HK1091509A - Human g-protein coupled receptor - Google Patents

Description

Novel human G protein-coupled receptor

Description of the invention

The present invention relates to novel polynucleotides which have been identified, polypeptides encoded by them, and the use and production of these polynucleotides and polypeptides. More particularly, the polynucleotides and polypeptides of the present invention relate to G-protein coupled receptors (GPCRs), hereinafter referred to as IGS 4. There are two polymorphic forms of IGS4, hereinafter referred to as IGS4A and IGS 4B. The invention also relates to inhibiting or activating the action of these polynucleotides and polypeptides, vectors comprising said polynucleotides, host cells comprising such vectors, and transgenic animals in which the IGS4 gene is overexpressed, misexpressed, underexpressed, or suppressed (knock-out animals). The invention also relates to methods for screening compounds capable of acting as agonists or antagonists of the G-protein coupled receptor IGS4, and to related ligands of IGS 4.

Background

It is well documented that many biological processes of medical importance are mediated by proteins involved in signal transduction pathways involving G proteins and/or second messengers such as cAMP (Lefkowitz, Nature 51: 353-354, 1991). These proteins are referred to herein as proteins involved in the G protein pathway. Examples of such proteins include GPC receptors such as adrenergic agents and dopamine receptors (B.K. Kobilka et al, Proc.Natl.Acad.Sci.USA 84: 46-50, 1987; B.K. Kobilka et al, Science 238: 650-; the G protein itself; effector proteins such as phospholipase C, adenylate cyclase, and phosphodiesterase; and activins (actuatorproteins) such as protein kinase A and protein kinase C (M.I. Simon et al Science 252: 802-.

For example, in one form of signal transduction, upon hormone binding to a GPCR, the receptor interacts with the heterotrimeric G protein and induces the dissociation of GDP from the guanine nucleotide binding site. At normal cellular concentrations of guanine nucleotides, GTP fills the site immediately. Binding of GTP to the G protein α subunit causes dissociation of the G protein from the receptor and the G protein dissociates into α and β γ subunits. The GTP-bearing form then binds to the activated adenylate cyclase. Hydrolysis of GTP to GDP is catalyzed by the G protein itself (the α subunit has intrinsic GTPase activity), which restores the G protein to its underlying inactive form. The gtpase activity of the alpha subunit is essentially the intrinsic clock that controls the on/off switching. The GDP-bound form of the alpha subunit has a high affinity for β γ, and subsequent re-binding of α GDP to β γ restores the system to its basal state. Thus, the G protein serves a dual role: as an intermediate, the signal is transmitted from the receptor to the effector (in this case adenylate cyclase); and as a clock, controls the duration of the signal.

It has been identified that the membrane-bound superfamily of G protein-coupled receptors has 7 putative transmembrane domains. These domains are thought to be transmembrane α -helices joined together by extracellular or cytoplasmic loops. G protein-coupled receptors include a wide range of biologically active receptors such as hormones, viruses, growth factors, and neuroreceptors.

The G protein-coupled receptor family includes dopamine receptors, which bind neuroleptic drugs used to treat CNS disorders. Other examples of members of this family include, but are not limited to, calcitonin, adrenergic, neuropeptide Y, somatostatin, neurotensin, neurokinin, capsaicin, VIP, CGRP, CRF, CCK, bradykinin, somatostatin (galanin), motilin, nociceptin, endothelin, cAMP, adenosine, muscarine, acetylcholine, 5-hydroxytryptamine, histamine, thrombin, kinin, follicle stimulating hormone, opsin, endothelial differentiation gene-1, rhodopsin, odorant (odorant), and receptors for cytomegalovirus.

Most G protein-coupled receptors have a single conserved cysteine residue in the first two extracellular loops, and the disulfide bonds they form are thought to stabilize the structure of functional proteins. The 7 transmembrane regions are designated TM1, TM2, TM3, TM4, TM5, TM6, and TM 7. The cytoplasmic loop linking TM5 with TM6 may be a major component of the G protein binding domain.

Most G protein-coupled receptors contain potential phosphorylation sites at the 3 rd cytoplasmic loop and/or the carboxy terminus. For several G protein-coupled receptors, such as the β -adrenoreceptor, phosphorylation by protein kinase a and/or specific receptor kinases mediates desensitization of the receptor.

It has recently been discovered that certain GPCRs, such as the calcitonin receptor-like receptor, may interact with small single transmembrane proteins known as Receptor Activity Modifying Proteins (RAMP). This interaction of a GPCR with a certain RAMP determines which natural ligand has the relevant affinity for the GPCR-RAMP combination and regulates the functional signaling activity of the complex (L.M. McLathie et al, Nature 393: 333-339, 1998).

For some receptors, the ligand binding site of G protein-coupled receptors is believed to comprise a hydrophilic groove formed by several transmembrane domains of the G protein-coupled receptor, surrounded by hydrophobic residues of the G protein-coupled receptor. It is postulated that the hydrophilic side of each transmembrane helix of G protein-coupled receptors faces inward and forms a polar ligand binding site. Among several G protein-coupled receptors, TM3 is involved in ligand binding sites, such as TM3 aspartic acid residues. Serine from TM5, asparagine from TM6, and phenylalanine or tyrosine from TM6 or TM7 are also involved in ligand binding.

G protein-coupled receptors can couple a variety of intracellular enzymes, ion channels, and transporters in cells via heterotrimeric G proteins (see Johnson et al, endocrine review (endo. Rev.) 10: 317-. Different G protein alpha subunits preferentially stimulate specific effectors, thereby modulating a variety of biological functions in a cell. Phosphorylation of cytoplasmic residues of G protein-coupled receptors has been identified as an important mechanism for the regulation of G protein coupling of certain G protein-coupled receptors. G protein-coupled receptors are found in many sites in mammalian hosts.

Of the known drugs, more than half are produced by receptors, mainly GPCR classes (Drews, Nature Biotechnology) 14: 1516, 1996). This suggests that these receptors have a proven, proven history as therapeutic targets. Clearly, there is a need to identify and characterize other receptors that may be used to prevent, ameliorate, or correct functional disorders or diseases, including but not limited to PNS disorders, psychosis, and CNS disorders, including schizophrenia, Episodic Paroxysmal Anxiety (EPA) disorders such as Obsessive Compulsive Disorder (OCD), post-traumatic stress disorder (PTSD), phobias, and panic, major depression, bipolar disorder, parkinson's disease, generalized anxiety disorder, autism, delirium, multiple sclerosis, alzheimer's disease/dementia, and other neurodegenerative disorders, severe mental retardation, movement disorders, huntington's disease, tourette's syndrome, tics, tremors, dystonia, spasticity, anorexia, bulimia, stroke, addiction/dependence/craving, sleep disorders, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension (such as essential hypertension, renal hypertension, or pulmonary hypertension), thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, raynaud's disease, renal disease (such as renal failure); dyslipidemias (dyslipdemiias); obesity; vomiting; gastrointestinal disorders including Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), gastroesophageal reflux disease (GERD), motility disorders (motility disorders), and conditions of delayed gastric emptying such as post-operative or diabetic gastroparesis, and diabetes, ulcers (e.g., gastric ulcers); diarrhea; other diseases, including osteoporosis; inflammation; infections, such as bacterial, fungal, protozoal, and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancer; damage induced by chemotherapy; tumor invasion; immune disorders; urinary retention; asthma; (ii) an allergic reaction; arthritis; benign prostatic hyperplasia; (ii) endotoxic shock; sepsis; diabetic complications; and gynecological diseases.

Summary of The Invention

One aspect of the invention relates to IGS4 polypeptides (including IGS4A and IGS4B polypeptide polymorphisms), polynucleotides, and recombinant materials and methods for their production. Another aspect of the invention relates to methods of using these IGS4 polypeptides, polynucleotides, and recombinant materials. These uses include, but are not limited to, as therapeutic targets for the treatment of PNS disorders, psychosis, and CNS disorders including schizophrenia, paroxysmal anxiety (EPA) disorders such as Obsessive Compulsive Disorder (OCD), post-traumatic stress disorder (PTSD), phobias, and panic, major depression, bipolar disorder, parkinson's disease, generalized anxiety disorder, autism, delirium, multiple sclerosis, alzheimer's disease/dementia, and other neurodegenerative disorders, severe mental retardation, movement disorders, huntington's disease, tourette's syndrome, tics, tremor, dystonia, spasticity, anorexia, bulimia, stroke, addiction/dependence/craving, sleep disorders, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension (such as essential hypertension, renal hypertension, or pulmonary hypertension), thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, raynaud's disease, renal disease (such as renal failure); dyslipidemias (dyslipdemiias); obesity; vomiting; gastrointestinal disorders including Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), gastroesophageal reflux disease (GERD), motility disorders (motility disorders), and conditions of delayed gastric emptying such as post-operative or diabetic gastroparesis, and diabetes, ulcers (e.g., gastric ulcers); diarrhea; other diseases, including osteoporosis; inflammation; infections, such as bacterial, fungal, protozoal, and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancer; damage induced by chemotherapy; tumor invasion; immune disorders; urinary retention; asthma; (ii) an allergic reaction; arthritis; benign prostatic hyperplasia; (ii) endotoxic shock; sepsis; diabetic complications; and gynecological diseases, etc. Preferred uses of the invention relate to disorders of the nervous system, including the Central Nervous System (CNS) and the Peripheral Nervous System (PNS), the gastrointestinal system, the cardiovascular system, skeletal muscles, and/or the thyroid, and/or lung diseases, immunological diseases, and genitourinary disorders.

Another aspect of the invention relates to methods of using the materials provided herein to identify agonists and antagonists, and to use the identified compounds to treat conditions associated with an imbalance of IGS 4. Another aspect of the invention relates to diagnostic assays for detecting diseases associated with abnormal IGS4 activity or levels. Another aspect of the invention relates to animal model systems for disorders caused by abnormal expression or activity of IGS 4. Preferred agonists or antagonists identified with reference to the present invention are useful in the treatment of disorders of the nervous system, including the Central Nervous System (CNS) and the Peripheral Nervous System (PNS), of the gastrointestinal system, of the cardiovascular system, of skeletal muscle, and/or of the thyroid, and/or of pulmonary diseases, immunological diseases, and genitourinary disorders.

The invention also relates to the identification of cognate ligands for the IGS4 polypeptides of the invention. A neuropeptide known as neuromedin U was found to have high affinity for binding to the IGS4 polypeptide.

Table 1: SEQ ID NO: 1 and SEQ ID NO: 3 IGS4A-DNA

5′-GGCTCAGCTTGAAACAGAGCCTCGTACCAGGGGAGGCTCAGGCCTTGGATTTTAATGTCAGGGATGGAAAAACTTCAGAATGCTTCCTGGATCTACCAGCAGAAACTAGAAGATCCATTCCAGAAACACCTGAACAGCACCGAGGAGTATCTGGCCTTCCTCTGCGGACCTCGGCGCAGCCACTTCTTCCTCCCCGTGTCTGTGGTGTATGTGCCAATTTTTGTGGTGGGGGTCATTGGCAATGTCCTGGTGTGCCTGGTGATTCTGCAGCACCAGGCTATGAAGACGCCCACCAACTACTACCTCTTCAGCCTGGCGGTCTCTGACCTCCTGGTCCTGCTCCTTGGAATGCCCCTGGAGGTCTATGAGATGTGGCGCAACTACCCTTTCTTGTTCGGGCCCGTGGGCTGCTACTTCAAGACGGCCCTCTTTGAGACCGTGTGCTTCGCCTCCATCCTCAGCATCACCACCGTCAGCGTGGAGCGCTACGTGGCCATCCTACACCCGTTCCGCGCCAAACTGCAGAGCACCCGGCGCCGGGCCCTCAGGATCCTCGGCATCGTCTGGGGCTTCTCCGTGCTCTTCTCCCTGCCCAACACCAGCATCCATGGCATCAAGTTCCACTACTTCCCCAATGGGTCCCTGGTCCCAGGTTCGGCCACCTGTACGGTCATCAAGCCCATGTGGATCTACAATTTCATCATCCAGGTCACCTCCTTCCTATTCTACCTCCTCCCCATGACTGTCATCAGTGTCCTCTACTACCTCATGGCACTCAGACTAAAGAAAGACAAATCTCTTGAGGCAGATGAAGGGAATGCAAATATTCAAAGACCCTGCAGAAAATCAGTCAACAAGATGCTGTTTGTCTTGGTCTTAGTGTTTGCTATCTGTTGGGCCCCGTTCCACATTGACCGACTCTTCTTCAGCTTTGTGGAGGAGTGGAGTGAATCCCTGGCTGCTGTGTTCAACCTCGTCCATGTGGTGTCAGGTGTCTTCTTCTACCTGAGCTCAGCTGTCAACCCCATTATCTATAACCTACTGTCTCGCCGCTTCCAGGCAGCATTCCAGAATGTGATCTCTTCTTTCCACAAACAGTGGCACTCCCAGCATGACCCACAGTTGCCACCTGCCCAGCGGAACATCTTCCTGACAGAATGCCACTTTGTGGAGCTGACCGAAGATATAGGTCCCCAATTCCCATGTCAGTCATCCATGCACAACTCTCACCTCCCAACAGCCCTCTCTAGTGAACAGATGTCAAGAACAAACTATCAAAGCTTCCACTTTAACAAAACCTGAATTCTTTCAGAGCTGACTCTCCTCTATGCCTCAAAACTTCAGAGAGGAACATCCCATAATGTATGCCTTCTCATATGATATTAGAGAGGTAGAATGGCTCTTACAACTCATGTACCCATTGCTAGTTTTTTTTTTTTAATAAACGTGAAAACTGAGAGTTAGATCTGGTTTCAAAACCCAAGACTGCCTGATTTTTAGTTATCTTTCCACTATCCTAACTGCCTCATGCCCCTTCACTAGTTCATGCCAAGAACGTGACTGGAAAGGCATGGCACCTATACCTTGATTAATTTCCATTAATGGAAATGGTTCGTCCTGAGTCATCTACGTTCCGAGTCAGGCTGTCACTCCTACTA-3′

Table 2: SEQ ID NO: 5 and SEQ ID NO: 7 IGS4B-DNA

5′-GGCTCAGCTTGAAACAGAGCCTCGTACCAGGGGAGGCTCAGGCCTTGGATTTTAATGTCAGGGATGGAAAAACTTCAGAATGCTTCCTGGATCTACCAGCAGAAACTAGAAGATCCATTCCAGAAACACCTGAACAGCACCGAGGAGTATCTGGCCTTCCTCTGCGGACCTCGGCGCAGCCACTTCTTCCTCCCCGTGTCTGTGGTGTATGTGCCAATTTTTGTGGTGGGGGTCATTGGCAATGTCCTGGTGTGCCTGGTGATTCTGCAGCACCAGGCTATGAAGACGCCCACCAACTACTACCTCTTCAGCCTGGCGGTCTCTGACCTCCTGGTCCTGCTCCTTGGAATGCCCCTGGAGGTCTATGAGATGTGGCGCAACTACCCTTTCTTGTTCGGGCCCGTGGGCTGCTACTTCAAGACGGCCCTCTTTGAGACCGTGTGCTTCGCCTCCATCCTCAGCATCACCACCGTCAGCGTGGAGCGCTACGTGGCCATCCTACACCCGTTCCGCGCCAAACTGCAGAGCACCCGGCGCCGGGCCCTCAGGATCCTCGGCATCGTCTGGGGCTTCTCCGTGCTCTTCTCCCTGCCCAACACCAGCATCCATGGCATCAAGTTCCACTACTTCCCCAATGGGTCCCTGGTCCCAGGTTCGGCCACCTGTACGGTCATCAAGCCCATGTGGATCTACAATTTCATCATCCAGGTCACCTCCTTCCTATTCTACCTCCTCCCCATGACTGTCATCAGTGTCCTCTACTACCTCATGGCACTCAGACTAAAGAAAGACAAATCTCTTGAGGCAGATGAAGGGAATGCAAATATTCAAAGACCCTGCAGAAAATCAGTCAACAAGATGCTGTTTGTCTTGGTCTTAGTGTTTGCTATCTGTTGGGCCCCGTTCCACATTGACCGACTCTTCTTCAGCTTTGTGGAGGAGTGGACTGAATCCCTGGCTGCTGTGTTCAACCTCGTCCATGTGGTGTCAGGTGTCTTATTCTACCTGAGCTCAGCTGTCAACCCCATTATCTATAACCTACTGTCTCGCCGCTTCCAGGCAGCATTCCAGAATGTGATCTCTTCTTTCCACAAACAGTGGCACTCCCAGCATGACCCACAGTTGCCACCTGCCCAGCGGAACATCTTCCTGACAGAATGCCACTTTGTGGAGCTGACCGAAGATATAGGTCCCCAATTCCTATGTCAGTCATCCGTGCACAACTCTCACCTCCCAACAGCCCTCTCTAGTGAACAGATGTCAAGAACAAACTATCAAAGCTTCCACTTTAACAAAACCTGAATTCTTTCAGAGCTGACTCTCCTCTATGCCTCAAAACTTCAGAGAGGAACATCCCATAATGTATGCCTTCTCATATGAAATTAGAGAGGTAGAATGGCTCTTACAACTCATGTACCCATTGCTAGTTTTTTTTTTTTAATAAACGTGAAAACTGAGAGTTAGATCTGGTTTCAAAACCCAAGACTGCCTGATTTTTAGTTATCTTTCCACTATCCTAACTGCCTCATGCCCCTTCACTAGTTCATGCCAAGAACGTGACTGGAAAGGCATGGCACCTATACCTTGATTAATTTCCATTAATGGAAATGGTTCGTCCTGAGTCATCTACGTTCCGAGTCAGGCTGTCACTCCTACTA-3′

Table 3: SEQ ID NO: 9 and SEQ ID NO: 11 IGS4A-64-DNA

5′-GGCTCAGCTTGAAACAGAGCCTCGTACCAGGGGAGGCTCAGGCCTTGGATTTTAATGTCAGGGATGGAAAAACTTCAGAATGCTTCCTGGATCTACCAGCAGAAACTAGAAGATCCATTCCAGAAACACCTGAACAGCACCGAGGAGTATCTGGCCTTCCTCTGCGGACCTCGGCGCAGCCACTTCTTCCTCCCCGTGTCTGTGGTGTATGTGCCAATTTTTGTGGTGGGGGTCATTGGCAATGTCCTGGTGTGCCTGGTGATTCTGCAGCACCAGGCTATGAAGACGCCCACCAACTACTACCTCTTCAGCCTGGCGGTCTCTGACCTCCTGGTCCTGCTCCTTGGAATGCCCCTGGAGGTCTATGAGATGTGGCGCAACTACCCTTTCTTGTTCGGGCCCGTGGGCTGCTACTTCAAGACGGCCCTCTTTGAGACCGTGTGCTTCGCCTCCATCCTCAGCATCACCACCGTCAGCGTGGAGCGCTACGTGGCCATCCTACACCCGTTCCGCGCCAAACTGCAGAGCACCCGGCGCCGGGCCCTCAGGATCCTCGGCATCGTCTGGGGCTTCTCCGTGCTCTTCTCCCTGCCCAACACCAGCATCCATGGCATCAAGTTCCACTACTTCCCCAATGGGTCCCTGGTCCCAGGTTCGGCCACCTGTACGGTCATCAAGCCCATGTGGATCTACAATTTCATCATCCAGGTCACCTCCTTCCTATTCTACCTCCTCCCCATGACTGTCATCAGTGTCCTCTACTACCTCATGGCACTCAGACTAAAGAAAGACAAATCTCTTGAGGCAGATGAAGGGAATGCAAATATTCAAAGACCCTGCAGAAAATCAGTCAACAAGATGCTGTCTTTGTGGAGGAGTGGAGTGAATCCCTGGCTGCTGTGTTCAACCTCGTCCATGTGGTGTCAGGTGTCTTCTTCTACCTGAGCTCAGCTGTCAACCCCATTATCTATAACCTACTGTCTCGCCGCTTCCAGGCAGCATTCCAGAATGTGATCTCTTCTTTCCACAAACAGTGGCACTCCCAGCATGACCCACAGTTGCCACCTGCCCAGCGGAACATCTTCCTGACAGAATGCCACTTTGTGGAGCTGACCGAAGATATAGGTCCCCAATTCCCATGTCAGTCATCCATGCACAACTCTCACCTCCCAACAGCCCTCTCTAGTGAACAGATGTCAAGAACAAACTATCAAAGCTTCCACTTTAACAAAACCTGAATTCTTTCAGAGCTGACTCTCCTCTATGCCTCAAAACTTCAGAGAGGAACATCCCATAATGTATGCCTTCTCATATGATATTAGAGAGGTAGAATGGCTCTTACAACTCATGTACCCATTGCTAGTTTTTTTTTTTTAATAAACGTGAAAACTGAGAGTTAGATCTGGTTTCAAAACCCAAGACTGCCTGATTTTTAGTTATCTTTCCACTATCCTAACTGCCTCATGCCCCTTCACTAGTTCATGCCAAGAACGTGACTGGAAAGGCATGGCACCTATACCTTGATTAATTTCCATTAATGGAAATGGTTCGTCCTGAGTCATCTACGTTCCGAGTCAGGCTGTCACTCCTACTA-3′

Table 4: SEQ ID NO: 2 and SEQ ID NO: 4 IGS 4A-protein

(3 amino acids not included in parentheses)

(MSG)MEKLQNASWIYQQKLEDPFQKHLNSTEEYLAFLCGPRRSHFFLPVSVVYVPIFVVGVIGNVLVCLVILQHQAMKTPTNYYLFSLAVSDLLVLLLGMPLEVYEMWRNYPFLFGPVGCYFKTALFETVCFASILSITTVSVERYVAILHPFRAKLQSTRRRALRILGIVWGFSVLFSLPNTSIHGIKFHYFPNGSLVPGSATCTVIKPMWIYNFIIQVTSFLFYLLPMTVISVLYYLMALRLKKDKSLEADEGNANIQRPCRKSVNKMLFVLVLVFAICWAPFHIDRLFFSFVEEWSESLAAVFNLVHVVSGVFFYLSSAVNPIIYNLLSRRFQAAFQNVISSFHKQWHSQHDPQLPPAQRNIFLTECHFVELTEDIGPQFPCQSSMHNSHLPTALSSEQMSRTNYQSFHFNKT

Table 5: SEQ ID NO: 6 and SEQ ID NO: 8 IGS 4B-protein

(3 amino acids not included in parentheses)

(MSG)MEKLQNASWIYQQKLEDPFQKHLNSTEEYLAFLCGPRRSHFFLPVSVVYVPIFVVGVIGNVLVCLVILQHQAMKTPTNYYLFSLAVSDLLVLLLGMPLEVYEMWRNYPFLFGPVGCYFKTALFETVCFASILSITTVSVERYVAILHPFRAKLQSTRRRALRILGIVWGFSVLFSLPNTSIHGIKFHYFPNGSLVPGSATCTVIKPMWIYNFIIQVTSFLFYLLPMTVISVLYYLMALRLKKDKSLEADEGNANIQRPCRKSVNKMLFVLVLVFAICWAPFHIDRLFFSFVEEWTESLAAVFNLVHVVSGVLFYLSSAVNPIIYNLLSRRFQAAFQNVISSFHKQWHSQHDPQLPPAQRNIFLTECHFVELTEDIGPQFLCQSSVHNSHLPTALSSEQMSRTNYQSFHFNKT

Table 6: SEQ ID NO: 10 and SEQ ID NO: 12 IGS 4A-64-protein

(3 amino acids not included in parentheses)

(MSG)MEKLQNASWIYQQKLEDPFQKHLNSTEEYLAFLCGPRRSHFFLPVSVVYVPIFVVGVIGNVLVCLVILQHQAMKTPTNYYLFSLAVSDLLVLLLGMPLEVYEMWRNYPFLFGPVGCYFKTALFETVCFASILSITTVSVERYVAILHPFRAKLQSTRRRALRILGIVWGFSVLFSLPNTSIHGIKFHYFPNGSLVPGSATCTVIKPMWIYNFIIQVTSFLFYLLPMTVISVLYYLMALRLKKDKSLEADEGNANIQRPCRKSVNKMLSLWRSGVNPWLLCSTSSMWCQVSSST

Detailed Description

Definition of

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

"IGS 4" refers to a polypeptide comprising SEQ ID NO: 2 or SEQ ID NO: 4(IGS4A), SEQ id no: 6 or SEQ ID NO: 8(IGS4B), or a variant thereof, and the like. IGS4B polypeptides are particularly preferred.

