WO2002088182A1 - Nove g protein-coupled receptor protein and dna thereof - Google Patents

Abstract

A mouse-origin protein and a polynucleotide encoding the same which are usable in: (1) determining a ligand to this protein; (2) preventives and/or remedies for diseases relating to the hypofunction of the above protein; (3) screening a compound (an agonist, an antagonist and so on) capable of altering the binding properties of the above protein to a ligand; etc.

Description

 Description New G-protein coupled receptor Yuichi protein and its DNA

 The present invention relates to a novel G protein-coupled receptor protein derived from mouse brain or a salt thereof, and DNA encoding the same. BACKGROUND ART '' Many physiologically active substances such as hormones and neurotransmitters regulate the functions of living organisms through specific receptor proteins present in cell membranes. Many of these receptor proteins transmit intracellular signals through the activation of the conjugated guanine nucleic acid-binding protein (hereinafter abbreviated as G protein). Since they have a common structure having a transmembrane region, they are collectively referred to as G protein-coupled receptor proteins or seven transmembrane receptors (7TMR).

 G protein-coupled receptor proteins are present on the surface of each functional cell in living cells and organs, and are physiologically targeted as molecules that regulate the functions of those cells and organs, such as hormones, neurotransmitters and bioactive substances. It plays an important role. The receptor transmits a signal into the cell via binding to a physiologically active substance, and this signal causes various reactions such as suppression of activation and activation of the cell. To clarify the relationship between substances that regulate complex functions in cells and organs of various organisms and their specific receptor proteins, especially G protein-coupled receptor proteins, It will provide a very important means for drug development closely related to these functions.

For example, in various organs of the living body, physiological functions are regulated under the control of many hormones, hormone-like substances, neurotransmitters or bioactive substances. In particular, physiologically active substances are present at various sites in the body, and regulate their physiological functions through the corresponding receptor proteins. There are many unknown hormones, neurotransmitters and other physiologically active substances in the living body, and the structure of these receptor proteins has not yet been reported. Furthermore, it is often unknown whether subtypes exist in known receptor proteins.

 Clarifying the relationship between substances that regulate complex functions in living organisms and their specific receptor proteins is a very important tool for the development of pharmaceuticals including agonists and antagonists against receptor proteins. . However, in the past, in order to efficiently screen agonists and angonists against receptor proteins and to develop pharmaceuticals, the functions of receptor protein genes expressed in vivo were elucidated, and these were clarified. It was necessary to express it in an appropriate expression system.

 In recent years, studies on random analysis of cDNA sequences have been actively conducted as a means of analyzing genes expressed in vivo. Registered in the database as a Sequence Tag (EST) and published. However, most ESTs contain only sequence information, and it is difficult to estimate their functions.

 Conventionally, substances that inhibit the binding between G protein-coupled receptors and their ligands and biologically active substances, and substances that bind and cause signal transduction similar to those of biologically active substances, are specific antagonists of these receptors. Or as an agonist, it has been used as a drug to regulate biological functions. Therefore, a novel G protein-coupled receptor protein that is not only important in physiological expression in vivo but also a target for drug development has been newly discovered, and its gene (eg, cDNA) has been cloned. This is a very important tool for finding specific ligands for novel G protein-coupled receptor protein I, agonist, and gonist.

However, not all G protein-coupled receptors have been found, and at this time there are many unknown G protein-coupled receptors and so-called orphan receptors whose ligands have not been identified. Therefore, there is a keen need to search for new G protein-coupled receptors and to elucidate their functions. The G protein-coupled receptor is useful for searching for a new ligand (a physiologically active substance) using its signal transduction action as an index, and for searching for an agonist or an agonist for the receptor. On the other hand, even if a physiological ligand is not found, by analyzing the physiological action of the receptor from an inactivation experiment of the receptor (experiment using a knockout animal), an agonist or an antagonist to the receptor is analyzed. It is also possible to make evening gonists. These ligands, agonists, and antagonists for the receptor can be expected to be used as preventive / therapeutic or diagnostic agents for diseases associated with dysfunction of G protein-coupled receptors.

 Furthermore, a decrease or enhancement of the function of the receptor in the living body based on a gene mutation of a G protein-coupled receptor often causes some disease. In this case, not only administration of an agonist or agonist to the receptor, but also introduction of the receptor gene into a living body (or a specific organ) or introduction of an antisense nucleic acid to the receptor gene. It can also be applied to gene therapy. In this case, the nucleotide sequence of the receptor is indispensable information for examining the presence or absence of a deletion or mutation in the gene, and the gene of the receptor is a prophylactic / therapeutic agent for a disease associated with dysfunction of the receptor. And can be applied to diagnostics.

The present invention provides a novel G protein-coupled receptor protein useful as described above. That is, a novel G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, a polynucleotide encoding the G protein-coupled receptor protein or a partial peptide thereof (DNA, RNA, and the like) (D, NA, RNA and derivatives thereof), a recombinant vector containing the polynucleotide, a transformant carrying the recombinant vector, the G protein A method for producing a conjugated receptor protein or a salt thereof, an antibody against the G protein-coupled receptor protein or a partial peptide or a salt thereof, a compound that changes the expression level of the G protein-coupled receptor protein, A method for determining a ligand for the G protein-coupled receptor, comprising: changing the binding between the ligand and the G protein-coupled receptor protein Method for screening a compound (antagonist, agonist) or a salt thereof, the binding between a ligand obtainable by using the screening kit, the screening method or the screening kit, and the G protein-coupled receptor protein (Antagonist, agonist) or a salt thereof, and a compound that changes the binding property between a ligand and the G protein-coupled receptor protein (antagonist, agonist) or the G protein-conjugated compound It is intended to provide a medicament or the like containing a compound that changes the expression level of a receptor protein or a salt thereof. Disclosure of the invention

