGACGGGCTATGCTGCTGTGACAGTGATGGAGAGCTGAAGAGAATCAGGGTCAGAGACTGCAGGCCGCTG TGATGGAGATGGATCACTCTGTGCAAGATGTTGCTTTTCACGTGCTGCTCTTCCAAGGTTGAAAACAAAGTT CCTCCATAAGATTGCCTCAAGGCTCCTTAGTA

SEQ ID NO:8

GTGTCTTCGTGCGCACGCCTTGGGGCTGCCTGACCGACTGCTCCAGCCCATACGTGCTGC TCCTCTTCGCGGTATACGCTTCCGCTTTCGGGCTCCTGGCGGTTCTCTCTGTCCCTCTCA CTCACCAGCTGCTGTGTTCGGAGGAACCGCTGAGACTCCATGCCAACTACCAGGAAATTT CTCGTGGCGCCTCTACTCCTGGGACCCCTGCTGCTGCGGGAAGAGTGCTCTGTCTGCCGC CAGAGGATGTTGAGATCCCTGCTCTGCGATGCACTGGGGGATGCTCCCCGAGCTCCGACG TGGTGTTTGCCCCGGGTCAGCCGGCTGCCTCCGGCGCTGGAGCCGGCAGGCGAGAGAATC CCGGGACTCCCCAGGGCACCAACAGCTTCCCCTTGAGCTTGGCACAGAAGCGCTTTTCTT TGATTCTAGCG

SEQ ID NO:9 VFVRTPWGCLTDCSSPYVLLLFAVYASAFGLLAVLSVPLTHQL CSEEPLR HANYQEIS RGASTPGTPAAAGRVLCLPPEDVE I PALRCTGGCS PS SDWFAPGQPAASGAGAGRRENP GTPQGTNS FP S AQKRFS ILA

SEQ ID NO:10

ATGTCTTTATTTCTCAGTAACTTATCAACAAATGACTCTAGCCTGTGGAAAGAGAATCAT AATTCTACGGACCTTTTAAATCCGCCAGGAACCCTGAATATCTATCTTTTTTGCTTGACA TGTCTCATGACTTTTGCAGCCTTGGTGGGCAGCATTTATTCACTAATTTCCCTGCTGAAA ATGCAGAACAGAACTGTTGTGTCCATGCTTGTGGCTTCCTGGTCTGTGGATGATCTCATG AGCGTCCTGTCGGTGACCATCTTCATGTTTTTGCAGTGGCCAAACGAGGTCCCCGGTTAC TTCCAATTTCTGTGCACCACCTCTGCCTTAATGTATTTATGCCAGGGCCTCTCTAGCAAC TTGAAGGCGACTCTCCTAGTCTCTTACAACTTTTATACGATGCACAGAGGTGTGGGGAGC CAGACAGCCTCCAGAAGATCGGGCCAGGTGCTCGGCGTGGTGCTGACCGTGTGGGCAGCC AGTCTGCTGCTCTCGGCGCTCCCGCTGTGCGGCTGGGGCGCCTCCGTGCGCACGCCCTGG GGCTGCCTGGTGGACTGCTCCAGCTCCTACGTACTATTCCTCTCTATCGTGTACGCTTTG GCCTTCGGACTCCTCGTGGGCCTCTCAGTCCCACTCACTCACCGATTGCTGTGTTCGGAG GAGCCGCCGAGACTCCACTCCAACTACCAGGAAATTTCCCGTGGAGCTTCAATTCCTGGG ACCCCTCCTACTGCGGGGAGAGTGGTTTCCCTGTCCCCAGAGGATGCTCCAGGCCCGAGT CTGCGGCGCTCTGGGGGATGCTCTCCGAGCTCCGACACCGTGTTCGGACCGGGTGCGCCC GCTGCCGCTGGGGCTGAAGCCTGCAGGCGTGAGAACCGGGGGACTCTCTATGGCACCAGG AGCTTCACCGTGAGCGTAGCGCAGAAGCGCTTCGCTTTGATCCTAGCGCTTACAAAAGTC GTCCTTTGGCTGCCCATGATGATGCACATGGTGGTCCAGAACGTCGTGGGGTTTCAGAGC CTTCCCTTGGAGACATTCAGCTTCCTACTTACCCTGCTGGCCACCACTGTAACCCCAGTG TTTGTCTTGTCCAAACGCTGGACCCACTTGCCCTGTGGCTGCATCATCAACTGCAGGCAG AATGCATATGCAGTGGCGTCCGATGGGAAAAAAATCAAGAGAAAAGGCTTTGAATTCAAT CTATCATTCCAAAAAAGTTATGGGATTTATAAAATAGCACATGAAGATTACTATGATGAT GATGAAAATTCCATATTCTATCACAACCTGATGAACTCTGAGTGTGAAACTACAAAAGAC CCTCAGAGAGACAACCGTAACATCTTCAATGCTATAAAAGTAGAAATCAGCACCACGCCC TCTCTGGACAGCTCCACACAAAGAGGCATCAACAAATGCACAAATACTGATATTACAGAA GCTAAACAGGATTCCAACAACAAAAAGGATGCGTTTTCTGACAAAACAGGAGGTGATATT AACTATGAAGAAACTACCTTTTCTGAAGGGCCAGAAAGAAGACTGTCTCATGAAGAGAGT CAGAAACCAGATCTTTCAGACTGGGAGTGGCGTAGGAGTAAATCAGAAAGAACCCCTCGT CAGCGTTCCGGTTATGCCCTTGCCATTCCCTTGTGTGCATTCCAGGGGACTGTGTCTCTC CATGCACCTACAGGGAAAACCCTATCTCTTTCTACCTATGAGGTAAGCGCAGAAGGGCAA AAAATAACTCCAGCCTCTAAGAAAATAGAAGTCTATCGATCCAAAAGTGTTGGCCATGAA CCAAACTCAGAAGATTCTTCATCC