"receptor activity" or "biological activity of a receptor" refers to the metabolic or physiological function of said IGS4, including similar activities or improved activities or those activities with reduced undesirable side effects. Also included are the antigenicity and immunogenicity of said IGS 4.

"IGS 4 gene" refers to a gene comprising SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7 or a variant thereof and/or the complement thereof.

As used herein, "antibody" includes polyclonal and monoclonal antibodies; chimeric, single chain, and humanized antibodies; and Fab fragments, including products of Fab or other immunoglobulin expression libraries.

"isolated" means "artificially" altered from a natural state and/or separated from the natural environment. Thus, if a composition or substance that occurs in nature is "isolated," it is altered or separated from its original environment, or both. For example, a polynucleotide or 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 that term is used herein.

"Polynucleotide" refers generally to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes but is not limited to single-and double-stranded DNA, single-and double-stranded region of DNA, single-and double-stranded RNA, single-and double-stranded region of RNA, DNA and RNA hybrid molecules (wherein DNA and RNA can be single-stranded, or more usually double-stranded, or single-and double-stranded region of mixed single-and double-stranded region). In addition, "polynucleotide" may also include triple-stranded regions comprising RNA or DNA or both. The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases, as well as 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). Various modifications have been made to DNA and RNA; thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides commonly found in nature, as well as DNA and RNA chemical forms characteristic of viruses and cells. "Polynucleotide" also encompasses relatively short polynucleotides, commonly referred to as oligonucleotides.

"polypeptide" refers to any peptide or protein comprising 2 or more amino acids linked to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres). "polypeptide" includes short chains, often referred to as peptides, oligopeptides, or oligomers; also included are long chains, commonly referred to as proteins; and/or combinations thereof. The polypeptide may comprise amino acids other than the 20 gene-encoded amino acids. "polypeptide" includes amino acid sequences that have been modified by natural processes (such as post-translational processing) or by chemical modification techniques well known in the art. These modifications are described in detail in basic texts, in more detailed monographs, and in voluminous research literature. Modifications may occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. It is understood that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Likewise, a given polypeptide may comprise many types of modifications. The polypeptide may be branched as a result of ubiquitination, and may be cyclic with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from post-translational natural processing, or may be produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent adsorption of flavin, covalent adsorption of a heme moiety, covalent adsorption of a nucleotide or nucleotide derivative, covalent adsorption of a lipid or lipid derivative, covalent adsorption of phosphatidylinositol, cross-linking, cyclization, formation of disulfide bonds, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenization, sulfurization, tRNA-mediated addition of amino acids to proteins (such as arginylation), and ubiquitination. See, e.g., protein-STRUCTURE AND MOLECULAR characterization (PROTEINS-STRUCTUREs AND moleculae PROPERTIES), 2 nd edition, t.e.creatton, w.h.freeman AND Company, new york, 1993; wold, "post-translational protein modification: points of view and Prospects "(Posttranslational protein modifications: Perspectives and sources)," Posttranslational COVALENT modification of proteins "(Posttranslational modification of proteins) pages 1-12, b.c. johnson, Academic press, new york, 1983; seifter et al, analysis of protein modifications and non-protein cofactors "(analysis for protein modifications and nonproteins cofactors), methods in enzymology (meth. enzymol.) 182: 626. 646, 1990; and Rattan et al, "protein synthesis: post-translational modification and senescence "(Protein Synthesis: Posttranslational Modifications and Aging), New York academy of sciences yearbook (Ann.NY Acad.Sci.) 663: 48-62, 1992.

As used herein, a "variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties, such as essential biological, structural, regulatory, or biochemical properties. A typical polynucleotide variant has a different nucleotide sequence than another reference polynucleotide. Changes in the variant nucleotide sequence may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference polynucleotide, as described below. A typical polypeptide variant has a different amino acid sequence than another reference polypeptide. In general, differences are limited, and thus the sequences of the reference polypeptide and the variant are closely similar overall, being identical in many regions. The amino acid sequences of a variant and a reference polypeptide may differ by one or more substitutions, additions, and deletions, and any combination thereof. The substituted or inserted amino acid residue may or may not be encoded by the genetic code. Variants of a polynucleotide or polypeptide may be naturally occurring, such as allelic variants, or non-naturally occurring. Non-naturally occurring variants of polynucleotides and polypeptides can be produced by mutagenesis techniques or by direct synthesis.

"identity" is a measure of the identity of a nucleotide or amino acid sequence. In general, the sequences are aligned to obtain the highest level of matching. "identity" essentially has the art-recognized meaning and can be calculated using published techniques. See, e.g., COMPUTATIONAL MOLECULAR BIOLOGY (COMPUTATIONAL MOLECULAR BIOLOGY), eds. a.m. lesk, oxford university press, new york, 1988; biological calculation: INFORMATICS AND GENOME project (BIOCOMPUTING: INFORMATICS AND GENEME PROJECTS), ed by D.W.Smith, Academic Press, New York, 1993; COMPUTER ANALYSIS of sequence information (COMPUTER ANALYSIS of sequence DATA), part 1, eds a.m. griffin and h.g. griffin, Humana press, new jersey, 1994; sequence analysis IN MOLECULAR BIOLOGY (SEQUENCEANALYSIS IN MOLECULAR BIOLOGY), G.von Heinje, Academic Press, 1987; and primers for SEQUENCE analysis (SEQUENCE ANALYSIS PRIMER), compiled by M.Gribskov and J.Devereux, M Stockton Press, New York, 1991. There are many methods available for measuring identity between two polynucleotide or polypeptide sequences, the term "identity" being well known to the skilled person (h. carillo and d. lipton, the journal of SIAM applied Math 48: 1073, 1988). Methods commonly used to determine identity or similarity between two sequences include, but are not limited to, those disclosed in "large computer instructions", edited by j. bishop, Academic press, san diego, 1994; and h.carillo and d.lipton, journal of SIAM applied math (SIAM j.applied math.) 48: 1073, 1988. Methods for determining identity and similarity have been compiled into computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program software package (J.Devereux et al, Nucleic Acids Research (Nucleic Acids Research)12 (1): 387, 1984), BLASTP, BLASTN, FASTA (S.F.Atschul et al, journal of molecular biology (J.Molec.biol.) 215: 403, 1990). The word "homology" may replace "identity".

By way of illustration, for example, a polypeptide having a sequence identical to the reference nucleotide sequence SEQ ID NO: 1 "identical" means that the nucleotide sequence of the polynucleotide is substantially identical to the reference sequence except that the nucleotide sequence of the polynucleotide is identical to the nucleotide sequence of SEQ ID NO: 1 may comprise up to 5 nucleotide differences per 100 nucleotides. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or any number of nucleotides up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence, or any number of nucleotides up to 5% of the total number of nucleotides in the reference sequence may be a combination of deletion, insertion, or substitution. These mutations of the reference sequence may occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between these terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

Similarly, e.g., having an amino acid sequence identical to the reference amino acid sequence SEQ ID NO: 2 "polypeptide having an amino acid sequence of at least 95%" identity "means that the amino acid sequence of the polypeptide is substantially identical to the reference sequence except that in the reference amino acid sequence SEQ ID NO: 2 may contain up to 5 amino acid changes per 100 amino acids. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acids in the reference sequence may be deleted or substituted with other amino acids, or up to 5% of the total amino acids in the reference sequence may be inserted. Such changes to the reference sequence can occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

Polypeptides of the invention

In one aspect, the invention relates to IGS4 polypeptides (or IGS4 proteins). The IGS4 polypeptide includes SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. and SEQ ID NO: 8, and a polypeptide having an amino acid sequence encoded by a DNA insert contained in deposit number CBS102221 or deposit number CBS102222 of the fungal species collection (Centraalbureau voor Schimmelcultures) deposited at Baarn, the netherlands on 24.9.1999; comprises the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, and a polypeptide comprising an amino acid sequence encoded by a DNA insert contained in deposit number CBS102221 or deposit number CBS102222 of the fungal species collection of Baarn, the netherlands; comprising a sequence identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, and/or a polypeptide having an amino acid sequence at least 80% identical to the amino acid sequence encoded by the DNA insert contained in fungal species collection deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands, still more preferably at least 90% identical to said amino acid sequence, even still more preferably at least 95% identical. Furthermore, it is highly preferred that there is at least 97%, especially at least 99% identity. IGS4 polypeptides also include polypeptides that are identical over their full length to the polypeptide having SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8 or a polypeptide having an amino acid sequence at least 80% identical to a polypeptide having an amino acid sequence encoded by a DNA insert contained in fungal species collection deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands, still more preferably a polypeptide having an amino acid sequence at least 80% identical to a polypeptide having an amino acid sequence encoded by a DNA insert contained in SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8 are at least 90% identical, even more preferably at least 95% identical. Furthermore, it is highly preferred that there is at least 97%, in particular at least 99%. Preferred IGS4 polypeptides exhibit at least one biological activity of the receptor.

In another embodiment of the invention, the IGS4 polypeptide may be part of a larger protein, such as a fusion protein. It is often advantageous to include additional amino acid sequences including secretory or leader sequences, pro-sequences, sequences that facilitate purification such as multiple histidine residues, sequences that facilitate detection such as antigenic peptide tags (such as hemagglutinin HA tags), or additional sequences that facilitate stability during recombinant production.

The invention also includes fragments of the IGS4 polypeptide. A fragment refers to a polypeptide having an amino acid sequence that is partially, but not completely, identical to the amino acid sequence of the IGS4 polypeptide described above. As with the IGS4 polypeptides, fragments may be "independent" or contained within a larger polypeptide to form a portion or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the present invention include, for example, the amino acid numbering of IGS4 polypeptide from about positions 1-20, 21-40, 41-60, 61-80, 81-100, and 101-terminal fragments. In this context, "about" includes a few, 5,4, 3, 2, or 1 amino acid greater or lesser than the specifically recited range at one or both ends.

Preferred fragments include, for example, truncated polypeptides having the amino acid sequence of IGS4 polypeptides, but with the deletion of a series of contiguous residues comprising the amino terminus or a series of contiguous residues comprising the carboxy terminus or the deletion of two contiguous residues, one comprising the amino terminus and the other comprising the carboxy terminus. Reference to the present invention is an example of a truncated polypeptide consisting of the polynucleotides SEQ ID NO: 9 and SEQ ID NO: 11 encoded SEQ ID NO: 10 and SEQ ID NO: 12. Also preferred are fragments characterized by structural or functional attributes, such as fragments comprising alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet forming regions, turn and turn forming regions, coil and coil forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface forming regions, substrate binding regions, and high antigen index regions. Other preferred fragments are biologically active fragments. Biologically active fragments are those that mediate the activity of the receptor, including those that have a similar activity or an improved activity, or a reduced undesirable activity. Also included are those fragments that are antigenic or immunogenic in animals, particularly humans.

Thus, the polypeptides of the invention include polypeptides having a sequence identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8 and/or a polypeptide having an amino acid sequence which is at least 80% identical to a polypeptide having an amino acid sequence encoded by a DNA insert contained in deposit no CBS102221 or deposit no CBS102222 of the fungal species collection of Baarn, the netherlands, or a fragment thereof having at least 80% identity to the corresponding fragment. Preferably, all of these polypeptide fragments retain the biological activity, including antigenicity, of the receptor. Variants of the defined sequences and fragments also form part of the invention. Preferred variants are those that differ from the reference by conservative amino acid substitutions (i.e., substitutions with residues having similar characteristics). Typical such substitutions are between Ala, Val, Leu, and Ile; ser and Thr; among the acidic residues Asp and Glu; between Asn and Gln; and between the basic residues Lys and Arg; or a substitution between the aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added, and any combination thereof.

With regard to the polypeptides of the present invention, they were also found to show a high affinity for binding neuromedin U, in particular neuromedin U8 (an oligopeptide of 8 amino acids), neuromedin U23 (an oligopeptide of 23 amino acids), and/or neuromedin U25 (an oligopeptide of 25 amino acids). In the context of the present invention, the term "high affinity" is understood to describe a protein exhibiting log EC₅₀Values of at least less than-6.00 (about 660nM), preferably log EC₅₀Values below-7.00 (about 55nM), more preferably logEC₅₀Values below-9.00 (approximately 500pM-1.2nM), most preferably log EC₅₀Values below-10.00 (approximately 50-100pM) for ligand binding.

In the literature, two forms of the neuropeptide neuromedin U, neuromedin U8 and neuromedin U25, are described as peptides that were originally isolated from porcine spinal cord, stimulate the uterus and are hypertensive (Minamino et al, Biochem.Biophys.Res.Commun.) 130: 1078-. For neuromedin U23 (an oligopeptide of 23 amino acids), see, e.g., Okimura et al, peptide chemistry (pept. chem.) 32: 321-324, 1995, vol.date 1994; salmon et al, J.Biol.chem.) -275 (7): 4549-4554, 2000. Neuromedin U (NMU) was subsequently isolated from a number of species, such as rat (NMU23), human (NMU25), frog (NMU25), dog (NMU8 and NMU25), rabbit (NMU25), and chicken (NMU 25). Thus, Domin et al describe the use of specific radioimmunoassay to identify neuromedin U immunoreactivity in rat, pig, guinea pig, and human tissue extracts (Biochem. Biophys. Res. Commun.) 140: 1127-1134, 1986). Conlon et al (neurochemistry 51: 988-. Minamino et al (Biochem. Biophys. Res. Commun.) 156: 355-360, 1988) isolated rat neuromedin U from the small intestine, determined the amino acid sequence of rat neuromedin U by microsequence analysis, and confirmed the structure by synthesis, using mainly immunoaffinity chromatography and radioimmunoassay for porcine neuromedin U8. Although the C-terminal heptapeptide amide structure of porcine neuromedin U is completely conserved in rat neuromedin U, the remainder of the peptide shows a 9 amino acid substitution and a 2 amino acid deletion compared to porcine neuromedin U25. Domin et al (J.biol.chem.) 264: 20881-20885, 1989 also determined the distribution, primary structure, and relative biological activity of neuromedin U in Rana temporaria of the frog, and found that the entire sequence was a 25 peptide showing significant sequence similarity to porcine and rat neuromedin U. In a further study, Domin et al (regulatory peptide (Regul. Pept.) 41: 1-8, 1992) purified avian homologs of neuromedin U from chickens. Microsequence analysis identified the peptide as 25 amino acid residues in length, and the chicken neuromedin U showed significant sequence similarity to porcine peptide at its biologically active C-terminal region. O' Harte et al (Peptides) 12: 11-15, 1991) describe the isolation, structural identification, and pharmacological activity of neuromedin U25 in dogs. Furthermore, for rabbit neuromedin U25, it was found to lack conservation of the postsynaptic processing site (Kage et al, regulatory peptides (Regul. Pept.) 33: 191-198, 1991); thus, in rabbit neuromedin U, the Arg16-Arg17 dibasic residue processing site found in pig and dog neuromedin U25 is replaced by Arg-Gly, but this potential monobasic processing site is not utilized by the cleavage enzymes in the intestine.

In the species studied, the 5 amino acids at the C-terminus of the peptide were found to be almost completely conserved, suggesting that this region is of major importance. Thus, mammalian neuromedins share the C-terminal sequence "-Phe-Leu-Phe-Arg-Pro-Arg-Asn-amide", which appears to be essential for biological activity. NMU is distributed in both the gastrointestinal tract and the Central Nervous System (CNS). In rats, Neuromedin (NMU) concentrations are highest in the ileum, followed by the pituitary, hypothalamus, spinal cord, thyroid, and genitourinary tract. Immunohistochemical studies showed that NMU immunoreactivity in the intestine was found only in nerve fibers (mainly the myenteric and submucosal plexus) and in the mucosa in all regions except the stomach, while no NMU immunoreactivity was found in endocrine cells. In rat brain, NMU immunoreactivity is found in fibers that are widely distributed throughout the brain except the cerebellum. Human and rat genes encoding the NMU precursor have been isolated. Both encode NMU at the C-terminus and other potential peptide products in the middle (Lo et al, J.Mol.Endocrinol.) -6: 1538-1544, 1992; Austin et al, J.Mol.Endocrinol.) -14: 157-169, 1995). High affinity NMU binding was identified in rat uterus and shown to be sensitive to GTP- -S (Nandha et al, Endocrinology 133: 482. sup. 486, 1993), suggesting that the receptor for NMU should be a G-protein coupled receptor. However, the physiological role of NMU remains largely unknown. Neuromedin U can cause forceful contraction of smooth muscle, increase arterial blood pressure, alter intestinal ion transport, and stimulate adrenocortical function and growth at low doses. NMU has also been shown to reduce blood flow in the upper intestinal artery and portal vein while slightly increasing blood flow in pancreatic tissue.

Furthermore, neuromedin U8 and U25 are useful in the treatment of gastrointestinal disorders, such as may be useful in selectively reducing blood flow to the gastrointestinal tract in the treatment of gastrointestinal bleeding and postprandial hypotension, according to International patent application WO 90/01330.

The IGS4 polypeptide of the invention has been identified as a G-protein coupled receptor that responds to neuromedin U or a ligand sufficiently similar thereto. Thus, the IGS4 receptor, and in particular the IGS4B receptor, that responds to neuromedin U would greatly aid in understanding the physiological mechanisms of neuromedin U and ligands sufficiently similar thereto, as well as understanding the associated diseases.

Tables 5-8 show the tissue distribution and expression levels of the polypeptides of the invention, whereby the skilled artisan can estimate the location and relevance of expression. For example, regarding the tissue distribution of the polypeptide of the present invention, it was found that the IGS4 polypeptide of the present invention is expressed at an intermediate level (relative to the expression in 100% of testis in MTE blot or 100% of spinal cord in quantitative RT-PCR, respectively) specifically in, for example, brain, skeletal muscle, cerebellum, thymus, medulla, thyroid gland, trachea, thalamus, substantia nigra, corpus callosum, caudate nucleus, pons, nucleus accumbens (nucleus accumbens), fetal brain, and stomach, as analyzed by, for example, MTE (multiple tissue expression) analysis, Northern blot analysis, and quantitative RT-PCR expression analysis; and at relevant levels, e.g., in the heart, lung, and prostate (if detectable by quantitative RT-PCR). For example, expression levels are considered moderate if they are at least 20% of the expression values found to date for the highest expression in the testis or spinal cord (set at 100%). For example, an expression level is considered relevant if it is detectable at least by quantitative RT-PCR analysis. It will be appreciated that the expression levels indicated for any organ are averages of the expression levels in the particular tissue and cell type comprising the organ. Thus, if expression levels are found to be only relevant in relation to an organ, this does not exclude a moderate or even high expression locally in a specific area (e.g. a specific tissue and/or cell type of the organ).

These results indicate that IGS4 polypeptides preferably act on the nervous system, including the Central Nervous System (CNS) and the Peripheral Nervous System (PNS), the gastrointestinal system, the cardiovascular system, skeletal muscle, and/or thyroid, and/or lung diseases, immunological diseases, and genitourinary disorders.

Thus, in another embodiment, the present invention relates to an isolated IGS4 polypeptide comprising an amino acid sequence of a neuromedin receptor protein, preferably a mammalian neuromedin receptor protein, which protein exhibits high affinity binding for neuromedin U, preferably for neuromedin U-8, for neuromedin U-23 and/or for neuromedin U-25. In particular, an isolated IGS4 polypeptide comprising an amino acid sequence of a neuromedin receptor protein is a protein which exhibits expression (at least detectable by Northern and/or MTE and/or quantitative RT-PCR analysis) in brain, skeletal muscle, cerebellum, testis, corpus callosum, spinal cord, substantia nigra, medulla, thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain, stomach, heart, thyroid gland, lung, thymus, prostate, and/or trachea. In a variant of this embodiment, the invention relates to an IGS4 polypeptide comprising an amino acid sequence of a neuromedin receptor protein, preferably a mammalian neuromedin receptor protein, said protein exhibiting high affinity binding for neuromedin U, preferably for neuromedin U-8, for neuromedin U-23 and/or for neuromedin U-25, said protein exhibiting expression in brain, skeletal muscle, cerebellum, testis, corpus callosum, spinal cord, substantia nigra, medulla, thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain, stomach, heart, thyroid gland, lung, thymus, prostate and/or trachea (detectable at least by Northern and/or MTE and/or quantitative RT-PCR analysis), and said amino acid sequence being selected from the amino acid sequences as defined above.

The IGS4 polypeptides of the invention may 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. Methods for preparing such polypeptides are well known in the art.

Polynucleotides of the invention

Another aspect of the invention relates to IGS4 polynucleotides. IGS4 polynucleotides include isolated polynucleotides encoding IGS4 polypeptides (including IGS4A and IGS4B) and fragments thereof, as well as polynucleotides closely related thereto. More specifically, the IGS4 polynucleotides of the invention comprise a polynucleotide comprising a sequence encoding SEQ ID NOs: 2 or SEQ ID NO: 4 or the IGS4A polypeptide of SEQ ID NO: 6 or SEQ ID NO: 8 of IGS4B polypeptide of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7, having the nucleotide sequence contained in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7, and a polynucleotide substantially corresponding to a DNA insert contained in deposit No. CBS102221 or deposit No. CBS102222 of the fungal species collection of Baarn, the netherlands.

IGS4 polynucleotides further include a polynucleotide comprising a sequence identical over its entire length to the sequence encoding SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, comprising a nucleotide sequence that is at least 80% identical over its entire length to the nucleotide sequence of the IGS4 polypeptide of seq id NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7, and a polynucleotide substantially corresponding to a DNA insert contained in deposit No. CBS102221 or deposit No. CBS102222 of the fungal species deposit center of Baarn, the netherlands.

In this regard, polynucleotides having at least 90% identity are particularly preferred, and polynucleotides having at least 95% identity are especially preferred. Furthermore, polynucleotides having at least 97% identity are highly preferred, with polynucleotides having at least 98-99% identity being most highly preferred, and in particular polynucleotides having at least 99% identity. IGS4 polynucleotides further include a sequence identical to SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7 or a nucleotide sequence which has sufficient identity to a DNA insert contained in deposit No. CBS102221 or deposit No. CBS102222 of the fungal species collection of Baarn, the netherlands, to hybridize under conditions which are useful for amplification or as a probe or label. The present invention also provides polynucleotides complementary to these IGS4 polynucleotides.