 The present inventors have conducted extensive research and have succeeded in isolating cDNA encoding a novel G protein-combined receptor Yuichi protein derived from mouse brain and analyzing its entire nucleotide sequence. . When this nucleotide sequence was translated into an amino acid sequence, the first to seventh transmembrane regions were identified on the hydrophobicity plot shown in FIG. 1, and the protein encoded by these cDNAs was transmembrane-seven times. Type G protein-coupled receptor. The present inventors have conducted further studies based on these findings, and as a result, have completed the present invention. That is, the present invention

 (1) a G protein-coupled receptor protein or a salt thereof, comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1;

 (2) a G protein-coupled receptor protein or a salt thereof, which comprises the amino acid sequence represented by SEQ ID NO: 1;

 (3) a partial peptide of the G protein-coupled receptor protein described in (1) or a salt thereof,

 (4) a polynucleotide comprising a polynucleotide encoding the G protein-coupled receptor protein described in (1) above,

(5) the polynucleotide according to (4), which is DNA; (6) the polynucleotide according to the above (5), having the nucleotide sequence represented by SEQ ID NO: 2;

 (7) a recombinant vector containing the polynucleotide according to (4),

(8) A transformant transformed by the recombinant vector according to (7) above, (9) A transformant described in (8) above is cultured, and the G protein-coupled receptor according to (1) is cultured. A method for producing a G protein-coupled receptor protein or a salt thereof according to the above (1), which comprises producing a protein;

 (10) a medicine comprising the G protein-coupled receptor protein according to (1) or the partial peptide according to (3) or a salt thereof;

 (11) a medicine comprising the polynucleotide according to the above (4),

 (12) an antibody against the G protein-coupled receptor protein according to the above (1) or the partial peptide or a salt thereof according to the above (3);

 (13) the antibody according to (12), which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to (1);

 (14) a diagnostic agent comprising the antibody according to the above (12),

 (15) The G protein-coupled receptor protein described in (1) above, which can be obtained by using the G protein-coupled receptor protein described in (1) or the partial peptide described in (3) or a salt thereof. A ligand for the salt,

 (16) a medicament comprising the ligand according to the above (15),

 (17) The G protein-coupled receptor protein according to (1), wherein the G protein-coupled receptor protein according to (1) or the partial peptide according to (3) or a salt thereof is used. (18) A method for determining a ligand for a salt thereof, wherein (18) the G protein-coupled receptor protein described in the above (1) or the partial peptide described in the above (3) or a salt thereof is used. A method for screening a compound or a salt thereof that alters the binding property between a G protein-coupled receptor protein or a salt thereof and a ligand;

(19) The G protein-coupled receptor protein described in (1) above, which comprises the G protein-coupled receptor protein described in (1) or the partial peptide or salt thereof described in (3). The binding between the salt and the ligand A kit for screening a compound to be changed or a salt thereof,

 (20) A ligand obtainable by using the screening method described in (18) or the screening kit described in (19) and the G protein-coupled receptor protein or salt thereof described in (1). Or a salt thereof, which changes the binding property of

 (21) The ligand obtainable by using the screening method described in (18) or the screening kit described in (19) above and the G protein-conjugated receptor protein or its salt described in (1) above (3) A medicament comprising a compound that changes the binding property or a salt thereof,

 (22) a polynucleotide that hybridizes with the polynucleotide of (4) above under high stringency conditions.

 (23) a polynucleotide comprising a nucleotide sequence complementary to the polynucleotide according to (4) or a part thereof,

 (24) The method for quantifying mRNA of G protein-coupled receptor protein according to (1), which comprises using the polynucleotide or a part thereof according to (4);

 (25) The method for quantifying the G protein-coupled receptor Yuichi protein according to (1), which comprises using the antibody according to (12);

 (26) The method for diagnosing a disease associated with the function of a G protein-coupled receptor according to (1), which comprises using the quantification method according to (24) or (25) above,

 (27) The method for screening a compound or a salt thereof that alters the expression level of a G protein-coupled receptor protein according to (1), wherein the method comprises using the quantification method according to (24).

 (28) A method for screening a compound or a salt thereof that alters the amount of a G protein-coupled receptor protein according to (1) in a cell membrane, which comprises using the quantification method according to (25);

(29) A compound that alters the expression level of the G protein-coupled receptor protein according to (1), which can be obtained by using the screening method according to (27) or Its salt,

 (30) The compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein described in (1) above in a cell membrane obtainable by using the screening method described in (28) above,

 (31) a pharmaceutical comprising a compound or a salt thereof that alters the expression level of the G protein-coupled receptor protein according to (1), which can be obtained by using the screening method according to (27);

 (32) A medicine comprising a compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein described in (1) above in a cell membrane obtainable by using the screening method described in (28).

 (33) The medicament according to any of (21), (31) or (32), which is a preventive or therapeutic agent for central disease, endocrine disease, metabolic disease, cancer or heart disease.

(34) administering to the mammal an effective amount of the compound or salt thereof according to any of (20), (29) or (30) above, Prevention and treatment of central diseases, endocrine diseases, metabolic diseases, cancer or heart diseases,

 (35) The compound according to any one of (20), (29) or (30) above, or a compound thereof, for producing a prophylactic or therapeutic agent for central disease, endocrine disease, metabolic disease, cancer or heart disease. Related to the use of salt. '

 Moreover,

(36) (1) the amino acid sequence represented by SEQ ID NO: 1; (2) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30, more preferably 1 to 10) An amino acid sequence in which several (1 to 5) amino acids have been deleted, and 3 or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30) More preferably about 1 to 10, more preferably several (1 to 5) amino acids; ④ one or two or more (preferably An amino acid sequence in which about 1 to 30, more preferably about 1 to 10, and still more preferably several (1 to 5) amino acids have been substituted with other amino acids; or The G protein-coupled receptor Yuichi protein or a salt thereof according to the above (1), comprising an amino acid sequence obtained by combining them.

