Patent Publication Number: US-2004053812-A1

Title: Methods

Description:
METHODS  
       [0001] This invention relates to the regulation of metabolism and in particular to human genes involved in appetite control or obesity. The invention also relates to the identification of ligands that interact with receptors encoded by such genes and the provision of therapeutic agents.  
       [0002] Obesity is now a major health problem. Currently 22.5% of the US population is considered to be clinically obese, 18.5% in the UK and with many other developed countries following this trend. It has been described as the most extensive non-communicable disease of the 21 st  century. Currently available treatments are reviewed by M. Lean in Exp. Clin. Endocrinol. Diabetes, 1998, 106, Suppl. 2, 22-26. These include diet and, in extreme cases, surgery.  
       [0003] It is only in recent years that the genetic basis and influences on obesity have been studied in detail. By some estimates 40-70% of the variation in obesity-related phenotypes in humans is heritable. The search for human obesity genes is conveniently summarised by Comuzzie et al (Science, 1998, 280, 1374-1377). In particular, leptin (LEP), the product of the ob gene, and the leptin receptor (LEPR) have now been studied in detail. Leptin is a hormone secreted by adipose tissue which, along with its receptor, are integral parts of the complex physiological system which has evolved to regulate and control energy balance and storage at an optimal level (Freidman J M and Halaas J L 1998 Nature 395, 763-769). Leptin also appears to play an important role in relaying nutritional status to several other physiological systems. The relevance of leptin to the pathogenesis of obesity in general is the subject of much study and underlines the complex nature of human obesity. A human obesity gene map is now available and the number of genes and other markers that have been associated or linked with human obesity phenotypes now approaches 200.  
       [0004] A large number of products are being developed for the treatment of obesity and eating disorders and these are targeted against a wide range of biological targets. The chosen targets include enzymes, hormones, neurotransmitters as well as the so-called G-protein-coupled-receptors (GPCRs). The GPCRs represent one of the largest families of genes so far identified. Over 800 family members have been cloned to date from a wide variety of species.  
       [0005] Our investigations have now revealed that the mRNA coding for a G-protein-coupled-receptor, GPR22, is differentially expressed in rodent appetite/obesity models. It follows that peptidic and non-peptidic compounds which modulate the biological activity of GPR22, will have utility in controlling food intake and metabolic processes.  
       [0006] Human GPR22 is a receptor with no known physiological ligand—i.e. it is an “Orphan Receptor”. The gene encoding GPR22 is located on human chromosome 7 q22-q31.1 (O&#39;Dowd, et al., 1997, Gene, 187, 75-81). The receptor with closest identity is the receptor for cholecystokinin-B, with which it shares 34% identity in the transmembrane domains (ibid). GPR22 is expressed in discrete brain regions including thalamus, caudate, frontal cortex and putamen (ibid).  
       [0007] A cDNA encoding GPR22 was cloned by O&#39;Dowd B F et al., 1997, Gene 187, 75-81. The cDNA sequence has been submitted to the Genbank database under accession number U66581. The amino acid sequence is published in the EMBL database under accession number Q99680.  
       [0008] Therefore in a first aspect of the present invention we provide a method for the provision of an appetite control agent which method comprises using one or more agonists and/or antagonists of the G protein coupled receptor GPR22 as test compounds in one or more appetite control test procedures, and selecting an active compound for use as an appetite control agent.  
       [0009] Convenient appetite control test procedures include the use of animal models to test the role of the test compound in appetite control and obesity. These will typically involve the administration of compounds by intra peritoneal injection, subcutaneous injection, intravenous injection, oral gavage or direct injection via canullae into the CNS of experimental animals. The effects on food intake, body temperature, metabolic rate, behavioural activities and body weight changes may all be measured using standard procedures.  
       [0010] Suitable antagonists or agonists may be firstly identified by screening for agonists and/or antagonists of GPR22.  
       [0011] Therefore in a further aspect of the present invention we provide a method for the provision of an appetite control agent which method comprises (i) screening for agonists and/or antagonists of GPR22 and (ii) using one or more agonists and/or antagonists so identified as test compounds in one or more appetite control test procedures, and selecting an active compound for use as an appetite control agent.  
       [0012] GPR22 is from any mammalian species, including human, rat, mouse, monkey, and dog. For screening purposes GPR22 is conveniently human GPR22.  
       [0013] Mammalian GPR22 may be conveniently isolated from commercially available RNA, brain cDNA libraries, genomic DNA, or genomic DNA libraries using conventional molecular biology techniques such as library screening and/or polymerase chain reaction (PCR). These techniques are extensively detailed in Molecular Cloning—A Laboratory Manual, 2 nd  edition, Sambrook, Fritsch &amp; Maniatis, Cold Spring Harbor Press.  
       [0014] The resulting cDNA&#39;s encoding mammalian GPR22 are then cloned into commercially available mammalian expression vectors such as pcDNAIII (In Vitrogen Ltd etc. see below). An alternative mammalian expression vector is disclosed by Davies et al., J of Pharmacol &amp; Toxicol. Methods, 33, 153-158. Standard transfection technologies are used to introduce these DNA&#39;s into commonly available cultured, mammalian cell lines such as CHO, HEK293, HeLa and clonal derivatives expressing the receptors are isolated. An alternative expression system is the MEL cell expression system claimed in our UK patent no. 2251622.  