SEQ ID NO:11

MSLFLSNLSTNDSSL KENHNSTDLLNPPGTLNIYLFCLTCLMTFAALVGSIYSLISLLK QNRTWSMLVAS SVDDLMSVLSVTIF FLQ PNEVPGYFQFLCTTSALMYLCQGLSSN LKATLLVSYNFYT HRGVGSQTASRRSGQVLGWLTV AASLLLSALPLCG GASVRTPW GCLVDCSSSYVLFLSIVYALAFGLLVGLSVPLTHRLLCSEEPPRLHS YQEISRGASIPG TPPTAGRWSLSPEDAPGPSLRRSGGCSPSSDTVFGPGAPAAAGAEACRRENRGTLYGTR SFTVSVAQKRFALILALTKWL LPMMMHMWQNWGFQSLPLETFSFLLTLLATTVTPV FVLSKR THLPCGCI INCRQNAYAVASDGKKIKRKGFEFNLSFQKSYGIYKIAHEDYYDD DENSIFYHNLMNSECETTKDPQRDNR IFNAIKVEISTTPSLDSSTQRGINKCTNTDITE AKQDSNNKKDAFSDKTGGDI YEETTFSEGPERRLSHEESQKPDLSDWE RRSKSERTPR

QRSGYALAIPLCAFQGTVSLHAPTGKTLSLSTYEVSAEGQKITPASKKIEVYRSKSVGHE PNSEDSSS

Claims

Claims

1. An isolated polypeptide comprising a polypeptide sequence which has at least 95% identity to the polypeptide sequence of SEQ ID NO:2, 4, 6 or 1 1.

2. The polypeptide as claimed in claim 1 comprising the polypeptide sequence of SEQ ID NO:2, 4, 6 or 11.

3. The isolated polypeptide of SEQ ID NO:2, 4, 6 or 1 1.

4. An isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide sequence which has at least 95% identity to a polypeptide of SEQ ID NO:2, 4, 6 or 11 ; or a polynucleotide sequence complementary to said isolated polynucleotide.

5. An isolated polynucleotide which comprises a polynucleotide sequence which has at least 95% identity to that of SEQ ID NO: 1, 3, 5 or 10; or a polynucleotide sequence complementary to said isolated polynucleotide.

6. An isolated polynucleotide selected from: (a) a polynucleotide comprising a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2, 4, 6 or l l;

(b) a polynucleotide encoding the polypeptide of SEQ ID NO:2, 4, 6 or 11 ;

(c) the polynucleotide of SEQ ID NO:l, 3, 5 or 10; and

(d) a polynucleotide obtainable by screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1, 3, 5, 10 or a fragment thereof; or a nucleotide sequence complementary to said isolated polynucleotide over the entire length thereof.

7. An expression system comprising a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present in a compatible host cell.

8. A host cell comprising the expression system of claim 7 or a membrane thereof expressing the polypeptide of claim 1.

9. A process for producing a polypeptide of claim 1 comprising the step of culturing a host cell of claim 8 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture medium.

10. An antibody immunospecific for the polypeptide of any one of claims 1 to 3.

1 1. A method for screening to identify compounds which stimulate or which inhibit the function of the polypeptide of claim 1 which comprises a method selected from the group consisting of:

(a) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes expressing the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound;

(b) measuring the binding of a candidate compound to the polypeptide (or to the cells or membranes expressing the polypeptide) or a fusion protein thereof in the presense of a labeled competitior; (c) testing whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells or cell membranes expressing the polypeptide;

(d) mixing a candidate compound with a solution containing a polypeptide of claim 1, to form a mixture, measuring the activity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard; or

(e) detecting the effect of a candidate compound on the production of mRNA encoding said polypeptide and said polypeptide in cells, using for instance, an ELISA assay.

12. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of the polypeptide of claim 1 in a subjectcomprising:

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in the genome of said subject; and/or

(b) analyzing for the presence or amount of said polypeptide expression in a sample derived from said subject.