 (37) The ligand according to (17), wherein the G protein-coupled receptor protein or the partial peptide or salt thereof according to (1) is contacted with a test compound. Decision method,,

(38) The ligand is, for example, angiotensin, bombesin, cannapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasoprescin, oxitocin, PAC ΑΡ (eg, PACAP 27, PACAP 38) , Secretin, glucagon, calcitonin, adrenomedullin, somatostin, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal Polypeptide), somatostin, dopamine, motilin, amylin, bradykinin, CGRP (Calcitonin gene relayed peptide), leukotriene, pancreatin, prostaglandin, tropoxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GR◦a, GROi3, GROa, NAP) -2, ENA-78, GCP-2, PF4, IP10, Mig, CXC chemokine subfamily such as PBSF / SDF-1; MCAF / MCP1, MCP_2, MCP-3, MCP-4, eotaxin, RANTES , MI P-1, MI P- l i3, HCC-1, MIP-3 α / LARC, MIP-3iB / ELC, I-309, TARC, MI PF-1, MIPF-2 / eotaxin-2, MD CC chemokine subfamily such as C, DC-CK1 / PARC, SLC; C chemokine subfamily such as lymphotactin; CX3 C chemokine subfamily such as iractalkine etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, Pancreatic polypeptides, galanin, lysophosphatidic acid (LPA), sphingosin monophosphate, lysophosphatidylserine, salts, bile acids, isoprenide, arachidonic acid metabolites, amines, amino acids, nucleotides, nucleosides, saturation Fatty acids or Method of determining the saturated fatty acid above (37), wherein the ligand,> (39) (i) contacting the ligand with the G protein-coupled receptor protein described in (1) or the partial peptide described in (3) or a salt thereof,

(ii) comparing the G protein-coupled receptor protein described in (1) above or the partial peptide described in (3) or a salt thereof with a ligand and a test compound when they are brought into contact with each other. The screening method described in (18) above,

 (40) (0 The case where the labeled ligand is brought into contact with the G protein-coupled receptor-protein described in (1) above or the partial peptide described in (3) or a salt thereof, and (ii) the labeled ligand and the test When the compound is brought into contact with the G protein-coupled receptor protein described in (1) above or the partial peptide described in (3) above or a salt thereof, the G protein-coupled receptor described in (1) above of the labeled ligand is obtained. Measuring the amount of binding to one protein or the partial peptide described in (3) or a salt thereof and comparing the ligand with the G protein-coupled receptor protein or the salt thereof described in (1). A method of screening for a compound or a salt thereof that changes the binding property,

 (41) (i) when the labeled ligand is brought into contact with cells containing the G protein-coupled receptor protein described in (1) above; and (ii) when the labeled ligand and test compound are as described in (1) above. (2) measuring the amount of the labeled ligand bound to the cell when the cell is contacted with the cell containing the G protein-coupled receptor protein described in (1), and comparing the ligand with the G in (1). A method of screening for a compound or a salt thereof that alters the binding to a protein-coupled receptor protein or a salt thereof,

(42) (i) when the labeled ligand is brought into contact with the membrane fraction of the G protein-coupled receptor-protein-containing cells described in (1) above, and (ii) when the labeled ligand and test compound are (1) measuring the amount of binding of a labeled ligand to the membrane fraction of the cell when the cell is contacted with the membrane fraction of the cell containing the G protein-coupled receptor protein described in (1), and comparing the measured amounts. A method for screening a compound or a salt thereof that alters the binding property between a ligand that binds to a G protein-coupled receptor protein or a salt thereof according to (1) above, (43) (i) contacting the labeled ligand with a G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to (8) above; (Ii) Labeling in the case where the labeled ligand and the test compound were brought into contact with the G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to (8) above; The amount of binding of the ligand to the G protein-coupled receptor protein is measured and compared, and the binding between the ligand and the G protein-coupled receptor protein or salt thereof described in (1) above is determined. A method of screening the compound to be changed or a salt thereof,

 (44) (0) The case where the compound that activates the G protein-coupled receptor protein or the salt thereof described in (1) above is brought into contact with the cell containing the G protein-coupled receptor protein described in (1) above. (Ii) a compound that activates the G protein-coupled receptor protein or the salt thereof described in (1) above and a test compound are brought into contact with cells containing the G protein-coupled receptor protein described in (1) above. In this case, binding of the liquid to the G protein-coupled receptor protein or the salt thereof according to (1) above, wherein the cell stimulating activity mediated by the G protein-coupled receptor protein is measured and compared. A method for screening a compound or a salt thereof,

 (45) A compound that activates the G protein-coupled receptor protein or its salt according to (1) was expressed in the cell membrane of the transformant by culturing the transformant according to (8). When the G protein-coupled receptor protein is brought into contact with the G protein-coupled receptor protein described in (1) above, the compound that activates the G protein-coupled receptor protein or a salt thereof and a test compound are transformed with the transformant described in (7) above. Measuring and comparing the cell stimulating activity via the G protein-coupled receptor 1 protein when the transformant is brought into contact with the G protein-coupled receptor 1 protein expressed on the cell membrane of the transformant. A method for screening a compound that changes the binding property between a ligand that binds to a G protein-coupled receptor protein or a salt thereof according to the above (1) or a salt thereof,

(46) The compound that activates the G protein-coupled receptor protein described in (1) above. If the substance is angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, pudding, vasoprescin, oxitosine, PACAP (eg, PACAP27, PACAP 38), secretin, glucagon, 7, calcitonin Drenomedullin, Somatos, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal Polypeptide), Somatos, Dipamine, Motilin, Amylin, Bradykinin, CGRP (Calcitonin Renewed) Peptide), leukotriene, pancreatin, prostaglandin, tropoxane, adenosine, adrenaline, chemokine inspa family 1 (eg, IL-8, GROa, GROi3, GROa, NAP-2, ENA_78, GCP-2 , PF4, IP 10, Mi g, PBSF / SDF-l and other CXC chemokine subfamilies; MCAF / MCP_1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, MIP-1α, MIP-1 / 3, HCC- 1, MIP-3 «/ LARC, MIP-3j3 / ELC, 1—309, TARC, MIPF—1, MIPF-2 / eotaxin-2, MDC, DC-CK1 / PAR3 (: etc. (3 (chemokine Subfamily 1; C chemokine subfamily such as lymphotactin; CX3C chemokine subfamily such as iractalkine, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA) ), Sphingosine 1-phosphate, lysophosphatidylserine, sphingosylphosphorylcholine, lysophosphatidylcholine, steroids, bile acids, isoprenoids, arachidonic acid metabolites, amines, amino acids Nucleotide, nucleoside, a saturated fatty acid or unsaturated fatty acid above (44) or (45) the screening method described in