       [0015] Application of a natural ligand to these cells causes activation of the transfected receptor that will cause changes in the levels of intracellular signalling molecules such as cyclic-AMP, intracellular calcium ions or arachidonic acid metabolite release. These may all be measured using standard published procedures and commercially available reagents. In addition, the receptor cDNA&#39;s may be transfected into derivatives of these cells lines that have previously been transfected with a “reporter” gene such as bacterial LacZ, Luciferase, aequorin or green fluorescent protein that will “report” these intracellular changes.  
       [0016] The natural ligand for GPR22 is not yet known. The cells transfected with GPR22 may be used to find natural ligands that will activate GPR22. Ligands may be sourced commercially or synthesised chemically (Lembo et al., 1999, Nature Cell Biol., 1, 267-271) or may be purified from mammalian sources such as animal brain extracts (Saurai et al., 1998, Cell, 92, 573-585). Once identified, purified, radiolabelled or fluorescently labelled material (eg. Amersham PLC &amp; Advanced Bioconcept Ltd) may be used as a ligand to detect ligand binding to the transfected receptors using standard published ligand binding assay technologies.  
       [0017] The transfected cell lines may be used to identify low molecular weight compounds that activate the receptors and cause changes in intracellular signalling molecules which mimic the effects of the natural ligand, these are defined as “agonists”.  
       [0018] In addition or alternatively, the same assays can be used to identify low molecular weight compounds that prevent activation of the receptors and suppress the effects of the natural ligand, these are defined as “antagonists”.  
       [0019] The test compound may be a polypeptide of equal to or greater than 2 amino acids, such as up to 6 amino acids, up to 10 or 12 amino acids, up to 20 amino acids or greater than 20 amino acids such as up to 50 amino acids. For drug screening purposes, preferred compounds are chemical compounds of low molecular weight and potential therapeutic agents. They are for example of less than about 2000 daltons, such as less than 1500, 1000, 800, 600 or 400 daltons in weight. If desired the test compound may be a member of a chemical library. This may comprise any convenient number of individual members, for example tens to hundreds to thousands to millions of suitable compounds, for example peptides, peptoids and other oligomeric compounds (cyclic or linear), and template-based smaller molecules, for example benzodiazepines, hydantoins, biaryls, carbocyclic and polycyclic compounds (eg. naphthalenes, phenothiazines, acridines, steroids etc.), carbohydrate and amino acids derivatives, dihydropyridines, benzhydryls and heterocycles (eg. triazines, indoles, thiazolidines etc.). The numbers quoted and the types of compounds listed are illustrative, but not limiting. Preferred chemical libraries comprise chemical compounds of low molecular weight and potential therapeutic agents.  
       [0020] In a further aspect of the invention we provide the use of an agonist of GPR22 as an appetite control agent.  
       [0021] In a further aspect of the invention we provide the use of an antagonist of GPR22 as an appetite control agent.  
       [0022] It will be appreciated that the present invention includes the use of orthologues and homologues of human GPR22.  
       [0023] By the term “orthologue” we mean the functionally equivalent receptor in other species.  
       [0024] By the term “homologue” we mean a substantially similar and/or related receptor in the same or a different species.  
       [0025] For either of the above definitions we believe the receptors may have for example at least 30%, such as at least 40%, at least 50%, at least 60%, and in particular at least 70%, such as at least 80%, for example 85%, or 90% or 95% peptide sequence identity. It is appreciated that homologous receptors may have substantially higher peptide sequence identity over small regions representing functional domains. We include receptors having greater diversity in their DNA coding sequences than outlined for the above amino acid sequences but which give rise to receptors having peptide sequence identity falling within the above sequence ranges. Convenient versions of GPR22 include the published sequence (ref al.ibid) and the sequence identities set out in tables 1 to 3.  
       [0026] Fragments and partial sequences of the GPR22 may be useful substrates in the assay and analytical methods of the invention. It will be appreciated that the only limitation on these is practical, they must comprise the necessary functional elements for use in the relevant assay and/or analytical procedures.  
       [0027] In a further aspect of the present invention we provide a method of appetite control which method comprises administering to an individual a pharmaceutically effective amount of an appetite control agent identified using one or more of the methods of this invention.  
       [0028] The appetite control agent of this invention may be administered in standard manner for the condition that it is desired to treat, for example by oral, topical, parenteral, buccal, nasal, or rectal administration or by inhalation. For these purposes the compounds of this invention may be formulated by means known in the art into the form of, for example, tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely divided powders or aerosols for inhalation, and for parenteral use (including intravenous, intramuscular or infusion) sterile aqueous or oily solutions or suspensions or sterile emulsions.  
       [0029] Knowledge of the GPR22 gene provides the ability to regulate its expression in vivo by for example the use of antisense oligonucleotides. Thus, according to a further aspect of the invention we provide an appetite control agent comprising an antisense oligonucleotide which is complementary to all or a part of a polynucleotide sequence shown in table 1. By complementary we mean that the two molecules can hybridise to form a double stranded molecule through nucleotide base pair interactions. U.S. Pat. No. 5,639,595, Identification of Novel Drugs and Reagents, issued Jun. 17, 1997, wherein methods of identifying oligonucleotide sequences that display in vivo activity are thoroughly described, is herein incorporated by reference.  