As shown by the BLSAT search results from published databases, the IGS4 of the present invention is structurally related to other proteins of the G-protein coupled receptor family. The amino acid sequence of Table 1(SEQ ID NO: 2) is approximately 46% identical over most of its length (316 amino acid residues) to the human orphan G protein-coupled receptor (accession No. 043664, Tan et al, Genomics (Genomics)52 (2): 223-229, 1998) (using BLAST, S.F. Altschul et al, Nucleic Acids research (Nucleic Acids Res.) 25: 3389-3402, 1997). There is 27% homology (amino acid residues 61-349) to the rat neurotensin 1 receptor (accession number P20789, K. tanaka et al, Neuron (Neuron) 4: 847-854, 1990). The nucleotide sequence of Table 1(SEQ ID NO: 1) is 63% identical to the orphan G protein-coupled receptor at nucleotide residue 120-864 (accession number AF044600, corresponding to protein sequence 043664). Furthermore, 53% identity to the human growth hormone secretagogue receptor at residues 94-1137 (A.D. Howard et al, Science 273: 974-. Thus, it is expected that the IGS4 polypeptides and polynucleotides of the invention will have similar biological functions/properties to their cognate polypeptides and polynucleotides, and their utility will be apparent to those skilled in the art.

The polynucleotides of the invention may be obtained from natural sources such as genomic DNA. In particular, degenerate PCR primers can be designed that encode conserved regions within a particular GPCR gene subfamily. PCR amplification reactions using degenerate primers on genomic DNA or cDNA will lead to the amplification of several members (known and novel) of the gene family under consideration (when using genomic templates, the degenerate primers must be located within the same exon) (Libert et al, Science 244: 569-. The polynucleotides of the present invention can be synthesized using well-known commercial techniques (e.g., F.M. Ausubel et al, Current Protocols in Molecular Biology, 2000).

Encoding the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: the nucleotide sequence of IGS4 polypeptide of SEQ ID NO: 1 (nucleotides 55 to 1299) or SEQ ID NO: 3 (nucleotides 64 to 1299) or SEQ ID NO: 5 (nucleotides 55 to 1299) or SEQ ID NO: 7 (nucleotides 64 to 1299), or it may be a different nucleotide sequence which, due to the redundancy (degeneracy) of the genetic code, is identical to the polypeptide sequence contained in SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7, but also encodes the polypeptide of seq id NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8.

In another embodiment, the invention relates to an isolated nucleotide sequence encoding an IGS4 neuromedin receptor protein, preferably encoding a mammalian neuromedin receptor protein, which protein exhibits high affinity binding for neuromedin U, preferably for neuromedin U-8, for neuromedin U-23 and/or for neuromedin U-25. In particular, the isolated nucleotide sequence encodes an IGS4 neuromedin receptor protein that exhibits expression (at least detectable by Northern and/or MTE and/or quantitative RT-PCR analysis) in brain, skeletal muscle, cerebellum, testis, corpus callosum, spinal cord, substantia nigra, medulla, thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain, stomach, heart, thyroid gland, lung, thymus, prostate, and/or trachea. In a variant of this embodiment, the invention relates to an isolated nucleotide sequence encoding an IGS4 neuromedin receptor protein, preferably encoding a mammalian neuromedin receptor protein, said protein exhibiting high affinity binding for neuromedin U, preferably for neuromedin U-8, for neuromedin U-23 and/or for neuromedin U-25, said protein exhibiting expression in brain, skeletal muscle, cerebellum, testis, corpus callosum, spinal cord, substantia nigra, medulla, thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain, stomach, heart, thyroid gland, lung, thymus, prostate and/or in trachea (detectable at least by Northern and/or MTE and/or quantitative RT-PCR analysis), and said nucleotide sequence being selected from the group consisting of the nucleotide sequences defined above.

When the polynucleotides of the invention are used in the recombinant production of an IGS4 polypeptide, the polynucleotide may itself comprise the coding sequence for the mature polypeptide or a fragment thereof; the coding sequence for the mature polypeptide or fragment thereof is in frame with other coding sequences, such as coding for a leader or secretory sequence, a proprotein sequence, a preproprotein sequence, or other fusion peptide moieties. For example, a marker sequence can be encoded that facilitates purification of the fusion polypeptide. 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 corporation) and described in Gentz et al, U.S. national academy of sciences (proc.natl.acad.sci.usa) 86: 821-824, 1989), or HA-tags. Polynucleotides may also comprise 5 'and 3' non-coding sequences, such as transcribed but not translated sequences, splicing and polyadenylation signals, ribosome binding sites, and sequences that stabilize mRNA.

Another preferred embodiment is a polypeptide encoding a polypeptide comprising SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, but wherein several, 5-10, 1-5, 1-3, 1-2, or 1 amino acid residue is substituted, deleted, or added, or any combination thereof, of an IGS4 variant.

The polynucleotides of the present invention may be engineered using methods generally known in the art to alter the IGS4 coding sequence for a variety of purposes, including but not limited to modifying the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments with synthetic oligonucleotides can be used to engineer nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis can be used to introduce mutations, thereby creating amino acid substitutions, creating new restriction sites, altering modification (e.g., glycosylation or phosphorylation) patterns, altering codon preferences, creating splice variants, and the like.

The present invention also relates to polynucleotides that hybridize to the sequences described herein. In this respect, the invention is particularly directed to polynucleotides which hybridize under stringent conditions to the polynucleotides described above. As used herein, the term "stringent conditions" means that hybridization will occur only if there is at least 80% (preferably at least 90%, more preferably at least 95%, even more preferably at least 97%, and especially at least 99%) identity between the sequences.

And SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7 or fragments thereof, can be used as hybridization probes for cDNA and genomic DNA, for isolating full-length cDNA and genomic clones encoding IGS4, and for isolating cDNA and genomic clones of other genes with high sequence similarity to the IGS4 gene, including genes encoding homologs and orthologs from non-human species. Those skilled in the art are fully aware of these hybridization techniques. Typically, these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to the reference sequence. The probe will typically comprise at least 5 nucleotides, preferably at least 8 nucleotides, more preferably at least 10 nucleotides, even more preferably at least 12 nucleotides, especially at least 15 nucleotides. Most preferably, these probes will have at least 30 nucleotides, and may have at least 50 nucleotides. Particularly preferred probes range from 30 to 50 nucleotides.

One embodiment for obtaining a polynucleotide encoding an IGS4 polypeptide (including homologs and orthologs from non-human species) comprises the steps of: using a nucleic acid having the sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7 or fragments thereof, and isolating full-length cDNA and genomic clones comprising the polynucleotide sequences. Such hybridization techniques are well known to those skilled in the art. Stringent hybridization conditions are as defined above, or incubation at 42 ℃ overnight in a solution containing 50% formamide, 5 XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 XDenhardt's solution, 10% dextran sulfate (w/v), and 20. mu.g/ml denatured, sheared salmon sperm DNA, followed by washing the filters with 0.1 XSSC at about 65 ℃.

The polynucleotides and polypeptides of the invention are useful as research reagents and materials for the development of therapeutics and diagnostics for human and animal diseases.

Vectors, host cells, expression

The invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered with the vectors of the invention, and to the production of the polypeptides of the invention by recombinant techniques. Cell-free translation systems may also be used to produce these proteins using RNA derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the invention. Polynucleotides can be introduced into host cells by METHODS described IN many standard LABORATORY guidelines (such as, for example, Davis et al, BASIC METHODS IN MOLECULAR BIOLOGY based METHODS, 1986, and Sambrook et al, MOLECULAR CLONING: LABORATORY guidelines, 2 nd edition, Cold spring harbor LABORATORY Press, Cold spring harbor, N.Y., 1989), such as calcium phosphate transfection, DEAE-dextran mediated transfection, translocation (transfection), microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading (scrape loading), ballistic introduction, or infection.

Representative examples of suitable hosts include bacterial cells, such as streptococci, staphylococci, E.coli, Streptomycete, 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, HEK293, and Bowes melanoma cells; and plant cells.

A wide variety of expression systems can be used. These systems include chromosome-, episome-, and virus-derived systems, such as vectors derived from bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papovaviruses (such as SV40), vaccinia viruses, adenoviruses, fowlpox viruses, pseudorabies viruses, and retroviruses, as well as vectors derived from combinations of the above, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system may comprise control regions that regulate as well as cause expression. In general, any system or vector suitable for maintaining, propagating, or expressing a polynucleotide in a host to produce a polypeptide may be used. The molecular cloning can be performed by a variety of well-known conventional techniques, such as Sambrook et al molecular cloning: appropriate nucleotide sequences are inserted into the expression system as described in the laboratory guide (see above).

For secretion of the translationally produced protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, a suitable secretion signal may be incorporated into the polypeptide of interest. These signals may be endogenous or exogenous, i.e. derived from different species.

In general, if an IGS4 polypeptide is to be expressed for use in a screening assay, it is preferred that the polypeptide be produced on the cell surface. At this point, the cells may be harvested prior to use in the screening assay. In the case where the affinity or functional activity of the IGS4 polypeptide is modified by a Receptor Activity Modifying Protein (RAMP), it is most likely preferred to co-express the relevant RAMP on the cell surface, and this is often desirable. It is also desirable to harvest cells expressing the IGS4 polypeptide and related RAMP prior to use in screening assays. If the IGS4 polypeptide is secreted into the culture medium, the culture medium may be recovered to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed prior to recovery of the polypeptide. Membranes expressing IGS4 polypeptides can be recovered by methods well known to those skilled in the art. Generally, these methods involve harvesting cells expressing IGS4 polypeptide and homogenizing the cells by methods such as, but not limited to, pottering. The membrane may be recovered by washing the suspension once or several times.

IGS4 polypeptides 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 used for purification. When the polypeptide is denatured during isolation and/or purification, well-known techniques for refolding proteins may be employed to regenerate the active conformation.

Diagnostic test

The present invention also relates to the use of the IGS4 polynucleotides as diagnostic agents. Detection of a mutant IGS4 gene associated with dysfunction would provide a diagnostic tool to increase or clarify the diagnosis of a disease or its susceptibility caused by under-expression, over-expression, or altered expression of IGS 4. Also, co-expression of the relevant receptor activity modifying proteins may be required at this point to obtain the desired quality of the diagnostic assay. Individuals carrying mutations within the IGS4 gene can be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis can be obtained from cells of a subject, such as from blood, urine, saliva, biopsy samples, or autopsy material. Genomic DNA may be used directly for detection or may be enzymatically amplified by PCR or other amplification techniques prior to analysis. RNA or cDNA can also be used in a similar manner. Deletions and insertions can be detected by a change in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to a labeled IGS4 nucleotide sequence. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or differences in melting temperatures. DNA sequence differences can also be detected by changes in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents, or by direct DNA sequencing (see, e.g., Myers et al, Science 230: 1242, 1985). Sequence changes at specific positions can also be revealed by nuclease protection experiments (such as RNase and S1 protection) or chemical cleavage methods (see Cotton et al, Advance in national academy of sciences USA 85: 4397-E4401, 1985). In another embodiment, an oligonucleotide probe array comprising the IGS4 nucleotide sequence or a fragment thereof can be constructed for efficient screening, e.g., for genetic mutations. Methods of array technology are well known and have general applicability and can be used to address a variety of problems in molecular genetics, including gene expression, genetic linkage, and genetic variability (see, e.g., M.Chee et al, Science 274: 610-.

The diagnostic assay provides a method for diagnosing or determining susceptibility to the following diseases by detecting mutations in the IGS4 gene by the methods described above: PNS disorders, psychosis, and CNS disorders, including schizophrenia, Episodic Paroxysmal Anxiety (EPA) disorders such as Obsessive Compulsive Disorder (OCD), post-traumatic stress disorder (PTSD), phobias, and panic, major depressive disorders, bipolar disorders, parkinson's disease, generalized anxiety disorder, autism, delirium, multiple sclerosis, alzheimer's disease/dementia, and other neurodegenerative disorders, severe mental retardation, movement disorders, huntington's disease, tourette's syndrome, tics, tremor, dystonia, spasticity, anorexia, bulimia, stroke, addiction/dependence/craving, sleep disorders, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension (such as essential hypertension, renal hypertension, or pulmonary hypertension), thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, raynaud's disease, renal disease (such as renal failure); dyslipidemias (dyslipdemiias); obesity; vomiting; gastrointestinal disorders including Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), gastroesophageal reflux disease (GERD), motility disorders (motility disorders), and conditions of delayed gastric emptying such as post-operative or diabetic gastroparesis, and diabetes, ulcers (e.g., gastric ulcers); diarrhea; other diseases, including osteoporosis; inflammation; infections, such as bacterial, fungal, protozoal, and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancer; damage induced by chemotherapy; tumor invasion; immune disorders; urinary retention; asthma; (ii) an allergic reaction; arthritis; benign prostatic hyperplasia; (ii) endotoxic shock; sepsis; diabetic complications; and gynecological diseases. In particular, the diagnostic assays of the invention provide methods for diagnosing or determining susceptibility to: disorders of the nervous system, including the Central Nervous System (CNS) and the Peripheral Nervous System (PNS), of the gastrointestinal system, of the cardiovascular system, of skeletal muscles, and/or of the thyroid, and/or of pulmonary diseases, immunological diseases, and genitourinary disorders.

In addition, PNS disorders, psychosis, and CNS disorders, including schizophrenia, Episodic Paroxysmal Anxiety (EPA) disorders such as Obsessive Compulsive Disorder (OCD), post-traumatic stress disorder (PTSD), phobias, and panic, major depression, bipolar disorder, parkinson's disease, generalized anxiety disorder, autism, delirium, multiple sclerosis, alzheimer's disease/dementia, and other neurodegenerative disorders, severe mental retardation, movement disorders, huntington's disease, tourette's syndrome, tics, tremor, dystonia, spasticity, anorexia, bulimia, stroke, addiction/dependence/craving, sleep disorders, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension (such as essential hypertension, renal hypertension, or pulmonary hypertension), thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, raynaud's disease, renal disease (such as renal failure); dyslipidemias (dyslipdemiias); obesity; vomiting; gastrointestinal disorders including Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), gastroesophageal reflux disease (GERD), motility disorders (motility disorders), and conditions of delayed gastric emptying such as post-operative or diabetic gastroparesis, and diabetes, ulcers (e.g., gastric ulcers); diarrhea; other diseases, including osteoporosis; inflammation; infections, such as bacterial, fungal, protozoal, and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancer; damage induced by chemotherapy; tumor invasion; immune disorders; urinary retention; asthma; (ii) an allergic reaction; arthritis; benign prostatic hyperplasia; (ii) endotoxic shock; sepsis; diabetic complications; and gynecological diseases. In particular, disorders of the nervous system, including the Central Nervous System (CNS) and Peripheral Nervous System (PNS), the gastrointestinal system, the cardiovascular system, skeletal muscle, and/or thyroid, and/or pulmonary diseases, immunological diseases, and genitourinary disorders can be diagnosed by a method comprising determining from a sample derived from a subject an abnormal decrease or increase in the level of an IGS4 polypeptide or an IGS4 mRNA. The decrease or increase in expression can be measured at the RNA level using any technique well known in the art for polynucleotide quantification, such as PCR, RT-PCR, RNase protection, Northern blotting, and other hybridization methods. The experimental techniques that can be used to determine the levels of proteins, such as IGS4, in a sample derived from a host are well known to those skilled in the art. These assays include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

In another aspect, the invention relates to a diagnostic kit for the diagnosis of diseases or a predisposition thereto, in particular PNS disorders, psychosis, and CNS disorders including schizophrenia, paroxysmal anxiety (EPA) disorders such as Obsessive Compulsive Disorder (OCD), post-traumatic stress disorder (PTSD), phobias, and panic, major depressive disorder, bipolar disorder, parkinson's disease, generalized anxiety disorder, autistic disorder, delusions, multiple sclerosis, alzheimer's disease/dementia, and other neurodegenerative disorders, severe mental development, dyskinesias, huntington's disease, tourette's syndrome, tics, tremor, dystonia, spasticity, anorexia, bulimia, stroke, addiction/dependence/craving, sleep disorders, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension (such as essential hypertension, renal hypertension, or pulmonary hypertension), thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, raynaud's disease, renal disease (such as renal failure); dyslipidemias (dyslipdemiias); obesity; vomiting; gastrointestinal disorders including Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), gastroesophageal reflux disease (GERD), motility disorders (motility disorders), and conditions of delayed gastric emptying such as post-operative or diabetic gastroparesis, and diabetes, ulcers (e.g., gastric ulcers); diarrhea; other diseases, including osteoporosis; inflammation; infections, such as bacterial, fungal, protozoal, and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancer; damage induced by chemotherapy; tumor invasion; immune disorders; urinary retention; asthma; (ii) an allergic reaction; arthritis; benign prostatic hyperplasia; (ii) endotoxic shock; sepsis; diabetic complications; and gynecological diseases, comprising:

(a) IGS4 polynucleotide, preferably SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7 or a fragment thereof; and/or

(b) A nucleotide sequence complementary to (a); and/or

(c) IGS4 polypeptide, preferably SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8 or a fragment thereof; and/or

(d) Antibodies to IGS4 polypeptides, preferably to the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8; and/or

(e) RAMP polypeptides required for the relevant biological or antigenic properties of IGS4 polypeptides.

It will be appreciated that in any of these kits, (a), (b), (c), (d), or (e) may comprise substantial components. Preferably, the invention relates to a diagnostic kit for diagnosing or determining the following diseases or a predisposition therefor: diseases of the nervous system, including the Central Nervous System (CNS) and the Peripheral Nervous System (PNS), of the gastrointestinal system, of the cardiovascular system, of skeletal muscles, and/or of the thyroid, and/or lung diseases, immunological diseases, and genitourinary disorders.

Chromosome experiment

The nucleotide sequences of the invention are also useful for chromosome identification. Sequences are specifically targeted to, and capable of hybridizing to, specific locations on a single human chromosome. Mapping of related sequences to chromosomes according to the present invention is an important first step in linking those sequences to gene-related diseases. Once the sequence is located at a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. These data can be found, for example, in "human mendelian genetics" (available through the network from Welch medical library, university of Johns Hopkins) at v.mckusick. The relationship between the gene and the disease that has been localized to the same chromosomal region is then identified by linkage analysis (co-inheritance of physically adjacent genes).

Differences in 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, it is likely to be the cause of the disease.

Antibodies

The polypeptides of the invention or fragments or analogs thereof, or cells expressing them (if desired together with the relevant RAMP) may also be used as immunogens to generate immunospecific antibodies to IGS4 polypeptides. The term "immunospecific" means that the affinity of an antibody for a polypeptide of the invention is significantly higher than for other related polypeptides of the prior art.

Antibodies raised against IGS4 polypeptides can be obtained by administering the polypeptides or epitope-bearing fragments, analogs, or cells to an animal, preferably a non-human animal, using conventional protocols. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include hybridoma technology (G.Kohler and C.Milstein, Nature 256: 495-.

The above antibodies can be used to isolate or identify clones expressing the polypeptide, or to purify the polypeptide by affinity chromatography.

Antibodies directed to these IGS4 polypeptides or to the IGS4 polypeptide-RAMP complex are also useful in the treatment of PNS disorders, psychosis, and CNS disorders, including schizophrenia, paroxysmal anxiety (EPA) disorders such as Obsessive Compulsive Disorder (OCD), post-traumatic stress disorder (PTSD), phobias, and panic, major depression, bipolar disorder, parkinson's disease, generalized anxiety disorder, autism, delirium, multiple sclerosis, alzheimer's disease/dementia, and other neurodegenerative disorders, severe mental retardation, movement disorders, huntington's disease, tourette's syndrome, tic, tremor, dystonia, spasticity, anorexia, bulimia, stroke, addiction/dependence/craving, sleep disorders, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension (such as essential hypertension, renal hypertension, or pulmonary hypertension), thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, raynaud's disease, renal disease (such as renal failure); dyslipidemias (dyslipdemiias); obesity; vomiting; gastrointestinal disorders including Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), gastroesophageal reflux disease (GERD), motility disorders (motility disorders), and conditions of delayed gastric emptying such as post-operative or diabetic gastroparesis, and diabetes, ulcers (e.g., gastric ulcers); diarrhea; other diseases, including osteoporosis; inflammation; infections, such as bacterial, fungal, protozoal, and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancer; damage induced by chemotherapy; tumor invasion; immune disorders; urinary retention; asthma; (ii) an allergic reaction; arthritis; benign prostatic hyperplasia; (ii) endotoxic shock; sepsis; diabetic complications; and gynecological diseases, etc. Preferably, the antibodies of the invention are useful for treating disorders of the nervous system, including the Central Nervous System (CNS) and the Peripheral Nervous System (PNS), the gastrointestinal system, the cardiovascular system, skeletal muscle, and/or thyroid, and/or lung diseases, immunological diseases, and genitourinary disorders.

Animal(s) production

Another aspect of the invention relates to the expression of the protein as aberrantly expressed by IGS4 orNon-human animal system that is a model of activity-induced disorders. Non-human animal model systems may also be used to further identify the activity of the IGS4 gene. These systems may be part of a screening strategy designed to identify compounds capable of treating IGS 4-based disorders such as PNS disorders, psychosis, and CNS disorders including schizophrenia, Episodic Paroxysmal Anxiety (EPA) disorders such as Obsessive Compulsive Disorder (OCD), post-traumatic stress disorder (PTSD), phobias, and panic, major depression, bipolar disorder, parkinson's disease, generalized anxiety disorder, autism, delirium, multiple sclerosis, alzheimer's disease/dementia, and other neurodegenerative disorders, severe mental retardation, movement disorders, huntington's disease, tourette's syndrome, tics, tremor, dystonia, spasticity, anorexia, bulimia, stroke, addiction/dependence/craving, sleep disorders, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension (such as essential hypertension, renal hypertension, or pulmonary hypertension), thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, raynaud's disease, renal disease (such as renal failure); dyslipidemias (dyslipdemiias); obesity; vomiting; gastrointestinal disorders including Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), gastroesophageal reflux disease (GERD), motility disorders (motility disorders), and conditions of delayed gastric emptying such as post-operative or diabetic gastroparesis, and diabetes, ulcers (e.g., gastric ulcers); diarrhea; other diseases, including osteoporosis; inflammation; infections, such as bacterial, fungal, protozoal, and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancer; damage induced by chemotherapy; tumor invasion; immune disorders; urinary retention; asthma; (ii) an allergic reaction; arthritis; benign prostatic hyperplasia; (ii) endotoxic shock; sepsis; diabetic complications; and gynecological diseases. In particular, these systems are part of a screening strategy designed to identify therapeutic treatment of the IGS 4-based nervous system (including the central nervous system)Nervous System (CNS) and Peripheral Nervous System (PNS)), disorders of the gastrointestinal system, cardiovascular system, skeletal muscle, and/or thyroid, and/or pulmonary diseases, immunological diseases, and genitourinary disorders. Thus, animal models can be used to identify pharmaceutical compounds, therapies, and interventions that are effective in treating disorders caused by aberrant expression or activity of IGS 4. In addition, these animal models can be used to determine LD in animal subjects₅₀And ED₅₀. These data can be used to determine the in vivo efficacy of potential treatments for IGS4 disorders.

Animal model systems based on aberrant IGS4 expression or activity, IGS 4-based disorders may include non-recombinant animals as well as recombinantly engineered transgenic animals.

Animal models of IGS4 disease may include, for example, genetic models. Animal models displaying IGS 4-based disorder-like symptoms can be engineered using, for example, the IGS4 sequences (such as described above), in combination with techniques well known to those skilled in the art for producing transgenic animals. For example, the IGS4 sequences may be introduced into the genome of the animal of interest and overexpressed or misexpressed therein, or, if endogenous IGS4 sequences are present, they may be overexpressed, misexpressed, or disrupted so that under-expressed or inactivated IGS4 gene expression is obtained.

For overexpression or misexpression of the IGS4 gene sequence, the coding portion of the IGS4 gene sequence can be linked to regulatory sequences capable of driving high levels of gene expression in the animal type of interest or capable of expression in cell types in which the gene is not normally expressed. These control sequences are well known to those skilled in the art and their use does not require undue experimentation.