 (47) A compound or a salt thereof that alters the binding property between the ligand obtainable by the screening method according to (39) to (46) and the G protein-coupled receptor protein or salt thereof according to (1). ,

(48) The reaction between the ligand obtainable by the screening method described in (39) to (46) and the G protein-coupled receptor protein or salt thereof described in (1). A medicament comprising a compound that changes the binding property or a salt thereof,

 (49) The screening kit according to (19), which comprises a cell containing the G protein-coupled receptor protein according to (1).

 (50) The screening kit according to (19), which comprises a membrane fraction of a cell containing the G protein-coupled receptor-1 protein according to (1).

 (51) The screening method according to the above (19), which comprises a G protein-coupled receptor protein expressed in the cell membrane of the transformant by culturing the transformant according to the above (8). Kit,

 (52) A compound or a compound that alters the binding between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1), which can be obtained using the screening kit according to (49) to (51). Its salt,

 (53) A compound or a compound that alters the binding between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1), which can be obtained using the screening kit according to (49) to (51). A medicament comprising the salt,

 (54) The antibody according to (1), wherein the antibody according to (12) is contacted with the G protein-coupled receptor protein according to (1) or the partial peptide or salt thereof according to (3). ) G protein-coupled receptor protein or the above

(3) a method for quantifying the partial peptide or a salt thereof according to the above,

 (55) The antibody according to (12) and a test solution and a labeled G protein-coupled receptor protein according to (1) or the partial peptide or salt thereof according to (3) above. Competitively reacting and measuring the ratio of the labeled G protein-coupled receptor protein described in (1) or the partial peptide described in (3) or a salt thereof bound to the antibody. A method for quantifying the G protein-coupled receptor protein described in (1) above or the partial peptide or salt thereof described in (3) above in a test solution, and

(56) After reacting the test solution with the antibody of (12) insolubilized on the carrier and the labeled antibody of (12) simultaneously or continuously, the label on the insolubilized carrier is reacted. Measuring the activity of the agent in the test solution described in (1) above. And a method for quantifying the G protein-coupled receptor protein or the partial peptide described in (3) or a salt thereof. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a hydrophobicity plot of TGR38. BEST MODE FOR CARRYING OUT THE INVENTION

 The G protein-coupled receptor protein of the present invention (hereinafter sometimes abbreviated as receptor protein) may have an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1. Contains Recept Yuichi protein.

 The receptor protein of the present invention includes, for example, all cells (eg, spleen cells, neurons) of human mammals (eg, guinea pigs, rats, mice, rabbits, bushes, sheep, horsetails, monkeys, etc.) , Glial cells, kidney | 8 cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T cells , B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, breast cells Hepatocytes or stromal cells, or precursors of these cells, stem cells or cancer cells), blood cells, or any tissue in which these cells are present, such as , Brain, various parts of the brain (e.g., olfactory bulb, acrosomal nucleus, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital lobe, frontal lobe, temporal lobe, putamen , Caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary, stomach, kidney, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, colon, Small intestine), proteins derived from blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate gland, testis, testis, ovary, placenta, uterus, bone, joints, skeletal muscle, etc. Or a synthetic protein.

The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is, for example, about 50% or more, preferably about 60% or more, more preferably the amino acid sequence represented by SEQ ID NO: 1. Is about 70% or more, more preferably about 80% Above all, preferably, an amino acid sequence having about 90% or more, most preferably about 95% or more homology is mentioned.

 Examples of the protein of the present invention containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include, for example, an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 And a protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1.

 Such activities include, for example, ligand binding activity, signal transduction action and the like. Substantially the same means that their activities are the same in nature. Therefore, activities such as ligand binding activity and signal transduction activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times). However, quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.

 The measurement of the activity such as the ligand binding activity and the signal information transmission activity can be performed according to a known method.For example, the measurement can be performed according to a ligand determination method or a screening method described later. it can.

 Further, the receptor protein of the present invention includes: (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30, more preferably about 1 to 10, more preferably An amino acid sequence in which several (1 to 5) amino acids have been deleted; (2) one or more (preferably about 1 to 30, more preferably 1 to 10) amino acid sequences represented by SEQ ID NO: 1 An amino acid sequence to which about, more preferably several (1 to 5) amino acids have been added; ③ SEQ ID NO: 1 or 2 or more (preferably 1 to 30) in the amino acid sequence represented by SEQ ID NO: 1 , More preferably about 1 to 10, more preferably several (i to 5) amino acids in which the amino acid sequence is substituted with another amino acid, or a protein containing an amino acid sequence in which they are combined. Also used That.

In the present specification, the amino acid sequence of the receptor protein is N-terminal (amino terminal) at the left end and C-terminal (terminal end of lipoxyl) at the right end according to the convention of peptide labeling. The receptor protein of the present invention, including the receptor protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminal carboxy group. Or a carboxylate (one C00—), an amide (_C0NH ₂ ) or an ester (one C00R).

Here, as R in the ester, e.g., methyl, Echiru, n- propyl, C, such as isopropyl or n- butyl, _ ₆ alkyl groups, for example, shea Kuropenchiru, C ₃ _ ₈ cycloalkyl group such as cyclohexyl , for example, Hue sulfonyl, C ₆ _ ₁₂ Ariru groups, such as single-naphthyl, for example, benzyl, phenylene Lou C such as phenethyl, _ ₂ alkyl or α- naphthylmethyl α- Na Fuchiru C Bok ₂ alkyl groups, such as such as C ₇ _ ₁₄ other Ararukiru groups, such as pivaloyl Ruo carboxymethyl group which is generally used as an oral ester.