       [0030] The antisense oligonucleotide for co-operation with a polynucleotide sequence corresponding to all or a part of a GPR22 gene may be produced using conventional means, by standard molecular biology and/or by chemical synthesis. If desired, the antisense oligonucleotide may be chemically modified so as to prevent degradation in vivo or to facilitate passage through a cell membrane and/or a substance capable of inactivating mRNA, for example ribozyme, may be linked thereto. Examples of antisense molecules include but are not limited to DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2′-O-alkylRNA, or other oligonucleotide mimetics such as peptide nucleic acids. U.S. Pat. No. 5,652,355, Hybrid Oligonucleotide Phosphorothioates, issued Jul. 29, 1997, and U.S. Pat. No. 5,652,356, Inverted Chimeric and Hybrid Oligonucleotides, issued Jul. 29, 1997, which describe the synthesis and effect of physiologically-stable antisense molecules, are herein incorporated by reference.  
       [0031] The antisense oligonucleotide can be complementary to the full length GPR22 gene of the invention or to a fragment thereof. Antisense molecules which comprise oligomers in the range from about 12 to about 30 nucleotides which are complementary to the regions of the gene which are proximal to, or include, the protein coding region, or a portion thereof, are preferred embodiments of the invention. GPR22 gene antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence.  
       [0032] GPR22 may also be used as the basis for diagnosis, for example to determine expression levels in a human subject, by for example direct DNA sequence comparison or DNA/RNA hybridisation assays. Diagnostic assays may involve the use of nucleic acid amplification technology such as PCR and in particular the Amplification Refractory Mutation System (ARMS) as claimed in our European Patent No. 0 332 435. Such assays may be used to determine allelic variants of the gene, for example insertions, deletions and/or mutations such as one or more point mutations. Such variants may be heterozygous or homozygous. Other approaches have been used to identify mutations in genes encoding similar molecules in obese patients (Yeo et al., 1998, Nature Genetics, 20, 111-112).  
       [0033] In a further aspect of the invention GPR22 can be genetically engineered in such a way that its interactions with other intracellular and membrane associated proteins are maintained but its effector function and biological activity are removed. The genetically modified protein is known as a dominant negative mutant. Overexpression of the dominant negative mutant in an appropriate cell type down regulates the effect of the endogenous protein, thus revealing the biological role of the genes in appetite control.  
       [0034] Similarly, GPR22 may also be genetically engineered in such a way that its effector function and biological activity are enhanced. The resultant overactive protein is known as dominant positive mutant. Overexpression of a dominant positive mutant in an appropriate cell type amplifies the biological response of the endogenous, native protein, spotlighting its role in appetite control. This also has utility in a screen for detecting antagonists of the constitutively active receptor in the absence of a ligand.  
       [0035] Therefore, in a further aspect of the invention we provide dominant negative and dominant positive mutants of GPR22 and their use in evaluating the biological role of GPR22 in the control of appetite.  
       [0036] Transgenic animal technology is also contemplated, providing new experimental models, useful for evaluating the effects of test compounds on the control of obesity and eating disorders. GPR22 genes may be deleted, inactivated or modified using standard procedures as outlined briefly below and as described for example in “Gene Targeting; A Practical Approach”, IRL Press, 1993. The target gene or a portion of it, for example homologous sequences flanking the coding region, is preferably cloned into a vector with a selection marker (such as Neo) inserted into the gene to disrupt its function. The vector is linearised then transformed (usually by electroporation) into embryonic stem cells (ES) cells (eg derived from a 129/Ola strain of mouse) and thereafter homologous recombination events take place in a proportion of the stem cells. The stem cells containing the gene disruption are expanded and injected into a blastocyst (such as for example from a C57BL/6J mouse) and implanted into a foster mother for development. Chimaeric offspring may be identified by coat colour markers. Chimaeras are bred to ascertain the contribution of the ES cells to the germ line by mating to mice with genetic markers which allow a distinction to be made between ES derived and host blastocyst derived gametes. Half of the ES cell derived gametes will carry the gene modification. Offspring are screened (for example by Southern blotting) to identify those with a gene disruption (about 50% of the progeny). These selected offspring will be heterozygous and may therefore be bred with another heterozygote to produce homozygous offspring (about 25% of the progeny).  
       [0037] Transgenic animals with a target gene deletion (“knockouts”) may be crossed with transgenic animals produced by known techniques such as microinjection of DNA into pronuclei, sphaeroplast fusion or lipid mediated transfection of ES cells to yield transgenic animals with an endogenous gene knockout and a foreign gene replacement. ES cells containing a targeted gene disruption may be further modified by transforming with the target gene sequence containing a specific alteration. Following homologous recombination the altered gene is introduced into the genome. These embryonic stem cells may subsequently be used to create transgenics as described above.  
       [0038] The transgenic animals will display a phenotype which reflects the role of GPR6 in the control of appetite and obesity and will thus provide useful experimental models in which to evaluate the effects of test compounds. Therefore in a further aspect of the invention we provide transgenic animals in which GPR22 genes are deleted, inactivated or modified, and their use in evaluating the effects of test compounds in appetite control and obesity. 
     