To underexpress the endogenous IGS4 gene sequence, such a sequence can be isolated and engineered such that, upon reintroduction into the genome of the animal of interest, the allele of the endogenous IGS4 gene will be inactivated or "knocked out". Preferably, the engineered IGS4 gene sequence is introduced by gene targeting such that the endogenous IGS4 sequence is disrupted upon integration of the engineered IGS4 gene sequence into the genome of the animal. Gene targeting is discussed below in this section.

Animals of any species, including but not limited to mice, rats, rabbits, squirrels, guinea pigs, mini-pigs, goats, and non-human primates (e.g., baboons, monkeys, and chimpanzees) can be used to generate animal models of disorders associated with IGS 4.

The IGS4 transgene may be introduced into the animal using any technique known in the art to generate a transgenic animal's starting line (launcher line). These techniques include, but are not limited to, prokaryotic microinjection (p.c. hoppe and t.e. wagner, 1989, U.S. Pat. No. 4,873,191); retroviral-mediated gene transfer into the germline (van der Putten et al, Advance in the national academy of sciences of the United states (Proc. Natl. Acad. Sci. USA) 82: 6148-; gene targeting in embryonic stem cells (Thompson et al, Cell (Cell) 56: 313-321, 1989); electroporation of embryos (Lo, molecular cell biology (mol. cell. biol.) 3: 1803-1814, 1983); and sperm-mediated gene transfer (Lavitrano et al, Cell (Cell) 57: 717-723, 1989); and the like. Review of these techniques is found in Gordon, "Transgenic Animals" (Transgenic Animals), international cytology review (intl. rev. cytol.) 115: 171-.

The present invention provides transgenic animals carrying the IGS4 transgene in all of their cells, as well as animals carrying the transgene in some but not all of their cells, i.e., chimeric animals (see, e.g., the techniques described in Jakobovits, Current biology (Curr. biol.) 4: 761-763, 1994). The transgene may be integrated as a single transgene, or may be in concatamers, such as head-to-head or head-to-tail concatemers. Transgenes can also be selectively introduced and activated in specific cell types by, for example, the teachings of Lasko et al (M.Lasko et al, Advance in the national academy of sciences USA 89: 6232-. The regulatory sequences required for specific activation of such cell types will depend on the particular cell type of interest and will be apparent to those skilled in the art.

Gene targeting is preferred when it is desired to integrate the IGS4 transgene into the chromosomal site of the endogenous IGS4 gene. Briefly, when such a technique is to be used, a vector containing some nucleotide sequence homologous to the endogenous IGS4 gene of interest (e.g., the nucleotide sequence of the mouse IGS4 gene) is designed for integration by homologous recombination with a chromosomal sequence and disruption of the function of the nucleotide sequence of the endogenous IGS4 gene or an allele thereof. A transgene can also be selectively introduced into a particular cell type by, for example, the teachings of Gu et al (H.Gu et al, Science 265: 103-106, 1994) to inactivate an endogenous gene of interest only in that cell type. The regulatory sequences required for specific inactivation of such cell types will depend on the particular cell type of interest and will be apparent to those skilled in the art.

Once the transgenic animal has been produced, the expression of the recombinant IGS4 gene and protein can be determined using standard techniques. Animal tissues can be analyzed for the presence of transgene integration by Southern blot analysis or PCR techniques, thus performing a primary screen. The mRNA expression level of the IGS4 transgene in the tissue of the transgenic animal can also be assessed using techniques including, but not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization techniques, and RT-PCR. Tissue samples expressing the target gene can also be evaluated immunocytochemically using specific antibodies against the transgene product of the target gene of interest. IGS4 transgenic animals expressing IGS4 gene mRAN or IGS4 transgenic peptides (detected immunocytochemically using antibodies directed against epitopes of the target gene product) at readily detectable levels can then be further evaluated to identify those exhibiting symptoms characteristic of IGS 4-based disorders.

Once the IGS4 transgenic starting animals (i.e., those expressing the IGS4 protein in cells or tissues of interest, and preferably displaying symptoms based on IGS4 disorders) have been generated, they can be bred, inbred, outbred, or crossed to generate a population of that particular animal. Examples of such breeding strategies include, but are not limited to: crossing over starting animals containing more than one integration site to create different lines; inbreeding of different lines to produce a hybrid IGS4 transgenic line expressing the IGS4 transgene of interest at a higher level as a result of the additive expression of each IGS4 transgene; crossing the heterozygous transgenic animal to produce a homozygous animal for the designated integration site, thereby both improving expression and eliminating the potential need to screen the animal by DNA analysis; crossing different homozygous lines to produce a compound heterozygote or homozygous line; animals were bred with different inbred genetic backgrounds to examine the effect of the modified allele on IGS4 transgene expression and IGS 4-like symptom development. One such method is to cross an IGS4 transgenic starter animal with a wild-type strain to produce F1 generations that exhibit the above-described IGS 4-related disorder-like symptoms. Then, if the homozygous target gene transgenic animal is found fertile, the F1 generation can be inbred to establish a homozygous line.

Vaccine

Another aspect of the invention relates to a method for inducing an immunological response in a mammal comprising administering (e.g., by vaccination) an IGS4 polypeptide or fragment thereof, if necessary together with a RAMP polypeptide, to the mammal in an amount sufficient to generate an antibody and/or T cell immune response to protect the animal from PNS disorders, psychosis, and CNS disorders, including schizophrenia, Episodic Paroxysmal Anxiety (EPA) disorders such as Obsessive Compulsive Disorder (OCD), post-traumatic stress disorder (PTSD), phobias, and panic, major depressive disorder, bipolar disorder, parkinson's disease, generalized anxiety disorder, autism, delirium, multiple sclerosis, alzheimer's disease/dementia, and other neurodegenerative disorders, severe mental retardation, movement disorders, huntington's disease, tourette's syndrome, tic, tremor, dystonia, spasticity, anorexia, bulimia, stroke, addiction/dependence/craving, sleep disorders, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension (such as essential hypertension, renal hypertension, or pulmonary hypertension), thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, raynaud's disease, renal disease (such as renal failure); dyslipidemias (dyslipdemiias); obesity; vomiting; gastrointestinal disorders including Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), gastroesophageal reflux disease (GERD), motility disorders (motility disorders), and conditions of delayed gastric emptying such as post-operative or diabetic gastroparesis, and diabetes, ulcers (e.g., gastric ulcers); diarrhea; other diseases, including osteoporosis; inflammation; infections, such as bacterial, fungal, protozoal, and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancer; damage induced by chemotherapy; tumor invasion; immune disorders; urinary retention; asthma; (ii) an allergic reaction; arthritis; benign prostatic hyperplasia; (ii) endotoxic shock; sepsis; diabetic complications; and gynecological diseases, etc. Yet another aspect of the invention relates to a method for inducing an immunological response in a mammal comprising delivering an IGS4 polypeptide via a vector that directs the expression of an IGS4 polynucleotide in vivo, thereby inducing such an immunological response to produce antibodies that protect the animal from disease. The invention particularly relates to a method for inducing an immunological response in a mammal comprising vaccinating the mammal with an IGS4 polypeptide or fragment thereof, and if desired a RAMP polypeptide, in an amount sufficient to generate an antibody and/or T cell immune response to protect the animal from a disorder of the nervous system, including the Central Nervous System (CNS) and the Peripheral Nervous System (PNS), the gastrointestinal system, the cardiovascular system, skeletal muscle, and/or thyroid, and/or a lung disease, an immunological disease, and a genitourinary system disorder.

Yet another aspect of the invention relates to immunological/vaccine formulations (compositions) which, upon introduction into a mammalian host, will induce an immunological response in the mammal against an IGS4 polypeptide, wherein the composition comprises an IGS4 polypeptide or an IGS4 gene. These immunological/vaccine formulations (compositions) may be therapeutic immunological/vaccine formulations or prophylactic immunological/vaccine formulations. The vaccine formulation may also comprise a suitable carrier. Since IGS4 polypeptides may be degraded in the stomach, parenteral administration (including subcutaneous, intramuscular, intravenous, intradermal, etc. injections) is preferred. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules or vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. Vaccine formulations may also contain 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.

Screening experiments

IGS4 polypeptides of the invention are useful in methods of screening for compounds that bind to the receptor and activate (agonists) or inhibit activation (antagonists) of the receptor polypeptides of the invention. Thus, the polypeptides of the invention can also be used to assess binding of small molecule substrates and ligands, e.g., to cells, non-cellular preparations, chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands, or structural or functional mimetics.

IGS4 polypeptides are responsible for biological functions, including pathology. Therefore, it would be desirable to find compounds and drugs that stimulate IGS4 or that inhibit IGS4 function. In general, agonists are useful for the therapeutic and prophylactic purposes of conditions such as PNS disorders, psychosis, and CNS disorders including schizophrenia, paroxysmal anxiety (EPA) disorders such as Obsessive Compulsive Disorder (OCD), post-traumatic stress disorder (PTSD), phobias, and panic, major depressive disorder, bipolar disorder, parkinson's disease, generalized anxiety disorder, autism, delusions, multiple sclerosis, alzheimer's disease/dementia, and other neurodegenerative disorders, severe mental development, dyskinesias, huntington's disease, tourette's syndrome, tics, tremor, dystonia, spasticity, anorexia, bulimia, stroke, addiction/dependence/craving, sleep disorders, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension (such as essential hypertension, renal hypertension, or pulmonary hypertension), thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, raynaud's disease, renal disease (such as renal failure); dyslipidemias (dyslipdemiias); obesity; vomiting; gastrointestinal disorders including Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), gastroesophageal reflux disease (GERD), motility disorders (moti 1-bit disorder), and conditions of delayed gastric emptying such as post-operative or diabetic gastroparesis, and diabetes, ulcers (e.g., gastric ulcers); diarrhea; other diseases, including osteoporosis; inflammation; infections, such as bacterial, fungal, protozoal, and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancer; damage induced by chemotherapy; tumor invasion; immune disorders; urinary retention; asthma; (ii) an allergic reaction; arthritis; benign prostatic hyperplasia; (ii) endotoxic shock; sepsis; diabetic complications; and gynecological diseases. The antagonists may be used for the therapeutic and prophylactic purposes of a variety of conditions, such as PNS disorders, psychosis, and CNS disorders, including schizophrenia, Episodic Paroxysmal Anxiety (EPA) disorders such as Obsessive Compulsive Disorder (OCD), post-traumatic stress disorder (PTSD), phobias, and panic, major depression, bipolar disorder, parkinson's disease, generalized anxiety disorder, autism, delirium, multiple sclerosis, alzheimer's disease/dementia, and other neurodegenerative disorders, severe mental retardation, movement disorders, huntington's disease, tourette's syndrome, tics, tremor, dystonia, spasticity, anorexia, bulimia, stroke, addiction/dependence/craving, sleep disorders, epilepsy, migraine; attention deficit/hyperactivity disorder (ADHD); cardiovascular diseases, including heart failure, angina pectoris, arrhythmia, myocardial infarction, cardiac hypertrophy, hypotension, hypertension (such as essential hypertension, renal hypertension, or pulmonary hypertension), thrombosis, arteriosclerosis, cerebral vasospasm, subarachnoid hemorrhage, cerebral ischemia, cerebral infarction, peripheral vascular disease, raynaud's disease, renal disease (such as renal failure); dyslipidemias (dyslipdemiias); obesity; vomiting; gastrointestinal disorders including Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), gastroesophageal reflux disease (GERD), motility disorders (motility disorders), and conditions of delayed gastric emptying such as post-operative or diabetic gastroparesis, and diabetes, ulcers (e.g., gastric ulcers); diarrhea; other diseases, including osteoporosis; inflammation; infections, such as bacterial, fungal, protozoal, and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancer; damage induced by chemotherapy; tumor invasion; immune disorders; urinary retention; asthma; (ii) an allergic reaction; arthritis; benign prostatic hyperplasia; (ii) endotoxic shock; sepsis; diabetic complications; and gynecological diseases. In particular, the present invention is useful in methods of screening for compounds that bind to the receptor and either activate (agonists) or inhibit activation (antagonists) of the IGS4 neuromedin receptor protein, preferably mammalian IGS4 neuromedin receptor protein, which exhibits high affinity binding to neuromedin U, preferably neuromedin U-8, neuromedin U-23, and/or neuromedin U-25. These screening assays are particularly useful for screening compounds effective for disorders of the nervous system, including the Central Nervous System (CNS) and Peripheral Nervous System (PNS), the gastrointestinal system, the cardiovascular system, skeletal muscle, and/or thyroid, and/or lung diseases, immunological diseases, and genitourinary disorders.

In general, these screening procedures involve the generation of appropriate cells that express the receptor polypeptides of the invention on their surface and, if desired, also co-express RAMPs on their surface. These cells include cells from mammals, yeast, Drosophila, or E.coli. Cells expressing the receptor (or whose cell membrane contains the expressed receptor) are then contacted with the test compound to observe binding, or stimulation or inhibition of a functional response.

One screening technique involves the use of cells (e.g., transfected CHO cells) expressing a receptor of the invention in a system that measures changes in extracellular pH, intracellular pH, or intracellular calcium resulting from receptor activation. In such techniques, a compound may be contacted with a cell expressing a receptor polypeptide of the invention. Second messenger responses (such as signal transduction, changes in pH, or changes in calcium levels) are then measured to determine whether a potential compound activates or inhibits the receptor.

Another method involves screening for receptor inhibitors by measuring modulation of signals mediated by the receptor, such as cAMP accumulation and/or adenylate cyclase activity. Such methods comprise transfecting a eukaryotic cell with a receptor of the invention, thereby expressing the receptor on the cell surface. The cells are then exposed to an agonist of the receptor of the invention in the presence of a potential antagonist. If a potential antagonist binds to a receptor and thereby inhibits receptor binding, then the signal mediated by the agonist will be modulated.

Another method for detecting agonists or antagonists of the receptors of the invention is the yeast-based technique described in U.S. Pat. No. 5,482,835, incorporated herein by reference.

These assays may simply test for binding of the candidate compound by a label attached directly or indirectly to the candidate compound, or in assays involving competition with a labeled competitor, adhesion to cells bearing the receptor is detected. In addition, these assays can use detection systems suitable for cells bearing receptors on their surface to test whether a candidate compound results in a signal generated by receptor activation. Inhibitors of activation are typically identified in the presence of known agonists, and the effect of agonists on activation is observed in the presence of candidate compounds.

Thus, candidate compounds that exhibit ligand binding to the IGS4 receptor of the invention can be screened. In the context of the present invention, "ligand binding" is understood to describe the affinity of a compound for the IGS4 receptor showing log EC₅₀Values of at least less than-6.00 (about 660nM), preferably log EC₅₀Values below-7.00 (about 55nM), more preferably log EC₅₀Values below-9.00 (approximately 500pM-1.2nM), most preferably log EC₅₀Values below-10.00 (approximately 50-100 pM).

Accordingly, in one aspect the present invention relates to a method for determining whether a substance is a potential ligand of the IGS4 receptor, comprising:

(a) expressing one of the above defined IGS4 neuromedin receptors or SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. and SEQ ID NO: 8, or a cell comprising one of the receptors of IGS4 neuromedin receptor as defined above or SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. and SEQ ID NO: 8 contacting a receptor membrane preparation of one of the receptors of neuromedin U labeled; and are

(b) Binding of neuromedin U to IGS4 was measured.

Furthermore, these experiments may only comprise the following steps: the candidate compound is mixed with a solution containing an IGS4 polypeptide to form a mixture, the IGS4 activity in the mixture is measured, and the IGS4 activity of the mixture is compared to a standard.

IGS4cDNA, proteins, and antibodies to the proteins can also be used to establish experiments for detecting the effect of added compounds on IGS4mRNA and protein production in cells. For example, an ELISA can be established to measure the level of secretion or cell binding of IGS4 protein using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents (also known as antagonists or agonists, respectively) that inhibit or enhance production of IGS4 to appropriately manipulated cells or tissues. Standard methods for performing screening assays are well known in the art.

Examples of potential IGS4 antagonists include antibodies or, in some cases, oligonucleotides or proteins closely related to the ligand of IGS4, such as fragments of the ligand, or small molecules that bind to the receptor but do not elicit a response that can prevent receptor activity.

Thus, in another aspect, the invention relates to agonists, antagonists, ligands, receptors, substrates, enzymes, etc., for identifying IGS4 polypeptides; or a screening kit for compounds that decrease, increase, and/or enhance production of an IGS4 polypeptide, comprising:

(a) IGS4 polypeptide, preferably SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8;

(b) expresses an IGS4 polypeptide, preferably SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8;

(c) cell membrane expressing an IGS4 polypeptide, preferably expressing the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, IGS4 polypeptide; or

(d) Antibodies to IGS4 polypeptides, preferably to the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, an IGS4 polypeptide.

It will be appreciated that in any of these kits, (a), (b), (c), or (d) may comprise substantial components.

Prophylactic and therapeutic methods

The present invention provides methods for treating abnormal conditions involving excess or insufficient amounts of IGS4 activity.

If IGS4 is overactive, several methods may be used. One approach is to administer an inhibitor compound (antagonist) as described above to the subject in an amount effective to inhibit activation, together with a pharmaceutically acceptable carrier, by blocking binding of the ligand to IGS4, or by inhibiting interaction with a RAMP polypeptide or second signal, thereby alleviating the abnormal condition.

Another approach is to administer a soluble form of an IGS4 polypeptide that is capable of competing with endogenous IGS4 for binding to the ligand. Typical embodiments of such competitors include fragments of the IGS4 polypeptide.

Yet another approach is to use expression blocking techniques to inhibit the expression of the gene encoding endogenous IGS 4. Such techniques are known to include the use of antisense sequences, either generated internally or administered separately. See, e.g., O' Connor, journal of neurochemistry (J Neurochem) 56: 560, 1991; oligodeoxynucleotides as Antisense Inhibitors of gene expression (Oligodeoxynucleotides as Antisense Inhibitors of Gene expression), CRC Press, Boca Raton, Florida, USA, 1988. Alternatively, oligonucleotides that form triplexes with the gene may be used. See, e.g., Lee et al, Nucleic Acids research (Nucleic Acids Res) 6: 3073, 1979; cooney et al, Science 241: 456, 1988; dervan et al, Science 251: 1360, 1991). These oligomers may be administered per se, or the relevant oligomers may be expressed in vivo. Synthetic antisense or triplex oligonucleotides may comprise modified bases or modified backbones. Examples of the latter include methylphosphonate, phosphorothioate, or peptide nucleic acid backbones. To provide protection from nuclease degradation, these backbones are incorporated into antisense or triplex oligonucleotides, as is well known in the art. Antisense and triplex molecules synthesized with these or other modified backbones also form part of the invention.

In addition, ribozymes specific for the IGS1 mRNA sequence may be used to prevent expression of the IGS4 polypeptide. Ribozymes are catalytically active, natural or synthetic RNAs (see, e.g., N.Usman et al, Current opinion of structural biology (curr. opin. struct. biol.). 6 (4): 527-. Synthetic ribozymes can be designed to specifically cleave IGS1 mRNA at selected positions, thereby preventing translation of IGS1 mRNA into a functional polypeptide. Ribozymes can be synthesized with natural ribose phosphate backbones and natural bases, as is common in RNA molecules. Alternatively, ribozymes can be synthesized with non-natural backbones (e.g., 2' -O-methyl RNA) and can contain modified bases in order to provide protection against degradation by ribonucleases.

For the treatment of abnormal conditions involving insufficient expression and activity of IGS4, several approaches may also be used. One approach is to administer to a subject a therapeutically effective amount of a compound that activates IGS4 (i.e., an agonist as described above) in conjunction with a pharmaceutically acceptable carrier, thereby alleviating the abnormal condition. Alternatively, gene therapy may be employed to achieve endogenous production of IGS4 by the relevant cells in the subject. For example, as described above, a polynucleotide of the present invention may be engineered for expression in a replication-defective reverse transcription vector. The retroviral expression construct can then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing an RNA encoding a polypeptide of the present invention, such that the packaging cell produces infectious viral particles containing the gene of interest. These producer cells can be administered to a subject to engineer the cells in vivo and express the polypeptide in vivo. For a summary of gene therapy, see "Human Molecular Genetics", chapter 20 "gene therapy and other Molecular Genetics-based therapies" (and references cited therein), t.

Any of the above methods of treatment may be applied to any subject in need of such treatment, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, most preferably humans.

Formulation and application

Peptides, such as soluble forms of the IGS4 polypeptide, as well as peptides or small molecules that act as agonists and antagonists, may be formulated in combination with suitable pharmaceutical carriers. These formulations comprise a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable carrier or excipient. The formulation should be adapted to the mode of administration and is well within the skill of the art. The invention also relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the above-described components of the compositions of the invention.

The polypeptides and other compounds of the invention may be used alone or in combination with other compounds, such as therapeutic compounds.

Preferred forms of systemic administration of the pharmaceutical composition include injection, typically intravenous injection. Other routes of injection, such as subcutaneous, intramuscular, or intraperitoneal, may also be used. Alternative methods of systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acid or other detergents. In addition, oral administration is also possible if the formulation is correctly enteric or capsule.

The dosage range required will depend upon the peptide or compound selected, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending physician. A suitable dosage range is 0.1-100. mu.g/kg of subject body weight. However, given the variety of compounds available and the varying efficacy of different routes of administration, it is expected that the required dosage will vary widely. For example, oral administration is expected to require larger doses than intravenous injection. As is well understood in the art, standard empirical routine procedures can be used to adjust for variations in these dosage levels for optimization.

The polypeptides used in the treatment may also be produced endogenously in the subject, which is the form of treatment described above, often referred to as "gene therapy". Thus, for example, cells from a subject can be engineered using a polynucleotide (such as DNA or RNA), for example by using a retroviral plasmid vector, to encode a polypeptide ex vivo. The cells are then introduced into the subject.

Examples

The following examples are intended to illustrate the invention in further detail only and therefore should not be construed as limiting the scope of the invention in any way.

Example 1: cloning of cDNAs encoding novel G protein-coupled receptors

Example 1 a: homologous PCR cloning of genomic fragments encoding novel G protein-coupled receptors (GPCRs)

PCR-based homologous cloning strategies were used to isolate parts of the genomic DNA sequence encoding the novel G protein-coupled receptor. Forward (F22) and reverse (R44 and R46) degenerate PCR primers were designed based on the conserved regions of the neurotensin receptor gene family (N.Vita et al, FEBS letters 317: 139-142, 1993; N.Vita et al, European journal of pharmacology (Eur.J.Pharmacol.) 360: 265-272, 1998), i.e., the interior of transmembrane domains 1(TM1) and 3(TM3) and the boundary between TM3 and intracellular loop 2 (I2):

F22(TM1)：

5′-CTCATCTTCGCGGTGGGC(A or G)C(A，C，G or T)G(C or T)(A，C，G or T)GG-3’(SEQ ID NO：13)

R44(TM3/I2)：

5 '-GGCCAGGCAGCGCTCCGCGCT (C or inosine) A (A or G) (A, C, G or T) C (C or T) (A, C, G or T) GC (A, Gor T) -3' (SEQ ID NO: 14)

R46(TM3)：

5′-GAA(A or G)TA(A or G)TAGCC(A or G)CG(A or G)CAGCC(A or T)-3′ (SEQ ID NO：15)

To suppress amplification of known members of the neurotensin receptor family, the 3' terminal nucleotide position of primer R44 was selected to be non-complementary to the corresponding positions of both NTR1 and NTR2 cdnas. The PCR reaction was performed in a volume of 60. mu.l, and contained 100ng of human genomic DNA (Clontech) and 6. mu.l of GeneAmp^TM10 XPCR buffer II (100mM Tris-HCl pH8.3, 500mM KCl, Perkin Elmer), 3.6. mu.l 25mM MgCl₂0.36. mu.l dNTP mix (containing 25mM of each dNTP), 1.5U AmpliTaq-Gold^TMPolymerase (Perkin Elmer), and 30pmol of each degenerate forward (F22) and reverse (R44) primer. The reaction tube was heated at 95 ℃ for 10min, and then subjected to 35 cycles of denaturation (95 ℃, 1min), annealing (55 ℃, 2min), and extension (72 ℃,3 min). Finally, the reaction tube was heated at 72 ℃ for 10 min.