 When the receptor protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the receptor protein of the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.

Furthermore, the receptions evening one protein of the present invention is the protein mentioned above, Amino groups Mechionin residues of Ν-terminal protecting group (for example, C, such as formyl group, which C ₂ _ ₆ Arukanoiru group of Asechiru, _ _6- glycyl group, etc.), the glutamyl group generated by cleavage of the terminal end in vivo and the glutamine oxidation of the mouth, the substituent on the side chain of the amino acid in the molecule (eg, SH, amino group, imidazo Ichiru group, indole group, Guanijino group, etc.) a suitable protecting group (e.g., formyl group, C ₂ such Asechiru - such as C, such as ₆ Arukanoiru group, _ ₆ § Shi Le group) And complex proteins such as so-called glycoproteins to which sugar chains are bound.

 Examples of the receptor protein of the present invention include, for example, a receptor protein containing an amino acid sequence represented by SEQ ID NO: 1 and the like.

The partial peptide of the receptor protein of the present invention (hereinafter sometimes abbreviated as partial peptide) may be any partial peptide of the receptor protein of the present invention described above. For example, among the receptor protein molecules of the present invention, those which are exposed outside the cell membrane and have substantially the same activity can be used. Here, “substantially the same activity” indicates, for example, ligand binding activity. The measurement of the ligand binding activity can be performed in the same manner as described above.

 Specifically, the partial peptide of the receptor protein having the amino acid sequence represented by SEQ ID NO: 1 is an extracellular region (hydrophilic region) in the hydrophobic plot shown in FIG. It is a peptide that contains the part that was analyzed. Further, a peptide partially containing a hydrophobic (Hydrophobic) site can also be used. A peptide containing individual domains may be used, but a peptide containing a plurality of domains at the same time may be used.

 The number of amino acids in the amino acid sequence of the partial peptide of the present invention is at least 20 or more, preferably 50 or more, more preferably 100 or more of the amino acid sequences constituting the receptor protein of the present invention. .

 A substantially identical amino acid sequence refers to an amino acid sequence having about 50% or more, preferably about 60% or more, more preferably about 70% or more, still more preferably about 80% or more, and still more preferably about 90% or more. The amino acid sequence has the above, most preferably about 95% or more homology.

 In the amino acid sequence of the partial peptide of the present invention, 1) one or more (preferably about 1 to 10, and more preferably several (1 to 5)) amino acids in the above amino acid sequence are deleted; (2) One or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids are included in the above amino acid sequence. (3) One or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the above amino acid sequence are replaced with other amino acids May be substituted. Further, any two or more selected from the above (1) to (3) may be appropriately combined.

Further, C-terminal, the partial peptide of the present invention, the force Rupokishiru group rather - C00H), the force Rupokishireto (one C00-), amide (- C0Nh ₂₎ or an ester (which may be either single C00R) (R is The meaning is as defined above). When the partial peptide of the present invention has a carboxyl group (or carboxylate) at a position other than the C-terminus, those having a lipoxyl group amidated or esterified may also be used in the present invention. Included in partial peptides. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

 Furthermore, the partial peptide of the present invention has the same structure as the above-mentioned receptor protein of the present invention, in which the amino group of the N-terminal methionine residue is protected with a protecting group, but the N-terminal is cleaved in vivo. Gin generated by pyroglutamine oxidation, amino acids in which the substituent on the side chain of the amino acid is protected by an appropriate protecting group, or complex peptides such as so-called bran peptides to which sugar chains are bound are also included. It is.

 Examples of the salt of the receptor protein or its partial peptide of the present invention include physiologically acceptable salts with acids or bases, and especially physiologically acceptable salts.

Preferred are acid addition salts. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) Acids, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid) are used.

 The receptor protein of the present invention or a salt thereof can be produced from the above-described human or mammalian cells or tissues by a known method for purifying a receptor protein. Alternatively, the receptor protein of the present invention described later can be prepared. It can also be produced by culturing a transformant containing the encoding DNA. Also, the protein can be produced by the protein synthesis method described later or according to the method.

 When the receptor protein of the present invention or a salt thereof is produced from human or mammalian tissues or cells, the human or mammalian tissues or cells are homogenized and then extracted with an acid or the like, and the extract is subjected to chromatography. (Eg, reversed phase chromatography, ion exchange chromatography, or a combination thereof), the receptor protein of the present invention or a salt thereof can be purified and isolated.

For production of the receptor protein of the present invention, its partial peptide, its salt or its amide, a commercially available resin for protein synthesis can be used. Such resins include, for example, chloromethyl resin, hydroxymethyl Resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethylphenylacetamide methyl resin, polyacrylamide Resin, 4- (2 ′, 4′-dimethoxyphenylhydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-1-Fmocaminoethyl) phenoxy resin and the like. Using such a resin, amino acids having a suitably protected amino group and side chain functional group are condensed on the resin in accordance with the amino acid sequence of the target protein or peptide according to various known condensation methods. At the end of the reaction, the protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed.In addition, an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or its amide You.

 For the condensation of the protected amino acids described above, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. Examples of the sulfoimides include DCC, Ν, Ν'-diisopropylcarpo- imide, and Ν-ethyl_Ν '-(3-dimethylaminoprolyl) carbodiimide. When using these activating reagents, the protected amino acid may be added directly to the resin along with a racemization inhibitor (eg, HOBtHOOBt), or may be added to the symmetric acid anhydride or HOBt ester or HOOBt. The amino acid is condensed by adding the activated amino acid to the resin after activating the protected amino acid in advance as an ester.