    
    
     [0039] The invention will now be illustrated but not limited by reference to the following specific description and tables [Many of the specific techniques used are detailed in standard molecular biology textbooks such as Sambrook, Fritsch &amp; Maniatis, Molecular cloning, a Laboratory Manual, Second Edition, 1989, Cold Spring Harbor Laboratory Press. Consequently references to this will be made at the appropriate points in the text.]:  
     [0040] PCR Cloning of GPR22  
     [0041] Oligonucleotide primers of 30 nucleotides in length corresponding to sequences immediately 5′ of the initiating ATG codon and immediately 3′ of the termination codon for the coding sequences of human and rodent GPR22 (sequences below) are synthesised. Commercial sources of rodent and human brain RNA are used as templates in standard RT-PCR reactions with these primers. RT-PCR primers are designed to incorporate nucleotides coding for tag sequences e.g. myc, His 6 to facilitate purification of the proteins at a later stage. Commercially available RT-PCR kits are used in accordance with the suppliers instructions and as documented in the Sambrook reference cited above. Products of the PCR vector are cloned using standard technology (ibid) into the plasmid vector pBluescript (Stratagene Ltd.). Plasmid DNA is isolated (ibid) and subjected to DNA sequence analysis (ibid) to identify a clone containing the GPR22 sequence identical to those listed below. The inserts corresponding to GPR22 cDNA are released from this DNA using standard digestion procedures and with appropriate restriction endonuclease enzymes. The inserts are then cloned into suitably prepared plasmid DNA using standard technology (ibid). These plasmids are the expression vectors used in the studies described below.  
     [0042] Cloning Into Expression Vectors  
     [0043] (i) A variety of mammalian expression vectors may be used to express the recombinant GPR22 molecule as well as variants contemplated herein. Commercially available mammalian expression vectors which are suitable for recombinant expression, include but are not limited to, pcDNA3 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC 37565), pLXIN and pSIR (Clontech), pIRES-EGFP (Clontech). Plasmid DNA containing the GPR22 cDNA inserts is then purified (ibid) and introduced into appropriate host cells.  
     [0044] (ii) A vector is described for use with the Mouse Erythroleukaemia Cells (MEL) expression system using the human beta globin gene locus control region (Davies et al., J of Pharmacol &amp; Toxicol. Methods, 33, 153-158.). This vector system and derivatives thereof may also be used. Plasmid DNA containing the GPR22 cDNA inserts is then purified (ibid) and introduced into appropriate host cells.  
     [0045] Transfection/Selection of Host Cells  
     [0046] (i) Mammalian expression vector plasmid DNA is introduced (ibid) into cultured mammalian cells. Eukaryotic recombinant host cells are especially preferred. Examples include but are not limited to yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616),BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and HEK293 (ATCC CRL 1573). In addition the DNA is introduced into variants of these cell lines that have previously been transfected and selected to express other proteins such as β-galactosidase, or mutated G-proteins such as Gal6 (Milligan et al, 1996, TiPS, 17, 235-237). Clones of mammalian cells expressing GPR22 cDNA are identified by selecting mammalian cell clones that have been selected on the basis of their resistance to antbiotics due to the presence of appropriate resistance genes on the parental plasmids (See Maniatis, et al), by RT-PCR of the introduced sequences and by detection of protein using specific antibodies.  
     [0047] (ii) The DNA containing the beta-globin locus control region and GPR22 cDNA is introduced into MEL cells, clones are selected and analysed as described (Davies et al. op cit).  
     [0048] The expression vectors may be introduced into host cells expressing GPR22 via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation. Commercially available kits applicable for use with the present invention for hererologous expression, including well-characterised vectors, transfection reagents and conditions, and cell culture materials are well-established and readily available. [CLONTECH, Palo Alto, Calif.; INVITROGEN, Carlsbad, Calif.; PHARMINGEN, San Diego, Calif.; STRATAGENE, LaJolla, Calif.] 
     [0049] Identification of Ligands for GPR22.  
     [0050] Identification of the natural ligand for GPR22 entails successive purification and assay steps using rat, porcine, or other animal brain as starting material. Homogenised brain tissue is fractionated by conventional biochemical methods and fractions are screened for activity in the reporter cell assays described below. Detailed protocols for these methods are available (Sakurai, et al. 1998, Cell, 92:573-585). Successive purification procedures yield a purified ligand for GPR22 that is characterised by sequencing methodologies (ibid).  