For the semi-nested PCR reactions, 1. mu.l of 1/50 diluted primary PCR reaction was used as template, and F22 and R46 were used as degenerate forward and reverse primers, respectively. The semi-nested PCR reaction was performed under the same conditions as the primary PCR reaction.

The semi-nested PCR reaction products were size separated on an agarose gel and stained with ethidium bromide. Although expected to be + -220 bp fragments, only + -120 bp fragments were seen. Using Qiaex-II^TMPurification kit (Qiagen) the fragment was purified from the gel and ligated using the pGEM-T kit (Promega) according to the protocol recommended by the supplierInto pGEM-T plasmid. The recombinant plasmid thus produced was used to transform competent E.coli SURE^TM2 bacteria (Stratagene). The transformed cells were plated on LB agar plates containing 100. mu.g/ml ampicillin, 0.5mM IPTG, and 50. mu.g/ml X-gal. Plasmid DNA was purified from individual colony microcultures using the Qiagen-tip 20 miniprep kit (Qiagen). Using ABI Prism^TM BigDye^TMTerminator cycle sequencing simple reaction kit (PE-ABI), using insert flanking primers, DNA sequencing reaction was performed on the purified plasmid DNA.

Table 7: overview of the oligomer primers used

SEQ ID NO：13	F22：5′-CTCATCTTCGCGGTGGGC(A or G)C(A，C，G or T)G(C or T)(A，C，G orT)GG-3′
		SEQ ID NO：14	R44: 5 '-GGCCAGGCAGCGCTCCGCGCT (C or inosine) A (A or G) (A, C, G or T) C (Cor T) (A, C, G or T) GC (A, G or T) -3'
SEQ ID NO：15	R46：5′-GAA(A or G)TA(A or G)TAGCC(A or G)CG(A or G)CAGCC(A or T)-3′
		SEQ ID NO：16	AP1：5′-CCATCCTAATACGACTCACTATAGGGC-3′
SEQ ID NO：17	AP2：5′-ACTCACTATAGGGCTCGAGCGGC-3′
		SEQ ID NO：18	IGS4R1：5′GGATCCCAAATAAGAAAGGGTAGTTGC-3′
SEQ ID NO：19	IGS4R2：5′AAAGGGTAGTTGCGCCACATCTCATAGAC-3′
		SEQ ID NO：20	IGS4F5：5′AGGTCTATGAGATGTGGCGCAACTACCCT-3′
SEQ ID NO：21	IGS4F6：5′ATGTGGCGCAACTACCCTTTCTTATTTGGG-3′
		SEQ ID NO：22	R74：5′-CGGAAGTTGGCGGACACG(A or G)(A，C or G)(A or G)TT(A or G)TA-3′
SEQ ID NO：23	IGS4F7：5′-GCTCAGCTTGAAACAGAGCCTCGTACC-3′
		SEQ ID NO：24	IGS4F8：5′-CCATGTGGATCTACAATTTCATCATCC-3′
SEQ ID NO：25	IGS4F9：5′-AAGACAAATCTCTTGAGGCAGATGAAGGG-3′
		SEQ ID NO：26	IGS4F10：5′-GATGCTGTTTGTCTTGGTCTTAGTGTTTGC-3′
SEQ ID NO：27	IGS4R5：5′-GGATGATGAAATTGTAGATCCACATGGGC-3′
		SEQ ID NO：28	IGS4R6：5′-TGTGGAGAAGTCTCTCAAAGTGTGG-3′
SEQ ID NO：29	IGS4R7：5′-TAGTAGGAGTGACAGCCTGACTCGGAACG-3′
		SEQ ID NO：30	IGS4R8：5′-AACGTAGATGACTCAGGACGAACCATTTCC-3′
SEQ ID NO：31	IGS4F11：5′-TCGTACCAGGGGAGGCTCAGGC-3′

The sequencing reaction products were purified by EtOH/NaOAc precipitation and analyzed on an ABI 377 automated sequencer.

Sequence analysis of the insert of clone HNT1552 showed that it likely encodes a portion of a new member of the GPCR family. We call this new GPCR sequence IGS 4.

Example 1 b: cloning of a cDNA fragment comprising the entire IGS4 coding sequence

The complete coding sequence of the IGS4cDNA was obtained by both RACE (Rapid amplification of cDNA Ends) analysis and RT-PCR amplification. Use with Marathon^TMAdapter primers 1 and 2(AP 1: SEQ ID NO: 16; AP 2: SEQ ID NO: 17) provided with cDNA amplification kit (Clontech, Cat. No. K1802-1) together with IGS4 specific primers in Marathon-Ready from brain or testis^TM5 'and 3' RACE PCR reactions were performed on cDNA (Clontech, Cat. Nos. 7400-1 and 7414-1, respectively). Reference is made to Marathon-Ready supplied by Clontech^TMThe PCR RACE reaction was performed as instructed by the cDNA user manual. The RACE is producedThe material was separated on an agarose gel, stained with ethidium bromide, and transferred to Hybond N⁺On the membrane. The blot was prehybridized in modified Church buffer (0.5M phosphate, 7% SDS, 10mM EDTA) at 65 ℃ for 2 hours, then in a medium containing 2X 10⁶cpm/ml ³²P-labeled IGS4cDNA probe was hybridized overnight at 65 ℃ in the same buffer. Using Prime-It II kit^TM(Stratagene), incorporation [ alpha-³²P]dCTP radiolabeling IGS4cDNA Probe to a specific Activity > 10⁹cpm/. mu.g. The blots were washed at high stringency (2 XSSC/0.1% SDS, room temperature, 2X 30 min; followed by 0.1 XSSC/0.1% SDS, 65 ℃, 2X 40min) and autoradiographed overnight. The hybrid fragment was purified from the preparative gel, cloned into the pGEM-T vector, and sequenced as described above.

The first round of semi-nested 5' RACE analysis of human brain cDNA using IGS4 specific primers IGS4R1(SEQ ID NO: 18) and IGS4R2(SEQ ID NO: 19) (designed based on the DNA sequence of clone HNT 1552) yielded clones HNT1886 and HNT1887 (FIG. 1). These clones extend upstream of the IGS4cDNA sequence and past the putative translation initiation codon. Similarly, the first round of 3' RACE analysis of human brain cDNA using the IGS4 specific primers IGS4F5(SEQ ID NO: 20) and IGS4F6(SEQ ID NO: 21) yielded clones HNT1874-1878 and HNT1902-1903 (FIG. 1). These clones extended the 3' end of the known IGS4 cDNA.

All the sequences obtained at this point were assembled into a single contiguous group of sequences comprising a long open reading frame encoding a portion of a novel protein most similar to the human orphan receptor FM-3(Tan et al Genomics (Genomics) 52: 223-229, 1998; GenBank accession Nos. AF044600 and AF 044601). To analyze the RNA expression profile of IGS4, Master Blot containing RNA from different human tissues was used under conditions recommended by the supplier^TMFilm (Clontech, Cat. No. 7770-1) and³²the P-tagged cloned HNT1903 insert was hybridized. The strongest hybridization was obtained from testicular RNA, while the signals obtained from prostate, stomach, spinal cord, hippocampus, medulla oblongata, thyroid, thymus, lung, and trachea were much weaker.

Since the sequences of the contig do not yet contain the complete IGS4 coding sequence, we performed RT-PCR homology cloning experiments on human whole brain RNA using the specific primer IGS4 IGS4F6(SEQ ID NO: 21) and degenerate primer R74(SEQ ID NO: 22), the latter primers designed based on conserved regions of the GPCR subfamily (TM 7/C-terminal intracellular portion) including neurotensin receptors 1 and 2, growth hormone secretagogue receptor (A.D. Howard et al, Science 273: 974-19-977, 1996), and orphan GPCRs FM-3 and GPR38(K.K. McKee et al, Genomics 46: 426-434, 1997). RT-PCR reactions were performed on 500ng total RNA from human brain in 50. mu.l volumes, using Titan according to the supplier's recommendations^TMSingle tube RT-PCR system (Boehringer, Cat. No. 1,888,382). RT-PCR conditions were as follows: reverse transcription at 55 deg.C for 45 min; denaturation at 94 ℃ for 2min followed by 20 cycles of reduced temperature PCR reaction (denaturation at 94 ℃ for 30sec, annealing at 60 ℃ for 30sec [ -0.25 ℃/cycle)]And extension at 68 ℃ for 2min), and another 30 PCR cycles (denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, and extension at 68 ℃ for 3min [ +5 sec/cycle)]). Finally, an additional extension step of 7min at 68 ℃ was carried out as the end. Reaction products were analyzed by southern blot using radiolabeled insert of clone HNT 1903. Using QiaexII^TMThe + -690 bp fragment hybridized with the probe was gel purified from the kit (Qiagen) and cloned into pGEM-T vector, yielding clone HNT 2210-2212. Sequence analysis of these clones enabled extension of the existing IGS4cDNA contig population in the 3' direction.

Since the extended IGS4cDNA contig still did not contain a translation stop codon, an additional IGS4 specific 3' RACE primer was designed (IGS4F7-10, SEQ ID NO: 23-26). In Marathon Ready from human testis^TMNested or semi-nested 3' RACE reactions were performed on the cDNA (Clontech, Cat. No. 7414-1). The specific band of IGS4 (assessed by Southern blotting using an IGS4 specific probe) was cloned into pGEM-T, resulting in clones HNT2289-2290(AP1/IGS4F5- > AP2/IGS4F9), HNT2293-2295(AP1/IGS4F6- > AP2/IGS4F9), HNT2296-2297(AP1/IGS4F7- > AP2/IGS4F9), HNT2308-2310(AP1/IGS4F8- > AP 2-IGS4F10), and HNT2253(AP1/IGS4F7- > AP1/IGS4F 5). Marathon Ready in testis^TMAdditional 5' RACE PCR reactions performed on the cDNA yielded clone HNT2279-2281(AP2/IGS4R6- > AP2/IGS4R 5). (Note: e.g.AP 1/IGS4F5- > AP2/IGS4F9 means that the clone was generated from an IGS4 specific fragment obtained after a primary RACE PCR reaction using the primer pair Ap1/IGS4F5 and nesting with the primer pair AP2/IGS4F 9).

Sequence analysis of these clones allowed further extension of the existing IGS4cDNA contig population in the 3' direction, but still did not reach the end of the IGS4 coding sequence. Computer-assisted homology search of DNA sequences of the IGS4 contig against Expressed Sequence Tag (EST) database (dbest) (Blastn; S.F. Altschul et al, Nucleic Acids Res.). 25: 3389-3402, 1997) showed the presence of EST sequences overlapping the 3' end of the IGS4 contig (accession number N45474) (close to 100% identity in the overlap). EST N45474 further extends the contig of IGS4DNA in the 3 ' direction to a translational stop codon and a3 ' untranslated region (3 ' -UTR). In addition, another set of ESTs were identified that all covered the 3' -UTR of IGS4mRNA (FIG. 2). Additional IGS4 specific primers were designed based on the 3' -UTR of these ESTs (IGS4R7-8, SEQ ID NO: 29-30). Marathon Ready from human testis using various combinations of primers IGS4F7(SEQ ID NO: 23), IGS4F11(SEQ ID NO: 31), and IGS4R7-8(SEQ ID NO: 29-30)^TMPrimary PCR reactions were performed on the cDNA. The PCR tube was heated at 95 ℃ for 2min, and then subjected to 35 cycles of denaturation (95 ℃, 30sec), annealing (65 ℃, 30sec), and extension (72 ℃, 90 sec). Finally, the reaction tube was heated at 72 ℃ for 10 min. Nested PCR reactions were also performed under the same conditions. DNA fragments of. + -. 1630bp were gel purified and cloned into pGEM-T vector to obtain the following clones: HNT2311, HNT2312, and HNT2317(IGS4F7/IGS4R7- > IGS4F11/IGS4R 8); HNT2313, HNT2324, HNT2326, and HNT2328(IGS4F11- > IGS4R 8); HNT2314, HNT2315, and HNT2322(IGS4F11- > R7). Clone HNT2363 was obtained from a purified 1630bp PCR fragment derived from Marathon Ready, human testis^TMThe cDNA was amplified using the primer pair IGS4F11/R7 under slightly modified conditions as follows: first, after denaturation at 94 ℃ for 2min, PCR was performedThe tube was subjected to 15 cycles of denaturation (94 ℃, 15sec), annealing (65 ℃, 30sec), and extension (72 ℃, 2min), followed by 20 additional cycles of denaturation (94 ℃, 15sec), annealing (65 ℃, 30sec), and extension (72 ℃, 2 min; +10 sec/cycle), and finally an extension step at 72 ℃ for 7 min. Sequence analysis of these clones enabled the assembly of the IGS4cDNA consensus sequence (FIG. 1). A close examination of all clones revealed that they were in fact 2 sequence types, differing in 5 nucleotide positions. These variant sequences correspond to polymorphisms in the human population. We refer to these different cDNA types as IGS4ADNA (SEQ ID NO: 1 and SEQ ID NO: 3) and IGS4BDNA (SEQ ID NO: 5 and SEQ ID NO: 7). The consensus sequence comprises a long open reading frame comprising 2 start codons in the same reading frame (55-57 (SEQ ID NO: 1 and SEQ ID NO: 5) and 64-66 (SEQ ID NO: 3 and SEQ ID NO: 7) in IGS4ADNA and IGS4BDNA), predicting proteins of amino acids 415(SEQ ID NO: 2 and SEQ ID NO: 6) or 412(SEQ ID NO: 4 and SEQ ID NO: 8) that show better homology to the PGCR protein. Hydrophilicity analysis of proteins (J.Kyte et al, J.mol.biol.) 157: 105-132, 1982; P.Klein et al, Biochemicla and biophysics (Biochim. Biophys. acta) 815: 468-476, 1985) also indicated the presence of 7 transmembrane domains. Since the first ATG initiation codon is within the weak Kozak translation initiation sequence and the second is within the strong Kozak sequence, it is possible that the IGS4A/B protein is initiated by a second methionine and is 412 amino acids in length (M.Kozak, Gene (Gene) 234: 187-208, 1999). However, it cannot be excluded that some (or even all) of the ATGs start with the first ATG. Of the 5 polymorphic nucleotides, 4 (positions 947, 999, 1202, and 1216 in IGS 4A/BDNA) resulted in the conversion of the encoded amino acid residues, while the 5 (position 1381 in IGS 4A/BDNA) was located within the 3' -UTR. The respective predicted protein sequences are designated IGS4APROT (SEQ ID NO: 2 and SEQ ID NO: 4) and IGS4BPROT (SEQ ID NO: 6 and SEQ ID NO: 8). (Note 1: in this document, the IGS4APROT and IGS4BPROT sequences represent the longest possible (415 amino acid) sequence, but it will be understood that the actual protein may be 3 amino acids shorter at the amino terminus; therefore,the first 3 amino acids of IGS4APROT and IGS4BPROT in tables 4 and 5 are bracketed) (note 2: in this document IGS4 refers to IGS4 sequences in general, regardless of the specific allele type). Homology searches of the IGS4 protein sequence against published domain protein databases showed the best homology (46% identity in amino acids 26-342 of IGS4A) with human orphan GPCR FM-3 (accession No. 043664; C.P. Tan et al, Genomics (Genomics) 52: 223-229, 1998).

Homology searches of the IGS4cDNA sequence against DNA databases yielded numerous entries that were also derived from the IGS4 locus (see FIG. 2 for an overview):

10 EST sequence entries (accession numbers W61169, AI432384, W61131, AI023570, F01358, F03770, Z38158, R40869, R37725, and H11333), 2 STSs (sequence tag sites) (accession numbers G20615 and G05725), and 1 genomic sequence (accession number AQ078563) were found, all derived from the 3' -UTR of the IGS4 cDNA.

EST number N45474 encodes the 3 'end of the IGS4 coding sequence and a part of the 3' -UTR (see above).

A "draft work" high throughput genomic sequence was found assembled from a population of 42 unordered contiguous sequences in any order (accession number AC008571, version AC008571.1, deposited 3.8.1999), where we detected the complete IGS4cDNA sequence in 4 separate regions. These regions most likely correspond to different IGS4 exons, as they are flanked by canonical splice donor and acceptor sequences. Based on this analysis, the positions of different exons in the IGS4ADNA (or IGS4BDNA) sequence can be defined as follows: exon 1 (positions 1-780), exon 2 (positions 781-. The AC008571 genomic sequence belongs to the IGS4A allelic type.

6 overlapping EST entries were found (accession numbers H11359, R13890, R13353, F07531, F05108, and F05107), where the assembled DNA sequence overlapped at its 3' end with the front of exon 2 and exon 3 of IGS 4. However, the DNA sequence upstream of exon 2 is completely different from exon 1 of IGS 4. It is likely that these 6 ESTs were derived from transcripts derived from other promoters.

Finally, 2 genomic sequence entries encoding exon 2 were found (accession numbers AQ019411 and AQ 015065).

Among the numerous IGS4cDNA clones we isolated in the different experiments described above, we found numerous clones containing a 64bp (866-929 th position in IGS4 ADNA) deletion in addition to those derived from unspliced (or partially spliced) transcripts. To date, we have found only truncated transcripts of polymorphic type A. We call this splice variant cDNA sequence IGS4A-64DNA (SEQ ID NO: 9 and SEQ ID NO: 11). Since this deletion occurs exactly at the exon 2/exon 3 boundary, and the last 2 nucleotides of the deleted fragment are "AGs", it is likely that this deletion represents an additional splicing event in which the "AG" in exon 3 acts as a splicing acceptor. The IGS4A reading frame encoded by this splice variant is shifted in reading frame after the deletion point. The encoded (truncated) 296 amino acid protein is designated IGS4A-64PROT (SEQ ID NO: 10 and SEQ ID NO: 12). Hydropathic analysis of the IGS4A-64PROT sequence showed that this protein contained only 5 transmembrane domains (corresponding to TM domains 1-5 of IGS4 APROT). Such truncated receptors may be physiologically relevant.

After plating on LB agar plates containing 100. mu.g/ml ampicillin, the bacterial strain containing the plasmid HNT2322 (containing the IGS4ADNA insert) was again cloned and deposited on the Innogenetics N.V. strain table (ICCG4320) and Centraalbureau voor Schimmelculturen (CBS) of Baarn, the Netherlands (accession number CBS 102221). Plasmid DNA was prepared from the re-cloned isolate and the insert was re-sequenced and found to be identical to the IGS4ADNA sequence.

After plating on LB agar plates containing 100. mu.g/ml ampicillin, bacterial strains containing the plasmid HNT2363 (containing the IGS4BDNA insert) were again cloned and deposited on the Innogenetics N.V. Strain Table (ICCG4340) and Centraalbureau voor Schimmelculturen (CBS) of Baarn, the Netherlands (accession number CBS 102222). Plasmid DNA was prepared from the re-cloned isolate and the insert was re-sequenced and found to be identical to the IGS4BDNA sequence.

Example 2: specific changes in intracellular calcium concentration induced by neuromedin U in CHOG alpha 16-IGS4 cells

Example 2 a: the experimental process comprises the following steps: method and material

A. Methods and materials for IGS4 transfection of CHOG alpha 16 cells

The following materials were used in the experiments: a vector comprising the IGS4DNA sequence (IGS 4-pcDNA3.1); SuperFect transfection reagent (Qiagen); Nut-Mix F12(Gibco) with 10% FCS; 0.028mg/ml gentamicin (Gibco); 0.22mg/ml hygromycin (Gibco).

Materials used for clonal selection: Nut-Mix F12 with 10% FCS; 0.028mg/ml gentamicin; 0.22mg/ml hygromycin; and 0.55mg/ml Geneticin (Gibco).

The following method was applied: transfection was performed with SuperFect transfection reagent as described by the manufacturer (Qiagen). Cells were plated in 24-well plates to 50% confluence. Mu.g/. mu.l plasmid DNA and 1. mu.l SuperFect transfection reagent were added to each well. After 24 hours, the medium was changed and transfected cell clones were selected by selection medium containing geneticin. Clones of cells expressing IGS4 were identified by RT-PCR and Northern blotting.

B. Methods and materials for FLIPR experiments

Preparation of cells:

for cell preparation, the following materials were used: plate: clear, flat-bottomed, black-well 96-well plates (Costar); culture medium: growth medium: Nut-Mix F12(HAM) with Glutamax (Gibco) supplemented with 10% fetal bovine serum (Gibco); an incubator: 5% CO₂、37℃(Nuaire)。

The method is carried out as follows: cells were seeded into black-wall microplates 24 or 48 hours prior to the experiment. The cell density in 48 hours of culture was 0.8X 10^-4Cell/well, 24 hours cultureThe density of cultured cells was 2.2X 10^-4Cells/well. All steps are performed under sterile conditions.

Dye loading:

to observe changes in intracellular calcium levels, cells must be "loaded" with fluorescent dyes that are sensitive to calcium. This dye, called FLUO-4(Molecular Probes), is only excited at 488nm and emits light in the 500-560nm range when it forms a complex with calcium. The dye was used at a final concentration of 4. mu.M. Pluronic acid was added to increase the solubility of the dye and the uptake of the dye by the cells. Probenicid (an anion exchange protein inhibitor) is added to the dye medium to enhance the retention of the dye in the cells.

The following materials were used:

2mM dye stock: 1mg Fluo-4(Molecular Probes) was dissolved in 443. mu.l low water DMSO (Sigma). Subpackaging and storing at-20 ℃.

20% pluronic acid solution: 400mg of pluronic acid (Sigma) was dissolved in 2ml of low-water DMSO (Sigma) at 37 ℃. Stored at room temperature.

Dye/pluronic acid mixture: just prior to use, an equal volume of dye stock was mixed with 20% pluronic acid. The final concentrations of dye and pluronic acid were 1mM and 10%, respectively.

250mM Probenicid stock: 710mg Probenicid (Sigma) was dissolved in 5ml 1N NaOH and mixed with 5ml phenol red free Hank's BSS (Gibco) supplemented with 20mM HEPES.

Loading buffer: 10.5ml of phenol red free Hank's BSS (Gibco), 105. mu.l Probenicid, and 210. mu.l 1M HEPES supplemented with 20mM HEPES.

Wash buffer: add 20mM HEPES (Gibco) and 2.5mM Probenicid phenol red free Hank's BSS (Gibco).

The method is carried out as follows: just prior to addition to the loading buffer, 2mM dye stock was mixed with a 20% (w/v) Pluronic acid equal volumeAnd (6) mixing. The growth medium was aspirated from the wells without agitating the confluent cell layers. 100 μ l of loading medium was dispensed into each well using a Multidrop (Labsystems). Cells were incubated at 5% CO₂And incubated at 37 ℃ for 30 minutes in an incubator. To calculate background fluorescence, some wells were not loaded with dye. The background fluorescence of these wells is derived from the autofluorescence of the cells. After loading with dye, cells were washed 3 times with wash buffer (automated Denley cell washer) to reduce the basal fluorescence to counts above background of 20.000-25.000. Add 100. mu.l buffer and keep the cells at 37 ℃ until the experiment is started.

C. Preparation of Compound plates

Prepare 3 μ M (3 x final concentration) peptide for primary screening. For the concentration response curves, peptide solutions were prepared in the concentration range 30. mu.M-100 nM. All peptides were diluted in buffer containing 0.1% BSA (Sigma).