The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluorophenol Sulfoxides such as dimethylsulfoxide; amines such as pyridine; ethers such as dioxane and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; An appropriate mixture or the like is used. The reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually selected from the range of about 20 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5- to 4-fold excess. As a result of the test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, the unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

 Examples of the protecting group for the amino group of the starting material include Z, Boc, Yuichi Sharipentyloxycarbonyl, Isopolnylooxycarponyl, 4-methoxybenzyloxylponyl, C11Z, Br—Z, Damantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 212-trophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used. The carboxyl group can be, for example, alkyl esterified (for example, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.). Branched or cyclic alkyl esterification), aralkyl esterification (eg, benzyl ester, 412 trobenzyl ester, 4-methoxybenzyl ester, chlorobenzene ester, benzhydryl esterification), fenasi It can be protected by esterification, benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide, or the like.

 The hydroxyl group of serine can be protected, for example, by esterification or etherification. As a group suitable for this esterification, for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarponyl group, an ethoxycarbonyl group and the like are used. Examples of a group suitable for etherification include a benzyl group, a tetrahydroviranyl group, and a t-butyl group.

The protecting group of the phenolic hydroxyl group of tyrosine, 'for example, B z 1, G 1 ₂ -Bz 2-nitrobenzyl, Br-Ζ, tert-butyl and the like are used.

 As the protecting group for histidine imidazole, for example, Tos, 4-methoxy2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

 Examples of the activated form of the raw oxypoxyl group include, for example, a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4 —Esters with dinitrophenol, cyanomethyl alcohol, paranitrophenol, H—NB, N-hydroxysuccinimide, N-hydroxyphenolimide, HOB t)]. As the activated amino group of the raw material, for example, a corresponding phosphoric amide is used.

 As a method for removing (eliminating) a protecting group, for example,? 01—What is black? (1-catalytic reduction in a stream of hydrogen in the presence of a catalyst such as carbon; acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof; diisopropyl ester Base treatment with tilamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, etc. The elimination reaction by the above acid treatment is generally about -20 ° C to 40 ° C. It is carried out at the temperature of C, but in the acid treatment, for example, anisol, phenol, thioanisole, methacrylol, paracresol, dimethylsulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc. It is effective to add a cation scavenger such as the following: Also used as an imidazole protecting group of histidine 2 The, 4-dinitrophenyl group is removed by thiophenol treatment, and the formyl group used as a tryptophan indole protecting group is an acid in the presence of 1,2-ethanedithiol, 1,4-butanedithiol and the like described above. In addition to deprotection by treatment, it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.

The protection of functional groups that should not be involved in the reaction of the raw materials and the protecting groups, and the removal of the protecting groups, the activation of the functional groups involved in the reaction, etc. are known groups or known groups. It can be appropriately selected from the means.

 As a method for obtaining an amide form of a protein, for example, first, an α-functional propyloxyl group of the C-terminal amino acid is protected by amidation, and a peptide (protein) chain is extended to a desired chain length on the amino group side. A protein was prepared by removing only the protecting group of the α-amino group at the Ν-terminal of the peptide chain and a protein from which only the protecting group of the C-terminal lipoxyl group was removed. A method of condensing in water can also be used. The condensation is performed in the same manner as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein. The crude protein is purified using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.

 In order to obtain an ester of a protein, for example, after condensing the α-hydroxyl group of the C-terminal amino acid with a desired alcohol to form an amino acid ester, the desired ester is obtained in the same manner as in the case of the amide of the protein. An ester of protein can be obtained.

 The partial peptide of the present invention or a salt thereof can be produced according to a known peptide synthesis method, or by cleaving the receptor protein of the present invention with an appropriate peptide. Examples of the peptide synthesis method include a solid phase synthesis method and a liquid phase synthesis method. For example, the partial peptide or amino acid that can constitute the receptor protein of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is removed to produce the desired partial peptide. can do. Here, the condensation and the elimination of the protecting group are performed, for example, according to the methods described in the following ① to ⑤.

 (1) M. Bodanszky and M. A. Ondet ti, peptide synthesis (Pepti de Synthes is ;; Interscience Publisher, New York (1966))

 ② Schroeder and Luebke, The Peptide, Academic Press, New York (1965)

 (3) Nobuo Izumiya et al. Basics and experiments on peptide synthesis, Maruzen Co., Ltd. (1975)

治 Haruaki Yajima and Shunpei Sakakibara, Laboratory of Biochemistry 1, Protein Chemistry IV, 205, (1977)

 島 Supervised by Haruaki Yajima, Development of Pharmaceuticals, Vol. 14 Peptide Synthesis Hirokawa Shoten In addition, the partial peptides obtained in this way can be purified by conventional purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, It can be purified and isolated by recrystallization or a combination thereof. When the partial peptide obtained by the above method is free, it can be converted to an appropriate salt by a known method.On the contrary, when the partial peptide is obtained as a salt, it can be converted to a free form by a known method. Can be converted.

 The polynucleotide encoding the receptor protein of the present invention may be any polynucleotide containing a nucleotide sequence (DNA or RNA, preferably DNA) encoding the above-described receptor protein of the present invention. Good. Examples of the polynucleotide include RNA such as DNA and mRNA encoding the receptor protein of the present invention. These may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).

 Using a polynucleotide encoding the receptor protein of the present invention, a known method, for example, the method described in Experimental Medicine Special Edition “New PCR and Its Application” 15 (7), 1997 or a method analogous thereto, specifically Can quantify the mRNA of the receptor protein of the present invention by a method such as TaqManPCR.

Examples of the DNA encoding the receptor protein of the present invention include genomic DNA, genomic DNA library, cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. The DNA can also be directly amplified by the reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using the total RNA or mRNA fraction prepared from the above-mentioned cell'tissue. . , Specifically, as the DNA encoding the receptor protein of the present invention, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a DNA having the nucleotide sequence represented by SEQ ID NO: 2 And a DNA that hybridizes under high stringent conditions and has substantially the same activity (eg, ligand binding activity, signal transduction action, etc.) as the receptor protein of the present invention. And DNA encoding the same.