     [0051] Cell Binding Assay.  
     [0052] Mammalian cells isolated from the selection procedures described above are cultured by standard techniques and exposed to  125 [I] ligand. Following extensive washing of cells to remove unbound material the extent of ligand binding is quantitated in a Gammamaster counter (Packard) using the methods described in detail by Davies et al. (op cit). Cell clones showing the greatest binding of this ligand are progressed to the next phase of this process.  
     [0053] Membrane Preparation  
     [0054] The mammalian cell clones identified by the method described above are cultured, harvested and used as the source of membrane preparation. Membranes are prepared from these cell clones by standard biochemical techniques that are described in detail by Davies et al. (op cit).  
     [0055] Ligand Binding Assays  
     [0056] (i) Cell membranes isolated from these mammalian cell clones are used to establish conventional ligand binding assays as described in detail in Davies et al. (op cit). or:  
     [0057] (ii) The same membranes are used with the same radioligand or with GTPg[S]35 to develop a scintillation proximity assay (SPA) using proprietary SPA beads developed by Amersham Ltd. Licences and detailed protocols for this technique are available from Amersham Ltd.  
     [0058] Reporter Cell Assays:—cAMP/Ca++Flux/Arachidonic Acid Metabolite Release  
     [0059] Cells expressing GPR22 are identified as described above. These cells have also been engineered to express the LacZ gene coupled to the mammalian cyclic AMP response element (Egerton et al, J. Mol. Endocrinol, 1995, 14(2), 179-189). When cAMP levels increase in the cell the transcription of the LacZ gene is proportionately increased and may be measured by standard beta-galactosidase assays (Maniatis et al., ibid).  
     [0060] Cells expressing GPR22 are also engineered to express the G-protein Gal6 (Milligan et al., 1996, TiPS, 17, 235-237). Upon activation the cells respond by increasing intracellular calcium concentrations. This increase is measured after pre-exposure of the cells to a fluorescent compound such as, but not limited to, Fura2 Molecular Probes Ltd) by reading on any commercially available fluorescence analysing equipment (Lembo et al., 1999, Nature Cell Biol., 1, 267-271).  
     [0061] Cells expressing GPR22 are also assayed for the increased release of radiolabelled arachidonic acid metabolites following pre-incubation of the cells to  3 [H] arachidonic acid and stimulation by PrRP31 (Davies et al., ibid).  
     [0062] Compound Screening  
     [0063] Chemical compounds are tested for their ability to inhibit (antagonise) the biological activity of GPR22 and to increase (agonise) the activity of GPR22.  
     [0064] (i) The ligand binding and SPA assays described above are conducted in the presence of varying amounts of individual compounds that will reveal those compounds that have the ability to displace the natural ligands from GPR22.  
     [0065] (ii) These compounds are applied to the mammalian cells in the presence and absence of ligand and those compounds that influence the output of the assays described above are identified.  
     [0066] The following methods, which do not require the presence of any ligand, are also suitable for identifying chemical compounds having the desired properties. Agonists: The reporter cells containing GPR22 are exposed to chemical compounds in the absence of any ligand, and assayed, as described, for changes in intracellular cAMP, and Cat++ as well as for increased arachidonic acid metabolite release. Antagonists: The GPR22 cDNA is mutated using standard molecular biology techniques (Maniatis, ibid) and transfected into the mammalian reporter cells, as described. Cell lines harbouring mutated receptors that give increased reporter gene activity are then used to screen chemical compounds for their ability to suppress this reporter gene activity through antagonising the constitutively active receptors.  
     [0067] Compound Testing In Vivo  
     [0068] Compounds identified from the assays described above are considered for testing in animal models. Appropriately formulated compounds are administered by, but not limited to, oral gavage, intraperitoneal, intravenous, intramuscular or intracerebrovascular injection or infusion. Animals will include, but are not limited to, standard laboratory rodents, dogs and primates, obese Zucker rats, obese (ob/ob) mice, and diabetic (db/db) mice. The animals may be fed standard laboratory diets, or may be offered altered diets, including but not limited to, diets designed to induce hyperphagia and weight gain, for example high fat, high carbohydrate (Stock, 1998, Clinical Obesity, Oxford Press, 50-72). The effect of compound on the following, but not limited to, will be established: food intake parameters, water intake, body weight changes, body fat, protein and water composition, endocrine parameters, metabolic substrate concentrations, energy expenditure and behavioural activities, using standard physiological, biochemical and neurobiological methods (Halford et al, 1998, Pharmacol. Biochem. Behav., 61, 159-168, Shimada et al, 1998, Nature, 396, 670-674).  