The following materials were used: peptide: pig neuromedin U25, rat neuromedin U23, pig neuromedin U8 (Bachem); dilution buffer: hank's BSS (Gibco) without phenol red to which 20mM HEPES (Gibco) and 0.1% BSA (Sigma) were added; plate: clear, flat-bottomed 96-well plates (Costar).

D. Experiment of

The FLIPR settings were set to O.4sec exposure duration, filter 1, 50 μ l fluid addition, pipette height 125 μ l, dispense rate 40 μ l/sec, no mixing.

Example 2 b: results

To identify endogenous ligands for the orphan G protein-coupled receptor (GPCR) IGS4, IGS4 (both forms IGS4A and IGS4B) was stably transfected into Chinese Hamster Ovary (CHO) cells. Since the G-protein coupling mechanism of IGS4 is unknown, a specific CHO cell line was used. These CHO cells stably express the G protein G.alpha.16 (Molecular Devices), which is a "universal adaptor" for GPCRs (G.Milligan, F.Marshall, and S.Rees, & G.alpha.16 as a universal G protein adaptor: implications of agonist screening strategies.) (G.alpha.16 as an autoimmune G protein adaptor: implications for agonist screening strategies.), TIPS 17: 235-237, 1996.

The resulting CHOG α 16-IGS4 cells were functionally screened on a fluorescence imaging plate reader (FLIPR) to measure intracellular calcium mobilisation in response to putative peptide ligands. At a concentration of 10nM of neuromedin U23, a large, transient and strong calcium response was induced. In contrast, CHOG α 16 cells and CHOG α 16 cells expressing another unrelated orphan GPCR do not respond to neuromedin U23. The results of these experiments using IGS4B are shown in fig. 4.

Furthermore, the concentration dependence of IGS4 activation on the U isomer of porcine and rat neuromedin was investigated (for both IGS4A and IGS 4B). In the range of 10^-5-10^-12Within M, porcine neuromedin U25, rat neuromedin U23, porcine neuromedin U8 induced specific calcium mobilisation mediated by IGS4 in FLIPR experiments. All 3 neuromedin U isomers tested caused the same maximal activation, LogEC, of IGS4B₅₀Values of-10.09 ± 0.08 (neuromedin U8, n ═ 4; 80pM), -10.61 ± 0.08 (neuromedin U23, n ═ 10; 50pM), and-9.14 ± 0.09 (neuromedin U25, n ═ 3; 1.2 nM). Thus, all 3 peptides elicited potent activation of, inter alia, IGS4B, suggesting that neuromedin U is a natural agonist of this receptor. The results of these experiments using IGS4B are shown in FIG. 3a (neuromedin U8), FIG. 3b (neuromedin U23), and FIG. 3c (neuromedin U25).

For the IGS4A receptor, a slightly lower affinity was found, but still neuromedin U peptide was shown to be a better ligand for the general IGS4 receptor. LogEC of IGS4A₅₀The values are as follows: LogEC of neuromedin U8₅₀The value-9.3 ± 0.09 (n-1; 485 pM); LogEC of neuromedin U23₅₀The value-7.27 ± 0.16 (n-6; 53 nM); and LogEC of neuromedin U25₅₀The value is-6.18 ± 0.14 (n-3; 658 nM).

The calcium mobilization response seen after neuromedin U activated IGS4 indicates that the receptor is coupled to a G protein of the Gq/11 subfamily. In addition, the basal level of intracellular cAMP in CHOG 16-IGS4 cells was not regulated by porcine neuromedin U8(1 and 10. mu.M), indicating that the receptor is not coupled to G proteins of the Gs subfamily (data not shown).

Example 3: human multi-tissue expression array (MTE)^TM) Hybridization of IGS4

Using Prime-It II kit^TM(Stratagene) by random priming of [ alpha-³²P]dCTP human IGS4A DNA (from pGEMT-hIGS4A [ ICCG #4320 ]]. + -. 730bpBamHI/HindIII insert) to a specific activity > 10⁹cpm/. mu.g. Purifying the labeled probe and free label by Sephadex G-50 chromatography, denaturing at 95 deg.C for 5min, and adding ExpressHyb hybridization solution to a final concentration of 1-1.5X 10⁶pm/ml. Human multi-organization expression (MTE) with reference to supplier recommendations^TM) The array (Clontech, Cat. No. 7775-1) was prehybridized in ExpressHyb fluid at 65 ℃ for 30min and hybridized overnight.

Hybridizing the hybridized MTE^TMThe array was washed 5 times with 2 XSSC/1% SDS at 65 ℃ for 20 min; then washed 2 times with 0.1 XSSC/0.5% SDS at 55 ℃ for 20 min. After washing, the array was autoradiographed by phosphorescence imaging (Cycolone Storage Phosphor System, Packard) (FIG. 5). Quantitative analysis of MTE Using OptiQuant image analysis software (Packard)^TMHybridization data of the array. The average background signal obtained from blank positions was corrected for the signal intensity of the different spot positions containing RNA. The signal intensity obtained from spots containing E.coli DNA was taken as representative of samples not displaying IGS4 expression. Samples with lower signal intensity than E.coli DNA were considered negative.

The hybridization signal of different tissues on the RNA array was recalculated by subtracting the hybridization signal observed for E.coli DNA (which was considered to be a background signal) from each value. All tissues showing lower hybridization signals were considered below background and were negative for IGS 4. Expression levels in testis (100%) were plotted and are provided in figure 7.

Example 4: northern blot analysis of tissue distribution of IGS4

Using Prime-It II kit^TM(Stratagene) byRandom initiation of incorporation [ alpha-³²P]dCTP human IGS4A DNA (from pGEMT-hIGS4A [ ICCG #4320 ]]. + -. 730bpBamHI/HindIII insert) to a specific activity > 10⁹cpm/. mu.g. Purifying the labeled probe and free label by Sephadex G-50 chromatography, denaturing at 95 deg.C for 5min, and adding ExpressHyb hybridization solution to a final concentration of 1-1.5X 10⁶cpm/ml. Human Northern blots (Clontech, Cat. Nos. 7760-1, 7759-1, 7767-1, 7755-1, and 7769-1) were prehybridized in ExpressHyb solution at 65 ℃ for 30min and hybridized overnight, according to the supplier's recommendations.

After hybridization, Northern blots were washed 4 times with 2 XSSC/0.05% SDS at room temperature for 10 min; then washed 2 times with 0.1 XSSC/0.1% SDS at 50 ℃ for 40 min. After washing, Northern blots were autoradiographed using Phosphor Storage plates (Cycolone Storage Phosphor System, Packard) and X-ray film. The results of Northern blotting are shown in FIG. 6.

The results of Northern blot analysis appear to be largely consistent with array hybridization (example 3). The strongest signal to date was found in testis (2.4kb transcript). Weaker 2.4kb bands were found in thymus, spinal cord, medulla, thyroid, thalamus, and substantia nigra, and weaker bands were found in corpus callosum, caudate nucleus, and stomach. For some tissues, no 2.4kb band was seen on Northern, whereas strong to moderate hybridization signals were observed on MTE arrays (e.g., whole brain, cerebral cortex, lung, temporal and frontal lobes, amygdala, cerebellum, kidney, and hippocampus).

Example 5: quantitative RT-PCR analysis

LightCycler is also used^TMInstrument (Roche Diagnostics) and IGS4 specific TaqMan^TMProbes, the expression level of IGS4 in different human tissues was determined by real-time quantitative RT-PCR (Q-PCR).

Example 5 a: experimental procedure

A.cDNA Synthesis

Prior to reverse transcription, 3. mu.g of total RNA from human total RNA groups I to V (Clontech, Cat. No. K4000-1 to K4004-1) was treated with 3U of DNAse I (Life Technologies, Cat. No. 18068-015) in a 30. mu.l reaction volume (20mM Tris pH8.3, 50mM KCl, 2mM KCl) for 15 minutes at room temperature to destroy potentially contaminating genomic DNA. The reaction was stopped by adding 3. mu.l of 25mM EDTA and heating at 65 ℃ for 10 minutes. Mu.g of DNase-treated RNA was annealed to 1.3. mu.g of oligo (dT) (Life Technologies, Cat. No. 18418-012) and reverse transcribed using Omniscript reverse transcriptase (Qiagen, Cat. No. 205111) incubated for 1 hour at 37 ℃ in a 52. mu.l reaction volume using the protocol recommended by the enzyme supplier. Omniscript reverse transcriptase was inactivated by heating at 93 ℃ for 5 minutes.

B.Q-PCR

Quantitative PCR reactions were performed in 20. mu.l reaction mixtures containing 1 × TaqMan^TMUniversal PCR Mastermix (PE Applied Biosystems, Cat. No. 4304437), 0.12mg/ml BSA, 900nM forward and reverse primers specific for IGS4 (IP14, 963 and IP14, 964), 250nM TaqMan specific for IGS4^TMThe probe (IP14, 962), and 1.6. mu.l (IGS4) or 0.16. mu.l (GAPDH: glyceraldehyde-3-phosphate dehydrogenase) cDNA synthesis reaction solution were used as templates. To establish the IGS4 standard curve, serial dilutions (10) were used⁷-10¹Copy/reaction) of IGS4 plasmid ICCG 4320; for the GAPDH standard curve, serial dilutions of human brain cDNA synthesis reaction solutions (0.16. mu.l, 0.016. mu.l, and 0.0016. mu.l) were used as templates. 1 × TaqMan^TMUniversal PCR Mastermix containing AmpliTaq Gold^TMDNA polymerase, Amperase^TMUNG (uracil-N-glycosylase), dNTP mix with dUTP, passiveeference, and optimized buffer components. The IGS4 specific primers and TaqMan probes were used with Primer Express^TMDesigned by software (PE Applied Biosystems). Quantitative PCR reaction of human GADPH was performed under the same conditions as described for IGS4, except using the PCR product from TaqMan^TMGAPDH-specific primers and TaqMan of the GAPDH control kit (PE Applied Biosystems, Cat No. 402869; sequence information not available from PE Applied Biosystems)^TMAnd (3) a probe.

PCR reaction in LightCycler Instrument^TMIn the glass capillary of (1). First at 50 ℃ for 2 minutes to AmpErase^TMAfter UNG reaction (thus starting and activating AmpliTaq GoldDNA polymerase (95 ℃ C. for 10min)), the reaction mixture was subjected to 40 cycles of denaturation (95 ℃ C., 15sec) and annealing/extension (60 ℃ C. [ GAPDH ]]Or 68 ℃ [ IGS4]1 min). Quantification of experimental samples was performed using LightCycler software version 3.0. Within the copy range of IGS4 plasmid 10-107, a better linear correlation was obtained between the amount of IGS4 plasmid and the release of the reporter dye. We also used GAPDH TaqMan^TMLinear standard curves were obtained using serial dilutions of brain cDNA for the probe. Relative GAPDH expression levels ranged from 0.4-10.2% of skeletal muscle, which had the highest GAPDH expression level in all tested tissues. Relative IGS4 expression levels were expressed as a ratio to the levels detected in the spinal cord, which had the highest expression of IGS4 in all tested tissues (fig. 8). We also plotted the relative IGS4 expression levels after normalizing the expression of GAPDH housekeeping genes (fig. 8).

Example 5 b: results

Q-PCR using an IGS 4-specific TaqMan probe showed that the highest expression level was found in spinal cord (not normalized to GAPDH). The expression level of IGS4 in the spinal cord amounted to 11,467 copies of mRNA/ng pA RNA (assuming 100% efficiency of the cDNA synthesis reaction and assuming pA RNA to be 2% of the total RNA). High IGS4 expression levels were also found in brain (41% of spinal cord levels), skeletal muscle (37%), cerebellum (31%), testis (19%), lung (12%), and heart (11%). Lower levels were found in fetal brain (5%), trachea (4%), prostate (2%), and thyroid (1.4%). After normalization to GAPDH expression, the relative IGS4 expression pattern remained largely unchanged, with the exception of skeletal muscle, where the relative expression level dropped to 2% of the spinal cord. Since it is unclear whether normalization of GADPH is the correct procedure (GAPDH expression levels are expected to vary more or less in different cell/tissue types), we prefer to focus on non-normalized relative expression levels.

Recognizing that testis, spinal cord, and brain appear to be one of the most significant expression sites, these Q-PCR data appear to match expression data from RNA array (example 3) and Northern blot (example 4) hybridization experiments. However, Q-PCR analysis also showed important expression in many other tissues, such as skeletal muscle, cerebellum, lung, and heart.

Without knowledge of the specific receptor, it has been proposed that the ligand neuromedin U is a neuropeptide or neuromodulator (J.Domin, M.A.Ghatei, P.Chohan, and S.R.bloom, Peptides (Peptides) 8: 779-784, 1987). Our investigations show that IGS4 is a new member of the neuromedin U receptor family, expressed in the CNS and PNS areas, the gastrointestinal system, the immune system, the urogenital system, the cardiovascular system, skeletal muscle, thyroid gland, and lung.

Table 8: overview of PCR oligonucleotide primers and TaqMan probes for IGS 4Q-PCR reaction

SEQ ID NO：32	IP 14,963	5′-CCTCTTCAGCCTGGCGGTCTCTG-3′
			SEQ ID NO：33	IP 14,964	5′-GGAGGCGAAGCACACGGTCTCA-3′
SEQ ID NO：34	IP 14,962	5′(FAM)-AGATGTGGCGCAACTACCCTTTCTTGTTCGGGCC-(TAMRA)3′

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

Brief description of the drawings

FIG. 1: schematic representation of the relative positions of different cDNA clones isolated for generating consensus IGS4cDNA sequences. The 5 'and 3' RACE primers used (IGS4R # and IGS4F #, respectively) are also indicated, as well as the position of EST number N45474. Primer IGS4R6 is located within intron 1. Some clones (e.g., HNT2311, HNT2312, and HNT2253) were only partially sequenced (only the sequenced portions are indicated). Consensus A and consensus B represent the consensus sequences for the IGS4 allelic forms A and B, respectively. The nucleotides identified at each of the 4 polymorphic positions are indicated (shaded box) for each clone. "S" denotes the sequence difference between clones HNT2211 and HNT2212, and refers to "C" or "T". The coding regions of the IGS4A and IGS4B consensus sequences are indicated with an "×". The IGS4ADNA and IGS4BDNA sequences are only from position 86 to the end due to some residual sequence differences at the 5' end of the consensus sequence.

FIG. 2: schematic representation of the relative positions of different DNA database entries with respect to the IGS4cDNA sequence. The IGS4cDNA sequence is indicated with boxes (the position of the IGS4 coding sequence is indicated with a filled box). The relative positions of IGS4 exons 1-4 are indicated above ("=") the IGS4cDNA sequence. The parts of the genomic sequence AC008571 encoding exons 1- > 4 are indicated with AC008571a- > d, respectively. The positions of these fragments within the AC008571 sequence are: AC008571a (13129-13908 bits of AC008571 reverse complement), AC008571b (51676-51760 bits of AC 008571), AC008571c (79978-80103 bits of AC008571 reverse complement), and AC008571d (83060-83728 bits of AC008571 reverse complement). G05725 and G20615 are STS (sequence-identified sequence) entries, while F05107, F05108, F07531, R13353, R13890, H11359, N45474, W61169, AI432384, W61131, AI023570, F01358, F03770, Z38158, R40869, R37725, and H11333 are EST entries. The portions of genomic clones AQ019411 and AQ015065 containing exon 2 of IGS4 were used ": "indicates. The 5' portions of the EST sequences F05107, F05108, F07531, R13353, R13890, and H11359 which are completely different from the IGS4cDNA sequence are indicated with "+". AQ078563 is a genomic clone.

FIG. 3: activation of the IGS4 receptor by different neuromedin U isoforms. CHOG α 16-IGS4B cells were cultured overnight in 96-well plates and loaded with Fluo-4 AM. Measurement of receptor-mediated Ca by FLIPR (molecular sieves)²⁺And (4) changing. The maximum change in fluorescence detected by the CCD camera was normalized to 1 and expressed as a count.

FIG. 3 a: results for neuromedin U8;

FIG. 3 b: results for neuromedin U23;

FIG. 3 c: neuromedin U25.

FIG. 4: neuromedin U23 induces intracellular Ca in IGS 4B-expressing CHOG α 16 cells²⁺And (5) mobilizing. The cell lines CHOG α 16-IGS4, CHOG α 16, and CHOG α 16 transfected with another orphan GPCR were applied with 10nM neuromedin U23. Cells were cultured overnight in 96-well plates and loaded with Fluo-4 AM. Measurement of receptor-mediated intracellular Ca with FLIPR (molecular devices)²⁺The change, expressed as a count detected by the CCD camera.

FIG. 5: human multi-tissue expression arrays using human IGS4 probes.

FIG. 6: northern blot analysis using an IGS4 probe.

FIG. 7: IGS4 expression analysis (MTE blot).

FIG. 8: relative expression levels of IGS4mRNA relative to expression observed in the spinal cord. Two expression levels are shown, unnormalized and GAPDH normalized.

Sequence listing

<110>SOLVAY PHARMACEUTICALS B.V.

<120> novel human G protein-coupled receptor

<130>SPW99.06/HA 00.19

<140>

<141>

<160>34

<170>PatentIn Ver.2.1

<210>1

<211>1658

<212>DNA

<213> human (Homo sapiens)

<220>

<221>CDS

<222>(55)..(1299)

<223> IGS4A Long form

<400>1

ggctcagctt gaaacagagc ctcgtaccag gggaggctca ggccttggat ttta atg 57

Met

1

tca ggg atg gaa aaa ctt cag aat gct tcc tgg atc tac cag cag aaa 105

Ser Gly Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln Lys

5 10 15

cta gaa gat cca ttc cag aaa cac ctg aac agc acc gag gag tat ctg 153

Leu Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr Leu

20 25 30

gcc ttc ctc tgc gga cct cgg cgc agc cac ttc ttc ctc ccc gtg tct 201

Ala Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val Ser

35 40 45

gtg gtg tat gtg cca att ttt gtg gtg ggg gtc att ggc aat gtc ctg 249

Val Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val Leu

50 55 60 65

gtg tgc ctg gtg att ctg cag cac cag gct atg aag acg ccc acc aac 297

Val Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr Asn

70 75 80

tac tac ctc ttc agc ctg gcg gtc tct gac ctc ctg gtc ctg ctc ctt 345

Tyr Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu Leu

85 90 95

gga atg ccc ctg gag gtc tat gag atg tgg cgc aac tac cct ttc ttg 393

Gly Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe Leu

100 105 110

ttc ggg ccc gtg ggc tgc tac ttc aag acg gcc ctc ttt gag acc gtg 441

Phe Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr Val

115 120 125

tgc ttc gcc tcc atc ctc agc atc acc acc gtc agc gtg gag cgc tac 489

cys Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg Tyr

130 135 140 145

gtg gcc atc cta cac ccg ttc cgc gcc aaa ctg cag agc acc cgg cgc 537

Val Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg Arg

150 155 160

cgg gcc ctc agg atc ctc ggc atc gtc tgg ggc ttc tcc gtg ctc ttc 585

Arg Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu Phe

165 170 175

tcc ctg ccc aac acc agc atc cat ggc atc aag ttc cac tac ttc ccc 633

Ser Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe Pro

180 185 190

aat ggg tcc ctg gtc cca ggt tcg gcc acc tgt acg gtc atc aag ccc 681

Asn Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys Pro

195 200 205

atg tgg atc tac aat ttc atc atc cag gtc acc tcc ttc cta ttc tac 729

Met Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe Tyr

210 215 220 225

ctc ctc ccc atg act gtc atc agt gtc ctc tac tac ctc atg gca ctc 777

Leu Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala Leu

230 235 240

aga cta aag aaa gac aaa tct ctt gag gca gat gaa ggg aat gca aat 825

Arg Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala Asn

245 250 255

att caa aga ccc tgc aga aaa tca gtc aac aag atg ctg ttt gtc ttg 873

Ile Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Phe Val Leu

260 265 270

gtc tta gtg ttt gct atc tgt tgg gcc ccg ttc cac att gac cga ctc 921

Val Leu Val Phe Ala Ile Cys Trp Ala Pro Phe His Ile Asp Arg Leu

275 280 285

ttc ttc agc ttt gtg gag gag tgg agt gaa tcc ctg gct gct gtg ttc 969

Phe Phe Ser Phe Val Glu Glu Trp Ser Glu Ser Leu Ala Ala Val Phe

290 295 300 305

aac ctc gtc cat gtg gtg tca ggt gtc ttc ttc tac ctg agc tca gct 1017

Asn Leu Val His Val Val Ser Gly Val Phe Phe Tyr Leu Ser Ser Ala

310 315 320

gtc aac ccc att atc tat aac cta ctg tct cgc cgc ttc cag gca gca 1065

Val Asn Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala Ala

325 330 335

ttc cag aat gtg atc tct tct ttc cac aaa cag tgg cac tcc cag cat 1113

Phe Gln Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln His

340 345 350

gac cca cag ttg cca cct gcc cag cgg aac atc ttc ctg aca gaa tgc 1161

Asp Pro Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu Thr Glu Cys

355 360 365

cac ttt gtg gag ctg acc gaa gat ata ggt ccc caa ttc cca tgt cag 1209

His Phe Val Glu Leu Thr Glu Asp Ile Gly Pro Gln Phe Pro Cys Gln

370 375 380 385

tca tcc atg cac aac tct cac ctc cca aca gcc ctc tct agt gaa cag 1257

Ser Ser Met His Asn Ser His Leu Pro Thr Ala Leu Ser Ser Glu Gln

390 395 400

atg tca aga aca aac tat caa agc ttc cac ttt aac aaa acc 1299

Met Ser Arg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr

405 410 415

tgaattcttt cagagctgac tctcctctat gcctcaaaac ttcagagagg aacatcccat 1359

aatgtatgcc ttctcatatg atattagaga ggtagaatgg ctcttacaac tcatgtaccc 1419

attgctagtt tttttttttt aataaacgtg aaaactgaga gttagatctg gtttcaaaac 1479

ccaagactgc ctgattttta gttatctttc cactatccta actgcctcat gccccttcac 1539

tagttcatgc caagaacgtg actggaaagg catggcacct ataccttgat taatttccat 1599

taatggaaat ggttcgtcct gagtcatcta cgttccgagt caggctgtca ctcctacta 1658

<210>2

<211>415

<212>PRT

<213> human

<400>2

Met Ser Gly Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln

1 5 10 15

Lys Leu Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr

20 25 30

Leu Ala Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val

35 40 45

Ser Val Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val

50 55 60

Leu Val Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr

65 70 75 80

Asn Tyr Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu

85 90 95

Leu Gly Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe

100 105 110

Leu Phe Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr

115 120 125

Val Cys Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg

130 135 140

Tyr Val Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg

145 150 155 160

Arg Arg Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu

165 170 175

Phe Ser Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe

180 185 190

Pro Asn Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys

195 200 205

Pro Met Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe

210 215 220

Tyr Leu Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala

225 230 235 240

Leu Arg Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala

245 250 255

Asn Ile Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Phe Val

260 265 270

Leu Val Leu Val Phe Ala Ile Cys Trp Ala Pro Phe His Ile Asp Arg

275 280 285

Leu Phe Phe Ser Phe Val Glu Glu Trp Ser Glu Ser Leu Ala Ala Val

290 295 300

Phe Asn Leu Val His Val Val Ser Gly Val Phe Phe Tyr Leu Ser Ser

305 310 315 320

Ala Val Asn Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala

325 330 335

Ala Phe Gln Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln

340 345 350

His Asp Pro Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu Thr Glu

355 360 365

Cys His Phe Val Glu Leu Thr Glu Asp Ile Gly Pro Gln Phe Pro Cys

370 375 380

Gln Ser Ser Met His Asn Ser His Leu Pro Thr Ala Leu Ser Ser Glu

385 390 395 400

Gln Met Ser Arg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr

405 410 415

<210>3

<211>1658

<212>DNA

<213> human

<220>

<221>CDS

<222>(64)..(1299)