 Examples of the DNA that hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 70% or more, preferably about 80% of the nucleotide sequence represented by SEQ ID NO: 2. DNA containing a nucleotide sequence having a homology of at least about 90% or more, more preferably at least about 95% or more is used.

 Hybridization can be performed by a known method or a method analogous thereto, such as the method described in Molecular 'Cloning (Molecular Cloning) 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). It can be done according to. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. Hybridization is preferably performed according to high stringency conditions.

 Here, the high stringency conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. Is shown. In particular, a condition in which the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.

 More specifically, as the DNA encoding the receptor protein containing the amino acid sequence represented by SEQ ID NO: 1, DNA containing the base sequence represented by SEQ ID NO: 2, etc. is used.

 A part of the nucleotide sequence of DNA encoding the receptor protein of the present invention or a polynucleotide containing a part of a nucleotide sequence complementary to the DNA is a partial peptide of the present invention described below. It is used not only to include the encoding DNA, but also to include RNA.

According to the present invention, replication or expression of a G protein-coupled receptor protein gene is provided. Antisense 'polynucleotides (nucleic acids) that can inhibit expression can be designed and synthesized based on cloned or determined DNA sequence information encoding G protein-coupled receptor proteins. . Such a polynucleotide (nucleic acid) can hybridize with the RNA of the G protein-coupled receptor protein gene and inhibit the synthesis or function of the RNA, or it can inhibit the synthesis or function of the G protein-coupled receptor protein. The expression of G protein-coupled receptor protein gene can be regulated and controlled through the interaction with NA. Polynucleotides that are complementary to the selected sequence of the G protein-coupled receptor protein-related RNA and that can specifically hybridize to G-protein-coupled receptor protein-related RNA are those that are used in vivo and in vitro. It is useful for regulating and controlling the expression of protein-coupled receptor protein gene, and is also useful for treating or diagnosing diseases and the like. In addition, the 5'-end hairpin loop, 5'-end 6-base spare repeat, 5'-end untranslated region, 5'-end untranslated region, polypeptide translation termination codon, protein coding region, 0RF translation start of the G protein-coupled receptor protein protein gene Codons, 3 'untranslated regions, 3' palindromes, and 3 'hairpin loops may be selected as preferred regions of interest, but any region within the G protein-coupled receptor protein gene may be selected. sell.

The relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, that is, the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target can be said to be “antisense”. As antisense * polynucleotides (antisense nucleic acids), 2-doxy! ) Polynucleotides containing one liposome, polynucleotides containing D-liposome, other evening polynucleotides that are N-glycosides of purines or pyrimidine bases, or have a non-nucleotide backbone Other polymers (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds, provided that the polymers are paired with bases such as those found in DNA or RNA And nucleotides having a configuration permitting base attachment). They are double-stranded D NA, single-stranded DNA, double-stranded RNA, single-stranded RNA, or DNA: RNA hybrid, and may be an unmodified polynucleotide (or an unmodified oligonucleotide) or a known polynucleotide. Modified polynucleotides [e.g., labeled, capped, methylated, substituted for one or more natural nucleotides with analogs, known in the art, intramolecular Nucleotide-modified (eg, having an uncharged bond (eg, methyl phosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphorothioate, phosphophosphate, etc.) (Eg, protein (nuclease, nuclease / inhibitor, toxin, antibody) Those having side groups such as signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides), those having inter-current compounds (eg, acridine, psoralen), Compounds containing chelating compounds (eg, metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents, and those having modified bonds (eg, α-anomers) And the like). Here, “nucleoside”, “nucleotide”, and “nucleic acid” include, in addition to purine and pyrimidine bases, modified heterocyclic bases (eg, methylated purine and pyrimidine, acylated purine and pyrimidine bases). Pyrimidine). In addition, the "nucleoside", "nucleotide" and "nucleic acid" may have a modified sugar moiety, one or more hydroxyl groups are substituted with a halogen or an aliphatic group, or an ether or an amine. It may be converted to a functional group.

The antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, for example, a nucleic acid resistant to decomposition of a sulfur derivative ゃ thiophosphate derivative, polynucleoside amide ゃ oligonucleoside amide, and the like. The antisense nucleic acid of the present invention makes the antisense nucleic acid more stable in cells, enhances the cell permeability of the antisense nucleic acid, and increases the affinity for the target sense strand. That the toxicity of the antisense nucleic acid is smaller 421S

 26 It can be modified for the purpose of

 Many such modifications are known in the art, for example, J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke ei al. ed., Ant iSense Research and Applications, CRC Press, 1993.

 The antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, and may be provided in special forms such as ribosomes and microspheres, or applied by gene therapy. Or can be given in additional form.

 Here, the adduct used in the addition form may be a polycation such as polylysine, which acts to neutralize the charge of the phosphate skeleton; enhances interaction with the cell membrane, and increases nucleic acid uptake. Hydrophobic substances, such as lipids (for example, phospholipids, cholesterol, etc.), may be mentioned. The protein is preferably cholesterol or a derivative thereof (for example, cholesteryl chromate formate, cholic acid, etc.), and these can be attached to the 3 ′ end or 5 ′ end of nucleic acid. It can be attached via a base, sugar, or intramolecular nucleoside bond.

 As an adduct, a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid to prevent degradation by nucleases such as exonuclease and RNase. No. Examples of such capping groups include hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene glycol.

 The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of a G protein-coupled receptor protein. it can. The nucleic acid can be applied to cells by various known methods. '

A nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of a polynucleotide (eg, DNA) encoding the protein or partial peptide used in the present invention. 4215

The antisense polynucleotide having 27 columns has a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the polynucleotide (eg, DNA) of the present invention, and the polynucleotide (eg, DNA) Any polynucleotide may be used as long as it has an action capable of suppressing the expression of. As the antisense polynucleotide, antisense DNA is preferable.