     [0069] Antibody Production  
     [0070] The GPR22 polypeptide can be used to raise diagnostic antibodies to detect the receptor in cultured cells and in vivo. Therefore, in accordance with yet a further aspect of the present invention, there are provided antibodies against the GPR22 polypeptide which may be used as part of various diagnostic assays for detecting physiological eating disorders. An example for the production of effective polyclonal antibodies against peptides derived from the known amino acid sequences of GPR22 utilises a well established algorithm method developed by Jameson and Wolf,  The antigenic Index: A novel Algorithm for Predicting Antigenic Determinants , CABIOS, 4:181 (1988). Peptide molecules of typically between 10-20 amino acid residues are synthesised chemically and conjugated to keyhole limpet hemocyanin and used for antibody generation by Genosys Biotechnologies, 1442 Lake Front Circle, Suite 185, The Woodlands, Tex. 77380. Specific antibodies may be raised by immunising animals, with rabbits being preferred, with an appropriate concentration of the GPR22 peptides either with or without an immune adjuvant.  
     [0071] Monospecific antibodies to the polypeptide of the present invention are purified from mammalian antisera containing antibodies reactive against the GPR22 polypeptide using the technique of Kohler and Milstein, Nature, 256:495 (1975). Mono-specific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for the novel signal transduction molecule. Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with sequences set out in tables 2 and 3. Monoclonal antibodies are produced in vivo by injection of pristane primed Balb/c mice, approximately 0.5 ml per mouse, with about 2×10 6  to about 6×10 6  hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art. In vitro production of the anti-polypeptide mAb is carried out by growing the hydridoma in DMEM containing about 2% foetal calf serum to obtain sufficient quantities of the specific mAb. The mAb are purified by techniques known in the art.  
               TABLE 1                       Human GPR22 cDNA sequence                                        Genbank accession number U66581           gttatttcttcaaaaggaaaacacaattttcttttatatcaaaacaatgc               aaacttgatggttcttaattctacattttctattaatagtttacaaactt               aaaaattaaactaagtacacaattgaaagatttttttttcttacaaagaa               cacgttatacgtcatttaaattgccaaatatcaaatagtttattctattt               cactttctagggaaaaaaaccaactgctccaaaagaatgtgtttttctcc               cattctggaaatcaacatgcagtctgaatctaacattacagtgcgagatg               acattgatgacatcaacaccaatatgtaccaaccactatcatatccgtta               agctttcaagtgtctctcaccggatttcttatgttagaaattgtgttggg               acttggcagcaacctcactgtattggtactttactgcatgaaatccaact               taatcaactctgtcagtaacattattacaatqaatcttcatgtacttgat               gtaataatttgtgtgqgatgtattcctctaactatagttatccttctgct               ttcactggagagtaacactgctctcatttgctgtttccatgaggcttgtg               tatcttttgcaagtgtctcaacagcaatcaacgtttttgctatcactttg               gacagatatgacatctctgtaaaacctgcaaaccqaattctgacaatggg               cagagctgtaatgttaatgatatccatttggattttttcttttttctctt               tcctgattccttttattgagqtaaattttttcagtcttcaaagtggaaat               acctgggaaaacaagacacttttatgtqtcagtacaaatgaatactacac               tgaactgggaatgtattatcacctgttagtacagatcccaatattctttt               tcactgttgtagtaatgttaatcacatacaccaaaatacttcaggctctt               aatattcgaataggcacaagattttcaacagggcagaagaagaaagcaag               aaagaaaaag&amp;caatttctctaaccacacaacatgaggctacagacatgt               cacaaagcagtggtggqagaaatgtagtctttggtgtaagaacttcaqtt               tctgtaataattgccctccggcgagctgtgaaacgacaccgtgaacgacg               agaaagacaaaagagagtcttcaggatgtctttattgattatttctacat               ttcttctctgctggacaccaatttctgttttaaataccaccattttatgt               ttaggcccaagtgaccttttagtaaaattaagattgtgttttttagtcat               ggcttatggaacaactatatttcaccctctattatatgcattcactagac               aaaaatttcaaaaggtcttgaaaagtaaaatgaaaaagcgagttgtttct               atagtagaagctgatcccctgcctaataatgctgtaatacacaactcttg               qatagatcccaaaagaaacaaaaaaattacctttgaagatagtgaaataa               gagaaaaacgtttagtgcctcaggttgtcacagactagagaaaagtctca               gtttcaccaaatccacattcaaatgagttttaaatttaaattgtaaaaac               tgatattactgccaaatataagaaaaatattttaagtattggttatgttg               taaattttcaatqtgaaatgctaattagataggtcatatatattcaattt               cttcattacttaatgtatttgttgcatggcagtttgttaaagtactatca               tgtgtatattttgtcaatattatgtccaacagaaaatattcatgtaagtc               atattttttaaggaataaatacatagccttaaaacagtgtataactttaa               aatgtaaaaaaaaaaaaaaaaaaaaaaaaaa                  
 