<223> IGS4A short model

<400>3

ggctcagctt gaaacagagc ctcgtaccag gggaggctca ggccttggat tttaatgtca 60

ggg atg gaa aaa ctt cag aat gct tcc tgg atc tac cag cag aaa cta 108

Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln Lys Leu

1 5 10 15

gaa gat cca ttc cag aaa cac ctg aac agc acc gag gag tat ctg gcc 156

Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr Leu Ala

20 25 30

ttc ctc tgc gga cct cgg cgc agc cac ttc ttc ctc ccc gtg tct gtg 204

Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val Ser Val

35 40 45

gtg tat gtg cca att ttt gtg gtg ggg gtc att ggc aat gtc ctg gtg 252

Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val Leu Val

50 55 60

tgc ctg gtg att ctg cag cac cag gct atg aag acg ccc acc aac tac 300

Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr Asn Tyr

65 70 75

tac ctc ttc agc ctg gcg gtc tct gac ctc ctg gtc ctg ctc ctt gga 348

Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu Leu Gly

80 85 90 95

atg ccc ctg gag gtc tat gag atg tgg cgc aac tac cct ttc ttg ttc 396

Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe Leu Phe

100 105 110

ggg ccc gtg ggc tgc tac ttc aag acg gcc ctc ttt gag acc gtg tgc 444

Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr Val Cys

115 120 125

ttc gcc tcc atc ctc agc atc acc acc gtc agc gtg gag cgc tac gtg 492

Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg Tyr Val

130 135 140

gcc atc cta cac ccg ttc cgc gcc aaa ctg cag agc acc cgg cgc cgg 540

Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg Arg Arg

145 150 155

gcc ctc agg atc ctc ggc atc gtc tgg ggc ttc tcc gtg ctc ttc tcc 588

Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu Phe Ser

160 165 170 175

ctg ccc aac acc agc atc cat ggc atc aag ttc cac tac ttc ccc aat 636

Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe Pro Asn

180 185 190

ggg tcc ctg gtc cca ggt tcg gcc acc tgt acg gtc atc aag ccc atg 684

Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys Pro Met

195 200 205

tgg atc tac aat ttc atc atc cag gtc acc tcc ttc cta ttc tac ctc 732

Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe Tyr Leu

210 215 220

ctc ccc atg act gtc atc agt gtc ctc tac tac ctc atg gca ctc aga 780

Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala Leu Arg

225 230 235

cta aag aaa gac aaa tct ctt gag gca gat gaa ggg aat gca aat att 828

Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala Asn Ile

240 245 250 255

caa aga ccc tgc aga aaa tca gtc aac aag atg ctg ttt gtc ttg gtc 876

Gln Arg Pro cys Arg Lys Ser Val Asn Lys Met Leu Phe Val Leu Val

260 265 270

tta gtg ttt gct atc tgt tgg gcc ccg ttc cac att gac cga ctc ttc 924

Leu Val Phe Ala Ile cys Trp Ala Pro Phe His Ile Asp Arg Leu Phe

275 280 285

ttc agc ttt gtg gag gag tgg agt gaa tcc ctg gct gct gtg ttc aac 972

Phe Ser Phe Val Glu Glu Trp Ser Glu Ser Leu Ala Ala Val Phe Asn

290 295 300

ctc gtc cat gtg gtg tca ggt gtc ttc ttc tac ctg agc tca gct gtc 1020

Leu Val His Val Val Ser Gly Val Phe Phe Tyr Leu Ser Ser Ala Val

305 310 315

aac ccc att atc tat aac cta ctg tct cgc cgc ttc cag gca gca ttc 1068

Asn Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala Ala Phe

320 325 330 335

cag aat gtg atc tct tct ttc cac aaa cag tgg cac tcc cag cat gac 1116

Gln Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln His Asp

340 345 350

cca cag ttg cca cct gcc cag cgg aac atc ttc ctg aca gaa tgc cac 1164

Pro Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu Thr Glu Cys His

355 360 365

ttt gtg gag ctg acc gaa gat ata ggt ccc caa ttc cca tgt cag tca 1212

Phe Val Glu Leu Thr Glu Asp Ile Gly Pro Gln Phe Pro Cys Gln Ser

370 375 380

tcc atg cac aac tct cac ctc cca aca gcc ctc tct agt gaa cag atg 1260

Ser Met His Asn Ser His Leu Pro Thr Ala Leu Ser Ser Glu Gln Met

385 390 395

tca aga aca aac tat caa agc ttc cac ttt aac aaa acc tgaattcttt 1309

Ser Arg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr

400 405 410

cagagctgac tctcctctat gcctcaaaac ttcagagagg aacatcccat aatgtatgcc 1369

ttctcatatg atattagaga ggtagaatgg ctcttacaac tcatgtaccc attgctagtt 1429

tttttttttt aataaacgtg aaaactgaga gttagatctg gtttcaaaac ccaagactgc 1489

ctgattttta gttatctttc cactatccta actgcctcat gccccttcac tagttcatgc 1549

caagaacgtg actggaaagg catggcacct ataccttgat taatttccat taatggaaat 1609

ggttcgtcct gagtcatcta cgttccgagt caggctgtca ctcctacta 1658

<210>4

<211>412

<212>PRT

<213> human

<400>4

Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln Lys Leu Glu

1 5 10 15

Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr Leu Ala Phe

20 25 30

Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val Ser Val Val

35 40 45

Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val Leu Val Cys

50 55 60

Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr Asn Tyr Tyr

65 70 75 80

Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu Leu Gly Met

85 90 95

Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe Leu Phe Gly

100 105 110

Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr Val Cys Phe

115 120 125

Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg Tyr Val Ala

130 135 140

Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg Arg Arg Ala

145 150 155 160

Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu Phe Ser Leu

165 170 175

Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe Pro Asn Gly

180 185 190

Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys Pro Met Trp

195 200 205

Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe Tyr Leu Leu

210 215 220

Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala Leu Arg Leu

225 230 235 240

Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala Asn Ile Gln

245 250 255

Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Phe Val Leu Val Leu

260 265 270

Val Phe Ala Ile Cys Trp Ala Pro Phe His Ile Asp Arg Leu Phe Phe

275 280 285

Ser Phe Val Glu Glu Trp Ser Glu Ser Leu Ala Ala Val Phe Asn Leu

290 295 300

Val His Val Val Ser Gly Val Phe Phe Tyr Leu Ser Ser Ala Val Asn

305 310 315 320

Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala Ala Phe Gln

325 330 335

Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln His Asp Pro

340 345 350

Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu Thr Glu Cys His Phe

355 360 365

Val Glu Leu Thr Glu Asp Ile Gly Pro Gln Phe Pro Cys Gln Ser Ser

370 375 380

Met His Asn Ser His Leu Pro Thr Ala Leu Ser Ser Glu Gln Met Ser

385 390 395 400

Arg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr

405 410

<210>5

<211>1658

<212>DNA

<213> human

<220>

<221>CDS

<222>(55)..(1299)

<223> IGS4B Long form

<400>5

ggctcagctt gaaacagagc ctcgtaccag gggaggctca ggccttggat ttta atg 57

Met

1

tca ggg atg gaa aaa ctt cag aat gct tcc tgg atc tac cag cag aaa 105

Ser Gly Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln Lys

5 10 15

cta gaa gat cca ttc cag aaa cac ctg aac agc acc gag gag tat ctg 153

Leu Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr Leu

20 25 30

gcc ttc ctc tgc gga cct cgg cgc agc cac ttc ttc ctc ccc gtg tct 201

Ala Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val Ser

35 40 45

gtg gtg tat gtg cca att ttt gtg gtg ggg gtc att ggc aat gtc ctg 249

Val Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val Leu

50 55 60 65

gtg tgc ctg gtg att ctg cag cac cag gct atg aag acg ccc acc aac 297

Val Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr Asn

70 75 80

tac tac ctc ttc agc ctg gcg gtc tct gac ctc ctg gtc ctg ctc ctt 345

Tyr Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu Leu

85 90 95

gga atg ccc ctg gag gtc tat gag atg tgg cgc aac tac cct ttc ttg 393

Gly Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe Leu

100 105 110

ttc ggg ccc gtg ggc tgc tac ttc aag acg gcc ctc ttt gag acc gtg 441

Phe Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr Val

115 120 125

tgc ttc gcc tcc atc ctc agc atc acc acc gtc agc gtg gag cgc tac 489

Cys Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg Tyr

130 135 140 145

gtg gcc atc cta cac ccg ttc cgc gcc aaa ctg cag agc acc cgg cgc 537

Val Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg Arg

150 155 160

cgg gcc ctc agg atc ctc ggc atc gtc tgg ggc ttc tcc gtg ctc ttc 585

Arg Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu Phe

165 170 175

tcc ctg ccc aac acc agc atc cat ggc atc aag ttc cac tac ttc ccc 633

Ser Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe Pro

180 185 190

aat ggg tcc ctg gtc cca ggt tcg gcc acc tgt acg gtc atc aag ccc 681

Asn Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys Pro

195 200 205

atg tgg atc tac aat ttc atc atc cag gtc acc tcc ttc cta ttc tac 729

Met Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe Tyr

210 215 220 225

ctc ctc ccc atg act gtc atc agt gtc ctc tac tac ctc atg gca ctc 777

Leu Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala Leu

230 235 240

aga cta aag aaa gac aaa tct ctt gag gca gat gaa ggg aat gca aat 825

Arg Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala Asn

245 250 255

att caa aga ccc tgc aga aaa tca gtc aac aag atg ctg ttt gtc ttg 873

Ile Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Phe Val Leu

260 265 270

gtc tta gtg ttt gct atc tgt tgg gcc ccg ttc cac att gac cga ctc 921

Val Leu Val Phe Ala Ile Cys Trp Ala Pro Phe His Ile Asp Arg Leu

275 280 285

ttc ttc agc ttt gtg gag gag tgg act gaa tcc ctg gct gct gtg ttc 969

Phe Phe Ser Phe Val Glu Glu Trp Thr Glu Ser Leu Ala Ala Val Phe

290 295 300 305

aac ctc gtc cat gtg gtg tca ggt gtc tta ttc tac ctg agc tca gct 1017

Asn Leu Val His Val Val Ser Gly Val Leu Phe Tyr Leu Ser Ser Ala

310 315 320

gtc aac ccc att atc tat aac cta ctg tct cgc cgc ttc cag gca gca 1065

Val Asn Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala Ala

325 330 335

ttc cag aat gtg atc tct tct ttc cac aaa cag tgg cac tcc cag cat 1113

Phe Gln Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln His

340 345 350

gac cca cag ttg cca cct gcc cag cgg aac atc ttc ctg aca gaa tgc 1161

Asp Pro Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu Thr Glu Cys

355 360 365

cac ttt gtg gag ctg acc gaa gat ata ggt ccc caa ttc cta tgt cag 1209

His Phe Val Glu Leu Thr Glu Asp Ile Gly Pro Gln Phe Leu Cys Gln

370 375 380 385

tca tcc gtg cac aac tct cac ctc cca aca gcc ctc tct agt gaa cag 1257

Ser Ser Val His Asn Ser His Leu Pro Thr Ala Leu Ser Ser Glu Gln

390 395 400

atg tca aga aca aac tat caa agc ttc cac ttt aac aaa acc 1299

Met Ser Arg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr

405 410 415

tgaattcttt cagagctgac tctcctctat gcctcaaaac ttcagagagg aacatcccat 1359

aatgtatgcc ttctcatatg aaattagaga ggtagaatgg ctcttacaac tcatgtaccc 1419

attgctagtt tttttttttt aataaacgtg aaaactgaga gttagatctg gtttcaaaac 1479

ccaagactgc ctgattttta gttatctttc cactatccta actgcctcat gccccttcac 1539

tagttcatgc caagaacgtg actggaaagg catggcacct ataccttgat taatttccat 1599

taatggaaat ggttcgtcct gagtcatcta cgttccgagt caggctgtca ctcctacta 1658

<210>6

<211>415

<212>PRT

<213> human

<400>6

Met Ser Gly Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln

1 5 10 15

Lys Leu Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr

20 25 30

Leu Ala Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val

35 40 45

Ser Val Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val

50 55 60

Leu Val Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr

65 70 75 80

Asn Tyr Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu

85 90 95

Leu Gly Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe

100 105 110

Leu Phe Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr

115 120 125

Val Cys Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg

130 135 140

Tyr Val Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg

145 150 155 160

Arg Arg Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu

165 170 175

Phe Ser Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe

180 185 190

Pro Asn Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys

195 200 205

Pro Met Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe

210 215 220

Tyr Leu Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala

225 230 235 240

Leu Arg Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala

245 250 255

Asn Ile Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Phe Val

260 265 270

Leu Val Leu Val Phe Ala Ile Cys Trp Ala Pro Phe His Ile Asp Arg

275 280 285

Leu Phe Phe Ser Phe Val Glu Glu Trp Thr Glu Ser Leu Ala Ala Val

290 295 300

Phe Asn Leu Val His Val Val Ser Gly Val Leu Phe Tyr Leu Ser Ser

305 310 315 320

Ala Val Asn Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala

325 330 335

Ala Phe Gln Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln

340 345 350

His Asp Pro Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu Thr Glu

355 360 365

Cys His Phe Val Glu Leu Thr Glu Asp Ile Gly Pro Gln Phe Leu Cys

370 375 380

Gln Ser Ser Val His Asn Ser His Leu Pro Thr Ala Leu Ser Ser Glu

385 390 395 400

Gln Met Ser Arg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr

405 410 415

<210>7

<211>1658

<212>DNA

<213> human

<220>

<221>CDS

<222>(64)..(1299)

<223> IGS4B short model

<400>7

ggctcagctt gaaacagagc ctcgtaccag gggaggctca ggccttggat tttaatgtca 60

ggg atg gaa aaa ctt cag aat gct tcc tgg atc tac cag cag aaa cta 108

Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln Lys Leu

1 5 10 15

gaa gat cca ttc cag aaa cac ctg aac agc acc gag gag tat ctg gcc 156

Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr Leu Ala

20 25 30

ttc ctc tgc gga cct cgg cgc agc cac ttc ttc ctc ccc gtg tct gtg 204

Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val Ser Val

35 40 45

gtg tat gtg cca att ttt gtg gtg ggg gtc att ggc aat gtc ctg gtg 252

Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val Leu Val

50 55 60

tgc ctg gtg att ctg cag cac cag gct atg aag acg ccc acc aac tac 300

Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr Asn Tyr

65 70 75

tac ctc ttc agc ctg gcg gtc tct gac ctc ctg gtc ctg ctc ctt gga 348

Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu Leu Gly

80 85 90 95

atg ccc ctg gag gtc tat gag atg tgg cgc aac tac cct ttc ttg ttc 396

Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe Leu Phe

100 105 110

ggg ccc gtg ggc tgc tac ttc aag acg gcc ctc ttt gag acc gtg tgc 444

Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr Val Cys

115 120 125

ttc gcc tcc atc ctc agc atc acc acc gtc agc gtg gag cgc tac gtg 492

Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg Tyr Val

130 135 140

gcc atc cta cac ccg ttc cgc gcc aaa ctg cag agc acc cgg cgc cgg 540

Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg Arg Arg

145 150 155

gcc ctc agg atc ctc ggc atc gtc tgg ggc ttc tcc gtg ctc ttc tcc 588

Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu Phe Ser

160 165 170 175

ctg ccc aac acc agc atc cat ggc atc aag ttc cac tac ttc ccc aat 636

Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe Pro Asn

180 185 190

ggg tcc ctg gtc cca ggt tcg gcc acc tgt acg gtc atc aag ccc atg 684

Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys Pro Met

195 200 205

tgg atc tac aat ttc atc atc cag gtc acc tcc ttc cta ttc tac ctc 732

Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe Tyr Leu

210 215 220

ctc ccc atg act gtc atc agt gtc ctc tac tac ctc atg gca ctc aga 780

Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala Leu Arg

225 230 235

cta aag aaa gac aaa tct ctt gag gca gat gaa ggg aat gca aat att 828

Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala Asn Ile

240 245 250 255

caa aga ccc tgc aga aaa tca gtc aac aag atg ctg ttt gtc ttg gtc 876

Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Phe Val Leu Val

260 265 270

tta gtg ttt gct atc tgt tgg gcc ccg ttc cac att gac cga ctc ttc 924

Leu Val Phe Ala Ile Cys Trp Ala Pro Phe His Ile Asp Arg Leu Phe

275 280 285

ttc agc ttt gtg gag gag tgg act gaa tcc ctg gct gct gtg ttc aac 972

Phe Ser Phe Val Glu Glu Trp Thr Glu Ser Leu Ala Ala Val Phe Asn

290 295 300

ctc gtc cat gtg gtg tca ggt gtc tta ttc tac ctg agc tca gct gtc 1020

Leu Val His Val Val Ser Gly Val Leu Phe Tyr Leu Ser Ser Ala Val

305 310 315

aac ccc att atc tat aac cta ctg tct cgc cgc ttc cag gca gca ttc 1068

Asn Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala Ala Phe

320 325 330 335

cag aat gtg atc tct tct ttc cac aaa cag tgg cac tcc cag cat gac 1116

Gln Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln His Asp

340 345 350

cca cag ttg cca cct gcc cag cgg aac atc ttc ctg aca gaa tgc cac 1164

Pro Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu Thr Glu Cys His

355 360 365

ttt gtg gag ctg acc gaa gat ata ggt ccc caa ttc cta tgt cag tca 1212

Phe Val Glu Leu Thr Glu Asp Ile Gly Pro Gln Phe Leu Cys Gln Ser

370 375 380

tcc gtg cac aac tct cac ctc cca aca gcc ctc tct agt gaa cag atg 1260

Ser Val His Asn Ser His Leu Pro Thr Ala Leu Ser Ser Glu Gln Met

385 390 395

tca aga aca aac tat caa agc ttc cac ttt aac aaa acc tgaattcttt 1309

Ser Arg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr

400 405 410

cagagctgac tctcctctat gcctcaaaac ttcagagagg aacatcccat aatgtatgcc 1369

ttctcatatg aaattagaga ggtagaatgg ctcttacaac tcatgtaccc attgctagtt 1429

tttttttttt aataaacgtg aaaactgaga gttagatctg gtttcaaaac ccaagactgc 1489

ctgattttta gttatctttc cactatccta actgcctcat gccccttcac tagttcatgc 1549

caagaacgtg actggaaagg catggcacct ataccttgat taatttccat taatggaaat 1609

ggttcgtcct gagtcatcta cgttccgagt caggctgtca ctcctacta 1658

<210>8

<211>412

<212>PRT

<213> human

<400>8

Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln Lys Leu Glu

1 5 10 15

Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr Leu Ala Phe

20 25 30

Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val Ser Val Val

35 40 45

Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val Leu Val Cys

50 55 60

Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr Asn Tyr Tyr

65 70 75 80

Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu Leu Gly Met

85 90 95

Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe Leu Phe Gly

100 105 110

Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr Val Cys Phe

115 120 125

Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg Tyr Val Ala

130 135 140

Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg Arg Arg Ala

145 150 155 160

Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu Phe Ser Leu

165 170 175

Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe Pro Asn Gly

180 185 190

Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys Pro Met Trp

195 200 205

Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe Tyr Leu Leu

210 215 220

Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala Leu Arg Leu

225 230 235 240

Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala Asn Ile Gln

245 250 255

Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Phe Val Leu Val Leu

260 265 270

Val Phe Ala Ile Cys Trp Ala Pro Phe His Ile Asp Arg Leu Phe Phe

275 280 285

Ser Phe Val Glu Glu Trp Thr Glu Ser Leu Ala Ala Val Phe Asn Leu

290 295 300

Val His Val Val Ser Gly Val Leu Phe Tyr Leu Ser Ser Ala Val Asn

305 310 315 320

Pro Ile Ile Tyr Asn Leu Leu Ser Arg Arg Phe Gln Ala Ala Phe Gln

325 330 335

Asn Val Ile Ser Ser Phe His Lys Gln Trp His Ser Gln His Asp Pro

340 345 350

Gln Leu Pro Pro Ala Gln Arg Asn Ile Phe Leu Thr Glu Cys His Phe

355 360 365

Val Glu Leu Thr Glu Asp Ile Gly Pro Gln Phe Leu Cys Gln Ser Ser

370 375 380

Val His Asn Ser His Leu Pro Thr Ala Leu Ser Ser Glu Gln Met Ser

385 390 395 400

Arg Thr Asn Tyr Gln Ser Phe His Phe Asn Lys Thr

405 410

<210>9

<211>1594

<212>DNA

<213> human

<220>

<221>CDS

<222>(55)..(942)

<223> IGs4A truncated DNA long form

<400>9

ggctcagctt gaaacagagc ctcgtaccag gggaggctca ggccttggat ttta atg 57

Met

1

tca ggg atg gaa aaa ctt cag aat gct tcc tgg atc tac cag cag aaa 105

Ser Gly Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln Lys

5 10 15

cta gaa gat cca ttc cag aaa cac ctg aac agc acc gag gag tat ctg 153

Leu Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr Leu

20 25 30

gcc ttc ctc tgc gga cct cgg cgc agc cac ttc ttc ctc ccc gtg tct 201

Ala Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val Ser

35 40 45

gtg gtg tat gtg cca att ttt gtg gtg ggg gtc att ggc aat gtc ctg 249

Val Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val Leu

50 55 60 65

gtg tgc ctg gtg att ctg cag cac cag gct atg aag acg ccc acc aac 297

Val Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr Asn

70 75 80

tac tac ctc ttc agc ctg gcg gtc tct gac ctc ctg gtc ctg ctc ctt 345

Tyr Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu Leu

85 90 95

gga atg ccc ctg gag gtc tat gag atg tgg cgc aac tac cct ttc ttg 393

Gly Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe Leu

100 105 110

ttc ggg ccc gtg ggc tgc tac ttc aag acg gcc ctc ttt gag acc gtg 441

Phe Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr Val

115 120 125

tgc ttc gcc tcc atc ctc agc atc acc acc gtc agc gtg gag cgc tac 489

Cys Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg Tyr

130 135 140 145

gtg gcc atc cta cac ccg ttc cgc gcc aaa ctg cag agc acc cgg cgc 537

Val Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg Arg

150 155 160

cgg gcc ctc agg atc ctc ggc atc gtc tgg ggc ttc tcc gtg ctc ttc 585

Arg Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu Phe

165 170 175

tcc ctg ccc aac acc agc atc cat ggc atc aag ttc cac tac ttc ccc 633

Ser Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe Pro

180 185 190

aat ggg tcc ctg gtc cca ggt tcg gcc acc tgt acg gtc atc aag ccc 681

Asn Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys Pro

195 200 205

atg tgg atc tac aat ttc atc atc cag gtc acc tcc ttc cta ttc tac 729

Met Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr ser Phe Leu Phe Tyr

210 215 220 225

ctc ctc ccc atg act gtc atc agt gtc ctc tac tac ctc atg gca ctc 777

Leu Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala Leu

230 235 240

aga cta aag aaa gac aaa tct ctt gag gca gat gaa ggg aat gca aat 825

Arg Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala Asn

245 250 255

att caa aga ccc tgc aga aaa tca gtc aac aag atg ctg tct ttg tgg 873

Ile Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Ser Leu Trp

260 265 270

agg agt gga gtg aat ccc tgg ctg ctg tgt tca acc tcg tcc atg tgg 921

Arg Ser Gly Val Asn Pro Trp Leu Leu Cys Ser Thr Ser Ser Met Trp

275 280 285

tgt cag gtg tct tct tct acc tgagctcagc tgtcaacccc attatctata 972

Cys Gln Val Ser Ser Ser Thr

290 295

acctactgtc tcgccgcttc caggcagcat tccagaatgt gatctcttct ttccacaaac 1032

agtggcactc ccagcatgac ccacagttgc cacctgccca gcggaacatc ttcctgacag 1092

aatgccactt tgtggagctg accgaagata taggtcccca attcccatgt cagtcatcca 1152

tgcacaactc tcacctccca acagccctct ctagtgaaca gatgtcaaga acaaactatc 1212

aaagcttcca ctttaacaaa acctgaattc tttcagagct gactctcctc tatgcctcaa 1272

aacttcagag aggaacatcc cataatgtat gccttctcat atgatattag agaggtagaa 1332

tggctcttac aactcatgta cccattgcta gttttttttt tttaataaac gtgaaaactg 1392

agagttagat ctggtttcaa aacccaagac tgcctgattt ttagttatct ttccactatc 1452

ctaactgcct catgcccctt cactagttca tgccaagaac gtgactggaa aggcatggca 1512

cctatacctt gattaatttc cattaatgga aatggttcgt cctgagtcat ctacgttccg 1572

agtcaggctg tcactcctac ta 1594

<210>10

<211>296

<212>PRT

<213> human

<400>10

Met Ser Gly Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln

1 5 10 15

Lys Leu Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr

20 25 30

Leu Ala Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val

35 40 45

Ser Val Val TyT Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val

50 55 60

Leu Val Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr

65 70 75 80

Asn Tyr Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu

85 90 95

Leu Gly Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe

100 105 110

Leu Phe Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr

115 120 125

Val Cys Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg

130 135 140

Tyr Val Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg

145 150 155 160

Arg Arg Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu

165 170 175

Phe Ser Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe

180 185 190

Pro Asn Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys

195 200 205

Pro Met Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe

210 215 220

Tyr Leu Leu Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala

225 230 235 240

Leu Arg Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala

245 250 255

Asn Ile Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Ser Leu

260 265 270

Trp Arg Ser Gly Val Asn Pro Trp Leu Leu Cys Ser Thr Ser Ser Met

275 280 285

Trp Cys Gln Val Ser Ser Ser Thr

290 295

<210>11

<211>1594

<212>DNA

<213> human

<220>

<221>CDS

<222>(64)..(942)