     [0072]               TABLE 2                       Human GPR22 amino acid sequence                                        EMBL accession number Q99680           MCFSPILEINMQSESNITVRDDIDDINTNMYQPLSYPLSFQVSLTGFLML               EIVLGLGSNLTVLVLYCMKSNLINSVSNIITMNLHVLDVIICVGCIPLTI               VILLLSLESNTALICCFHEACVSFASVSTAINVFAITLDRYDISVKPANR               ILTMGRAVMLMISIWIFSFFSFLIPFIEVNFFSLQSGNTWENKTLLCVST               NEYYTELGMYYHLLVQIPIFFFTVVVMLITYTKILQALNTRIGTRFSTGQ               KKKARKKKTISLTTQHEATDMSQSSGGRNVVFGVRTSVSVIIALRRAVKR               HRERRERQKRVFRMSLLIISTFLLCWTPISVLNTTILCLGPSDLLVKLRL               CFLVMAYGTTIFHPLLYAFTRQKFQKVLKSKMKKRVVSIVEADPLPNNAV               IHNSWIDPKRNKKITFEDSEIREKRLVPQVVTD                    
     [0073] 
    
     
       
         1 
         
           
             2  
           
           
             1  
             1881  
             DNA  
             Human  
           
            1 

gttatttctt caaaaggaaa acacaatttt cttttatatc aaaacaatgc aaacttgatg     60 

gttcttaatt ctacattttc tattaatagt ttacaaactt aaaaattaaa ctaagtacac    120 

aattgaaaga tttttttttc ttacaaagaa cacgttatac gtcatttaaa ttgccaaata    180 

tcaaatagtt tattctattt cactttctag ggaaaaaaac caactgctcc aaaagaatgt    240 

gtttttctcc cattctggaa atcaacatgc agtctgaatc taacattaca gtgcgagatg    300 

acattgatga catcaacacc aatatgtacc aaccactatc atatccgtta agctttcaag    360 

tgtctctcac cggatttctt atgttagaaa ttgtgttggg acttggcagc aacctcactg    420 

tattggtact ttactgcatg aaatccaact taatcaactc tgtcagtaac attattacaa    480 

tgaatcttca tgtacttgat gtaataattt gtgtgggatg tattcctcta actatagtta    540 

tccttctgct ttcactggag agtaacactg ctctcatttg ctgtttccat gaggcttgtg    600 

tatcttttgc aagtgtctca acagcaatca acgtttttgc tatcactttg gacagatatg    660 

acatctctgt aaaacctgca aaccgaattc tgacaatggg cagagctgta atgttaatga    720 

tatccatttg gattttttct tttttctctt tcctgattcc ttttattgag gtaaattttt    780 

tcagtcttca aagtggaaat acctgggaaa acaagacact tttatgtgtc agtacaaatg    840 

aatactacac tgaactggga atgtattatc acctgttagt acagatccca atattctttt    900 

tcactgttgt agtaatgtta atcacataca ccaaaatact tcaggctctt aatattcgaa    960 

taggcacaag attttcaaca gggcagaaga agaaagcaag aaagaaaaag acaatttctc   1020 

taaccacaca acatgaggct acagacatgt cacaaagcag tggtgggaga aatgtagtct   1080 

ttggtgtaag aacttcagtt tctgtaataa ttgccctccg gcgagctgtg aaacgacacc   1140 

gtgaacgacg agaaagacaa aagagagtct tcaggatgtc tttattgatt atttctacat   1200 

ttcttctctg ctggacacca atttctgttt taaataccac cattttatgt ttaggcccaa   1260 

gtgacctttt agtaaaatta agattgtgtt ttttagtcat ggcttatgga acaactatat   1320 

ttcaccctct attatatgca ttcactagac aaaaatttca aaaggtcttg aaaagtaaaa   1380 

tgaaaaagcg agttgtttct atagtagaag ctgatcccct gcctaataat gctgtaatac   1440 

acaactcttg gatagatccc aaaagaaaca aaaaaattac ctttgaagat agtgaaataa   1500 

gagaaaaacg tttagtgcct caggttgtca cagactagag aaaagtctca gtttcaccaa   1560 

atccacattc aaatgagttt taaatttaaa ttgtaaaaac tgatattact gccaaatata   1620 

agaaaaatat tttaagtatt ggttatgttg taaattttca atgtgaaatg ctaattagat   1680 

aggtcatata tattcaattt cttcattact taatgtattt gttgcatggc agtttgttaa   1740 

agtactatca tgtgtatatt ttgtcaatat tatgtccaac agaaaatatt catgtaagtc   1800 

atatttttta aggaataaat acatagcctt aaaacagtgt ataactttaa aatgtaaaaa   1860 

aaaaaaaaaa aaaaaaaaaa a                                             1881 

 
           
             2  
             433  