<223> short forms of IGS4A truncated DNA

<400>11

ggctcagctt gaaacagagc ctcgtaccag gggaggctca ggccttggat tttaatgtca 60

ggg atg gaa aaa ctt cag aat gct tcc tgg atc tac cag cag aaa cta 108

Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln Lys Leu

1 5 10 15

gaa gat cca ttc cag aaa cac ctg aac agc acc gag gag tat ctg gcc 156

Glu Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr Leu Ala

20 25 30

ttc ctc tgc gga cct cgg cgc agc cac ttc ttc ctc ccc gtg tct gtg 204

Phe Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val Ser Val

35 40 45

gtg tat gtg cca att ttt gtg gtg ggg gtc att ggc aat gtc ctg gtg 252

Val Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val Leu Val

50 55 60

tgc ctg gtg att ctg cag cac cag gct atg aag acg ccc acc aac tac 300

Cys Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr Asn Tyr

65 70 75

tac ctc ttc agc ctg gcg gtc tct gac ctc ctg gtc ctg ctc ctt gga 348

Tyr Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu Leu Gly

80 85 90 95

atg ccc ctg gag gtc tat gag atg tgg cgc aac tac cct ttc ttg ttc 396

Met Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe Leu Phe

100 105 110

ggg ccc gtg ggc tgc tac ttc aag acg gcc ctc ttt gag acc gtg tgc 444

Gly Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr Val Cys

115 120 125

ttc gcc tcc atc ctc agc atc acc acc gtc agc gtg gag cgc tac gtg 492

Phe Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg Tyr Val

130 135 140

gcc atc cta cac ccg ttc cgc gcc aaa ctg cag agc acc cgg cgc cgg 540

Ala Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg Arg Arg

145 150 155

gcc ctc agg atc ctc ggc atc gtc tgg ggc ttc tcc gtg ctc ttc tcc 588

Ala Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu Phe Ser

160 165 170 175

ctg ccc aac acc agc atc cat ggc atc aag ttc cac tac ttc ccc aat 636

Leu Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe Pro Asn

180 185 190

ggg tcc ctg gtc cca ggt tcg gcc acc tgt acg gtc atc aag ccc atg 684

Gly Ser Leu Val Pro Gly Ser Ala Thr Cys Thr Val Ile Lys Pro Met

195 200 205

tgg atc tac aat ttc atc atc cag gtc acc tcc ttc cta ttc tac ctc 732

Trp Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe Tyr Leu

210 215 220

ctc ccc atg act gtc atc agt gtc ctc tac tac ctc atg gca ctc aga 780

Leu Pro Met Thr Val Ile Ser Val Leu tyr Tyr Leu Met Ala Leu Arg

225 230 235

cta aag aaa gac aaa tct ctt gag gca gat gaa ggg aat gca aat att 828

Leu Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala Asn Ile

240 245 250 255

caa aga ccc tgc aga aaa tca gtc aac aag atg ctg tct ttg tgg agg 876

Gln Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Ser Leu Trp Arg

260 265 270

agt gga gtg aat ccc tgg ctg ctg tgt tca acc tcg tcc atg tgg tgt 924

Ser Gly Val Asn Pro Trp Leu Leu Cys Ser Thr Ser Ser Met Trp Cys

275 280 285

cag gtg tct tct tct acc tgagctcagc tgtcaacccc attatctata 972

Gln Val Ser Ser Ser Thr

290

acctactgtc tcgccgcttc caggcagcat tccagaatgt gatctcttct ttccacaaac 1032

agtggcactc ccagcatgac ccacagttgc cacctgccca gcggaacatc ttcctgacag 1092

aatgccactt tgtggagctg accgaagata taggtcccca attcccatgt cagtcatcca 1152

tgcacaactc tcacctccca acagccctct ctagtgaaca gatgtcaaga acaaactatc 1212

aaagcttcca ctttaacaaa acctgaattc tttcagagct gactctcctc tatgcctcaa 1272

aacttcagag aggaacatcc cataatgtat gccttctcat atgatattag agaggtagaa 1332

tggctcttac aactcatgta cccattgcta gttttttttt tttaataaac gtgaaaactg 1392

agagttagat ctggtttcaa aacccaagac tgcctgattt ttagttatct ttccactatc 1452

ctaactgcct catgcccctt cactagttca tgccaagaac gtgactggaa aggcatggca 1512

cctatacctt gattaatttc cattaatgga aatggttcgt cctgagtcat ctacgttccg 1572

agtcaggctg tcactcctac ta 1594

<210>12

<211>293

<212>PRT

<213> human

<400>12

Met Glu Lys Leu Gln Asn Ala Ser Trp Ile Tyr Gln Gln Lys Leu Glu

1 5 10 15

Asp Pro Phe Gln Lys His Leu Asn Ser Thr Glu Glu Tyr Leu Ala Phe

20 25 30

Leu Cys Gly Pro Arg Arg Ser His Phe Phe Leu Pro Val Ser Val Val

35 40 45

Tyr Val Pro Ile Phe Val Val Gly Val Ile Gly Asn Val Leu Val Cys

50 55 60

Leu Val Ile Leu Gln His Gln Ala Met Lys Thr Pro Thr Asn Tyr Tyr

65 70 75 80

Leu Phe Ser Leu Ala Val Ser Asp Leu Leu Val Leu Leu Leu Gly Met

85 90 95

Pro Leu Glu Val Tyr Glu Met Trp Arg Asn Tyr Pro Phe Leu Phe Gly

100 105 110

Pro Val Gly Cys Tyr Phe Lys Thr Ala Leu Phe Glu Thr Val Cys Phe

115 120 125

Ala Ser Ile Leu Ser Ile Thr Thr Val Ser Val Glu Arg Tyr Val Ala

130 135 140

Ile Leu His Pro Phe Arg Ala Lys Leu Gln Ser Thr Arg Arg Arg Ala

145 150 155 160

Leu Arg Ile Leu Gly Ile Val Trp Gly Phe Ser Val Leu Phe Ser Leu

165 170 175

Pro Asn Thr Ser Ile His Gly Ile Lys Phe His Tyr Phe Pro Asn Gly

180 185 190

Ser Leu Val Pro Gly Ser Ala Thr Cys Tnt Val Ile Lys Pro Met Trp

195 200 205

Ile Tyr Asn Phe Ile Ile Gln Val Thr Ser Phe Leu Phe Tyr Leu Leu

210 215 220

Pro Met Thr Val Ile Ser Val Leu Tyr Tyr Leu Met Ala Leu Arg Leu

225 230 235 240

Lys Lys Asp Lys Ser Leu Glu Ala Asp Glu Gly Asn Ala Asn Ile Gln

245 250 255

Arg Pro Cys Arg Lys Ser Val Asn Lys Met Leu Ser Leu Trp Arg Ser

260 265 270

Gly Val Asn Pro Trp Leu Leu Cys Ser Thr Ser Ser Met Trp Cys Gln

275 280 285

Val Ser Ser Ser Thr

290

<210>13

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: degenerate primers

<220>

<221> variants

<222>(21)

<223> A, C, G, or T

<220>

<221> variants

<222>(24)

<223> A, C, G, or T

<400>13

ctcatcttcg cggtgggcrc ngyngg 26

<210>14

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: degenerate primers

<220>

<221> variants

<222>(22)

<223> C or inosine

<220>

<221> variants

<222>(25)

<223> A, C, G, or T

<220>

<221> variants

<222>(28)

<223> A, C, G, or T

<400>14

ggccaggcag cgctccgcgc tnarncyngc d 31

<210>15

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: degenerate primers

<400>15

gaartartag ccrcgrcagc cw 22

<210>16

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>16

ccatcctaat acgactcact atagggc 27

<210>17

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>17

<3>actcactata gggctcgagc ggc 23

<210>18

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>18

ggatcccaaa taagaaaggg tagttgc 27

<210>19

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>19

aaagggtagt tgcgccacat ctcatagac 29

<210>20

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>20

aggtctatga gatgtggcgc aactaccct 29

<210>21

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>21

atgtggcgca actacccttt cttatttggg 30

<210>22

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: degenerate primers

<400>22

cggaagttgg cggacacgrv rttrta 26

<210>23

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>23

gctcagcttg aaacagagcc tcgtacc 27

<210>24

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>24

ccatgtggat ctacaatttc atcatcc 27

<210>25

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>25

aagacaaatc tcttgaggca gatgaaggg 29

<210>26

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>26

gatgctgttt gtcttggtct tagtgtttgc 30

<210>27

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>27

ggatgatgaa attgtagatc cacatgggc 29

<210>28

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>28

tgtggagaag tctctcaaag tgtgg 25

<210>29

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>29

tagtaggagt gacagcctga ctcggaacg 29

<210>30

<211>30

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>30

aacgtagatg actcaggacg aaccatttcc 30

<210>31

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>31

tcgtaccagg ggaggctcag gc 22

<210>32

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>32

cctcttcagc ctggcggtct ctg 23

<210>33

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<400>33

ggaggcgaag cacacggtct ca 22

<210>34

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: primer and method for producing the same

<220>

<221>misc_binding

<222>(1)

<223> labeling with 6-carboxyfluorescein

<220>

<221>misc_binding

<222>(34)

<223> labeling with N, N, N ', N' -tetramethyl-6-carboxyrhodamine

<400>34

agatgtggcg caactaccct ttcttgttcg ggcc 34

Claims (40)

1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) encoding the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, a nucleotide sequence of the IGS4 polypeptide;

(b) a nucleotide sequence encoding a polypeptide encoded by a DNA insert contained in fungal species collection deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands, in particular a nucleotide sequence comprising the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7;

(c) a nucleotide sequence having at least 80% (preferably at least 90%) sequence identity to the nucleotide sequence (a) or (b) over its entire length;

(d) a nucleotide sequence complementary to the nucleotide sequence (a) or (b) or (c).

2. The polynucleotide of claim 1, wherein the polynucleotide comprises the sequence of SEQ ID NO: 1 encoding the amino acid sequence of SEQ ID NO: 2, the nucleotide sequence of IGS4 polypeptide of SEQ ID NO: 3 encoding the amino acid sequence of SEQ ID NO: 4, the nucleotide sequence of IGS4 polypeptide of SEQ ID NO: 5 encoding the amino acid sequence of SEQ ID NO: 6, or the nucleotide sequence of the IGS4 polypeptide of SEQ ID NO: 7 encoding the amino acid sequence of SEQ ID NO: 8, or a variant thereof, and a nucleotide sequence of the IGS4 polypeptide of 8.

3. The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence that is identical over its entire length to SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7, or a nucleotide sequence at least 80% identical to the sequence of a DNA insert contained in fungal species collection deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands.

4. The polynucleotide of claim 3 which is SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 5. or SEQ ID NO: 7 or a DNA insert contained in deposit No. CBS102221 or deposit No. CBS102222 of Baarn, the netherlands.

5. The polynucleotide of claims 1-4 which is DNA or RNA.

6. An isolated nucleotide sequence encoding an IGS4 neuromedin receptor protein, preferably a mammalian neuromedin receptor protein, which protein exhibits high affinity binding for neuromedin U, preferably for neuromedin U-8, for neuromedin U-23 and/or for neuromedin U-25.

7. An isolated nucleotide sequence according to claim 6 encoding an IGS4 neuromedin receptor protein which exhibits expression in brain, skeletal muscle, cerebellum, testis, corpus callosum, spinal cord, substantia nigra, medulla, thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain, stomach, heart, thyroid gland, lung, thymus, prostate and/or trachea.

8. An isolated nucleotide sequence encoding an IGS4 neuromedin receptor protein, preferably a mammalian neuromedin receptor protein, said protein exhibiting high affinity binding for neuromedin U, preferably for neuromedin U-8, for neuromedin U-23 and/or for neuromedin U-25, said protein exhibiting expression in brain, skeletal muscle, cerebellum, testis, corpus callosum, spinal cord, substantia nigra, medulla, thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain, stomach, heart, thyroid gland, lung, thymus, prostate and/or trachea and said nucleotide sequence being selected from the group consisting of the nucleotide sequences defined in claims 1 to 5.

9. A DNA or RNA molecule comprising an expression system, wherein said expression system is capable of producing a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, or an IGS4 polypeptide having an amino acid sequence at least 80% identical to a polypeptide encoded by a DNA insert contained in fungal species collection deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands.

10. An isolated DNA or RNA molecule comprising an expression system, wherein said expression system is capable of producing an IGS4 polypeptide comprising an amino acid sequence of a neuromedin receptor protein, preferably a mammalian neuromedin receptor protein, said protein exhibiting high affinity binding for neuromedin U, preferably for neuromedin U-8, for neuromedin U-23, and/or for neuromedin U-25, and exhibiting expression in brain, skeletal muscle, cerebellum, testis, corpus callosum, spinal cord, substantia nigra, medulla, thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain, stomach, heart, thyroid, lung, thymus, prostate, and/or trachea.

11. A host cell comprising the expression system of claim 9 or 10.

12. The host cell of claim 11 which is a yeast cell.

13. The host cell of claim 11 which is an animal cell.

14. An IGS4 receptor membrane preparation derived from the cells of claims 11-13.

15. A method for producing an IGS4 polypeptide, comprising culturing the host of claims 11-13 under conditions sufficient for the production of the polypeptide, and recovering the polypeptide from the culture.

16. A method for producing a cell producing an IGS4 polypeptide, comprising transforming or transfecting a host cell with the expression system of claim 9 or 10 such that the host cell produces an IGS4 polypeptide under suitable culture conditions.

17. Comprising a sequence identical to SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, or an IGS4 polypeptide having an amino acid sequence at least 80% identical to a polypeptide encoded by a DNA insert contained in fungal species collection deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands.

18. The polypeptide of claim 17, comprising SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, or an amino acid sequence encoded by a DNA insert contained in the fungal species collection deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands.

19. An isolated IGS4 polypeptide comprising an amino acid sequence of a neuromedin receptor protein, preferably a mammalian neuromedin receptor protein, said protein exhibiting high affinity binding to neuromedin U, preferably neuromedin U-8, neuromedin U-23 and/or neuromedin U-25.

20. The isolated IGS4 polypeptide of claim 19, comprising an amino acid sequence of a neuromedin receptor protein that exhibits expression in brain, skeletal muscle, cerebellum, testis, corpus callosum, spinal cord, substantia nigra, medulla, thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain, stomach, heart, thyroid, lung, thymus, prostate, and/or trachea.

21. An isolated IGS4 polypeptide comprising an amino acid sequence of a neuromedin receptor protein, preferably a mammalian neuromedin receptor protein, said protein exhibiting high affinity binding for neuromedin U, preferably for neuromedin U-8, for neuromedin U-23 and/or for neuromedin U-25, said protein exhibiting expression in brain, skeletal muscle, cerebellum, testis, corpus callosum, spinal cord, substantia nigra, medulla, thalamus, caudate nucleus, pons, nucleus accumbens, fetal brain, stomach, heart, thyroid gland, lung, thymus, prostate and/or trachea and said amino acid sequence being selected from the group consisting of the amino acid sequences as defined in claims 17 to 18.

22. An antibody immunospecific for the IGS4 polypeptide of claims 17-21.

23. A method for treating a subject in need of enhancement of activity or expression of IGS4 polypeptide of claims 17-21, comprising:

(a) administering to the subject a therapeutically effective amount of an agonist of the receptor; and/or

(b) Providing to the subject a polypeptide comprising a sequence identical over its entire length to a sequence encoding SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, or a polypeptide encoded by a DNA insert contained in fungal species collection deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands, and a nucleotide sequence having at least 80% identity to the nucleotide sequence of the polypeptide; or an isolated polynucleotide of a nucleotide sequence that is complementary in some form to one of the nucleotide sequences to cause production of the receptor activity in vivo; and/or

(c) Providing the subject with an isolated polynucleotide comprising a nucleotide sequence encoding a protein comprising an IGS4 neuromedin receptor, preferably a mammalian IGS4 neuromedin receptor, said protein exhibiting high affinity binding for neuromedin U, preferably for neuromedin U-8, for neuromedin U-23 and/or for neuromedin U-25.

24. A method for treating a subject in need of inhibition of activity or expression of IGS4 polypeptide of claims 17-21, comprising:

(a) administering to the subject a therapeutically effective amount of an antagonist of the receptor; and/or

(b) Administering to the subject a nucleic acid molecule that inhibits expression of a nucleotide sequence encoding the receptor; and/or

(c) Administering to the subject a therapeutically effective amount of a polypeptide that competes with the receptor for a ligand.

25. A method for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of IGS4 polypeptides of claims 17-21, comprising:

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said IGS4 polypeptide in the genome of said subject; and/or

(b) Analyzing the presence or absence of expression or amount of expression of said IGS4 polypeptide in a sample derived from said subject.

26. A method for identifying an agonist of IGS4 polypeptide of claims 17-21, comprising:

(a) contacting a cell producing an IGS4 polypeptide with a test compound; and are

(b) Determining whether the test compound causes a signal resulting from activation of the IGS4 polypeptide.

27. An agonist identified by the method of claim 26.

28. A method for identifying an agonist of an IGS4 neuromedin receptor protein, preferably a mammalian IGS4 neuromedin receptor protein, which protein exhibits high affinity binding to neuromedin U, preferably neuromedin U-8, neuromedin U-23 and/or neuromedin U-25, comprising:

(a) contacting a cell producing an IGS4 neuromedin receptor protein with a test compound; and are

(b) Determining whether the test compound causes a signal generated by activation of the IGS4 neuromedin receptor protein.

29. The method of claim 28 for identifying an agonist of the IGS4 neuromedin receptor protein wherein the agonist is effective for a disorder of the nervous system including the Central Nervous System (CNS) and Peripheral Nervous System (PNS), the gastrointestinal system, the cardiovascular system, skeletal muscle, and/or thyroid, and/or is also effective for a pulmonary disease, an immunological disease, and a genitourinary disorder.

30. Agonists identified by the method of claim 28 or 29, preferably agonists that are effective against disorders of the nervous system, including the Central Nervous System (CNS) and Peripheral Nervous System (PNS), the gastrointestinal system, the cardiovascular system, skeletal muscle, and/or thyroid, and/or also against pulmonary diseases, immunological diseases, and genitourinary disorders.

31. A method for identifying antagonists of IGS4 polypeptides of claims 17-21, comprising:

(a) contacting a cell producing an IGS4 polypeptide with an agonist; and are

(b) Determining whether the signal generated by said agonist decreases in the presence of the candidate compound.

32. An antagonist identified by the method of claim 31.

33. A method for identifying antagonists of IGS4 neuromedin receptor proteins, preferably mammalian IGS4 neuromedin receptor proteins, which exhibit high affinity binding for neuromedin U, preferably neuromedin U-8, neuromedin U-23, and/or neuromedin U-25, comprising:

(a) contacting a cell that produces an IGS4 neuromedin receptor protein with an agonist; and are

(b) Determining whether the signal generated by said agonist decreases in the presence of the candidate compound.

34. The method of claim 33 for identifying an antagonist of IGS4 neuromedin receptor protein wherein said antagonist is effective for a disorder of the nervous system including the Central Nervous System (CNS) and Peripheral Nervous System (PNS), the gastrointestinal system, the cardiovascular system, skeletal muscle, and/or thyroid, and/or is also effective for a pulmonary disorder, an immunological disorder, and a genitourinary disorder.

35. An antagonist identified by the method of claim 33 or 34, preferably an antagonist that is effective against a disorder of the nervous system, including the Central Nervous System (CNS) and Peripheral Nervous System (PNS), the gastrointestinal system, the cardiovascular system, skeletal muscle, and/or thyroid, and/or also a pulmonary disease, an immunological disease, and a genitourinary disorder.

36. A recombinant host cell produced by the method of claim 16 or a membrane thereof expressing an IGS4 polypeptide.

37. A method of producing a genetically modified non-human animal comprising:

(a) linking a coding portion of a nucleic acid molecule to a regulatory sequence capable of driving high levels of gene expression or expression in a cell type in which the gene is not normally expressed in said animal, wherein said coding portion consists essentially of a nucleic acid sequence encoding a polypeptide having the sequence set forth in SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8 or the nucleic acid sequence of a protein of the amino acid sequence encoded by the DNA insert contained in fungal species collection deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands, or a biologically active portion of one of said sequences; or

(b) Isolating and engineering a coding portion of a nucleic acid molecule consisting essentially of a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8 or the nucleic acid sequence of a protein having the amino acid sequence encoded by a DNA insert contained in fungal species collection deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands, or a biologically active part of one of said sequences, and reintroducing said sequences into the genome of said animal such that a polypeptide having the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8 or the amino acid sequence encoded by the DNA insert contained in deposit number CBS102221 or deposit number CBS102222 of Baarn, the netherlands, is completely or partially inactivated.

38. A method for determining whether a substance is a potential ligand of the IGS4 receptor, comprising:

(a) expressing the polypeptide of any one of claims 17 to 21 or SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8, or a cell comprising a receptor according to any one of claims 17 to 21 or SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 6. or SEQ ID NO: 8 contacting a receptor membrane preparation of one of said receptors with labeled neuromedin U; and are

(b) Binding of neuromedin U to the IGS4 was measured.

39. The polypeptide according to any of claims 17 to 21, characterized in that the polypeptide binds neuromedin U, preferably neuromedin U-8, neuromedin U-23 and/or neuromedin U-25 and exhibits an affinity of at least about log EC₅₀＝-6。

40. RightsThe polypeptide according to any one of claims 17 to 21, characterized in that the polypeptide binds neuromedin U, preferably neuromedin U-8, neuromedin U-23 and/or neuromedin U-25 and exhibits an affinity of at least about log EC₅₀＝-9。