             PRT  
             Human  
           
            2 

Met Cys Phe Ser Pro Ile Leu Glu Ile Asn Met Gln Ser Glu Ser Asn 
1               5                   10                  15 

Ile Thr Val Arg Asp Asp Ile Asp Asp Ile Asn Thr Asn Met Tyr Gln 
            20                  25                  30 

Pro Leu Ser Tyr Pro Leu Ser Phe Gln Val Ser Leu Thr Gly Phe Leu 
        35                  40                  45 

Met Leu Glu Ile Val Leu Gly Leu Gly Ser Asn Leu Thr Val Leu Val 
    50                  55                  60 

Leu Tyr Cys Met Lys Ser Asn Leu Ile Asn Ser Val Ser Asn Ile Ile 
65                  70                  75                  80 

Thr Met Asn Leu His Val Leu Asp Val Ile Ile Cys Val Gly Cys Ile 
                85                  90                  95 

Pro Leu Thr Ile Val Ile Leu Leu Leu Ser Leu Glu Ser Asn Thr Ala 
            100                 105                 110 

Leu Ile Cys Cys Phe His Glu Ala Cys Val Ser Phe Ala Ser Val Ser 
        115                 120                 125 

Thr Ala Ile Asn Val Phe Ala Ile Thr Leu Asp Arg Tyr Asp Ile Ser 
    130                 135                 140 

Val Lys Pro Ala Asn Arg Ile Leu Thr Met Gly Arg Ala Val Met Leu 
145                 150                 155                 160 

Met Ile Ser Ile Trp Ile Phe Ser Phe Phe Ser Phe Leu Ile Pro Phe 
                165                 170                 175 

Ile Glu Val Asn Phe Phe Ser Leu Gln Ser Gly Asn Thr Trp Glu Asn 
            180                 185                 190 

Lys Thr Leu Leu Cys Val Ser Thr Asn Glu Tyr Tyr Thr Glu Leu Gly 
        195                 200                 205 

Met Tyr Tyr His Leu Leu Val Gln Ile Pro Ile Phe Phe Phe Thr Val 
    210                 215                 220 

Val Val Met Leu Ile Thr Tyr Thr Lys Ile Leu Gln Ala Leu Asn Ile 
225                 230                 235                 240 

Arg Ile Gly Thr Arg Phe Ser Thr Gly Gln Lys Lys Lys Ala Arg Lys 
                245                 250                 255 

Lys Lys Thr Ile Ser Leu Thr Thr Gln His Glu Ala Thr Asp Met Ser 
            260                 265                 270 

Gln Ser Ser Gly Gly Arg Asn Val Val Phe Gly Val Arg Thr Ser Val 
        275                 280                 285 

Ser Val Ile Ile Ala Leu Arg Arg Ala Val Lys Arg His Arg Glu Arg 
    290                 295                 300 

Arg Glu Arg Gln Lys Arg Val Phe Arg Met Ser Leu Leu Ile Ile Ser 
305                 310                 315                 320 

Thr Phe Leu Leu Cys Trp Thr Pro Ile Ser Val Leu Asn Thr Thr Ile 
                325                 330                 335 

Leu Cys Leu Gly Pro Ser Asp Leu Leu Val Lys Leu Arg Leu Cys Phe 
            340                 345                 350 

Leu Val Met Ala Tyr Gly Thr Thr Ile Phe His Pro Leu Leu Tyr Ala 
        355                 360                 365 

Phe Thr Arg Gln Lys Phe Gln Lys Val Leu Lys Ser Lys Met Lys Lys 
    370                 375                 380 

Arg Val Val Ser Ile Val Glu Ala Asp Pro Leu Pro Asn Asn Ala Val 
385                 390                 395                 400 

Ile His Asn Ser Trp Ile Asp Pro Lys Arg Asn Lys Lys Ile Thr Phe 
                405                 410                 415 

Glu Asp Ser Glu Ile Arg Glu Lys Arg Leu Val Pro Gln Val Val Thr 
            420                 425                 430 

Asp