 The nucleotide sequence substantially complementary to the polynucleotide of the present invention (eg, DNA) is, for example, a nucleotide sequence complementary to the polynucleotide of the present invention (eg, DNA) (ie, the polynucleotide of the present invention). A base sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology with the entire base sequence or a partial base sequence of Is mentioned. In particular, of the entire nucleotide sequence of the complementary strand of the polynucleotide of the present invention (eg, DNA), the nucleotide sequence of the portion encoding the N-terminal portion of the protein of the present invention (eg, the nucleotide sequence near the initiation codon) Antisense polynucleotides having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more homology with the complementary strand are suitable.

 Specific examples include a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 2, or an antisense polynucleotide having a part thereof. Can be

 In addition, an antisense polynucleotide having a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the 5 ′ untranslated region or 3 ′ untranslated region (preferably 5 ′ untranslated region), or a part thereof And the like. Specifically, a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6 (preferably SEQ ID NO: 5), or a nucleotide sequence thereof Antisense polynucleotides having a portion are also included.

 The antisense polynucleotide is usually composed of about 10 to 40, preferably about 15 to 30 bases.

To prevent degradation by hydrolytic enzymes such as nuclease, phosphate residues (phosphate) of each nucleotide constituting the antisense polynucleotide May be substituted with a chemically modified phosphate residue such as, for example, phosphorothioate, methylphosphonate, or phosphorodithioate. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

 The DNA encoding the partial peptide of the present invention may be any as long as it contains the above-described nucleotide sequence encoding the partial peptide of the present invention.In addition, a genomic DNA, a genomic DNA library, Any of the above-described cDNA derived from cells and tissues, the above-described cDNA library derived from cells and tissues, and synthetic DNA may be used. The vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. The DNA can also be directly amplified by the RT-PCR method using the mRNA fraction prepared from the cells and tissues described above.

 Specifically, the DNA encoding the partial peptide of the present invention includes, for example, (1) a DNA having a partial nucleotide sequence of DNA having a base sequence represented by SEQ ID NO: 2, or (2) A protein having DNA that hybridizes under high stringent conditions with the DNA represented by SEQ ID NO: 2 and having substantially the same activity as the partial peptide of the present invention (eg, ligand binding activity, signal transduction activity, etc.) For example, a DNA having a partial nucleotide sequence of the DNA encoding DNA is used.

 Examples of the DNA that hybridizes with the DNA represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 70% or more, preferably about 80% or more, and more preferably the base sequence represented by SEQ ID NO: 2 For example, DNA containing a nucleotide sequence having a homology of about 90% or more, more preferably about 95% or more, may be used.

Cloning of a DNA encoding the receptor protein of the present invention or a partial peptide thereof (hereinafter, sometimes abbreviated as the receptor protein of the present invention) may be performed by encoding the peptide of the present invention. Amplification by PCR using synthetic DNA primers having a partial nucleotide sequence of the DNA nucleotide sequence, and DNA incorporated into an appropriate vector Examples include a method of hybridizing with a DNA fragment encoding a part or all of the region or a DNA labeled with a synthetic DNA. Hybridization can be carried out, for example, according to the method described in ui in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual.

 Substitution of the nucleotide sequence of DNA is performed using PCR or a known kit, for example, Mutan ™ -super Express Km (Takara Shuzo Co., Ltd.), Mutan ™ -K (Takara Shuzo Co., Ltd.), and the like. The method can be performed according to a known method such as the gapped duplex method, the Kunkel method, or the like, or a method analogous thereto.

 The DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or can be used after digestion with a restriction enzyme or addition of a linker if desired. The DNA may have ATG as a translation initiation codon at its 5 'end and may have TAA, TGA or TAG as a translation stop codon at its 3' end. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

 The expression vector of the receptor protein of the present invention is, for example, (i) excising a DNA fragment of interest from, for example, cDNA containing DNA encoding the receptor protein of the present invention; It can be produced by ligating downstream of a promoter in an expression vector.

 Here, examples of the expression vector include plasmids derived from Escherichia coli (eg, pCR4, pCR2.K PBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, UBHO, pTP5, pC194); Eg, pSH19, pSH15); bacteriophages such as human phage; animal viruses such as retrovirus, vaccinia virus, and baculovirus; PA11, pXTK pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. .

The promoter is not particularly limited, and may be appropriately selected depending on the host used for gene expression. As an example of the promotion, animal cells are used as hosts. When used, SR promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.

Of these, CMV promoter, SRo! Promo One Night, etc. are preferred. For example, when the host is Eshierihia genus bacterium, trp promoter Isseki one, lac promoter one, re cA promoter one, AP _L promoter, lpp promoter one coater is; when the host is Bacillus, S PO 1 promoter, SP02 promoter, penP promoter and the like; when the host is yeast, PH5 promoter, PGK promoter, GAP promoter, ADH promoter and the like are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

The expression vector may contain, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) and the like. Examples of the selectable marker include a dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene (methotrexate (MTX) resistance), an ampicillin resistance gene (hereinafter sometimes abbreviated as Amp ¹ ) , neomycin resistance gene (hereinafter sometimes abbreviated as Ne o ¹ ", G418 resistance).

 When CHO (dhfr-) cells are used as a host and the dhfr gene is used as a selection marker, the target gene can be selected using a thymidine-free medium.

If necessary, a signal sequence suitable for the host may be added to the N-terminal side of the receptor protein of the present invention. For example, if the host is a genus Escherichia, a PhoA signal sequence, a 〇mpA signal sequence, etc .; if the host is a bacterium, a monoamylase signal sequence, a subtilisin-signal sequence, etc. If the host is yeast, MFa signal sequence, SUC2 signal sequence, etc .; if the host is animal cells, inulin signal sequence, α-interferon signal sequence, antibody molecule A signal array and the like can be used. 4215

 31 A transformant can be produced by introducing the thus-produced vector containing DNA encoding the receptor protein of the present invention into a host.

 Here, as the host, for example, bacteria of the genus Escherichia, bacteria of the genus Bacillus, yeast, insect cells, insects, animal cells, and the like are used.

 Examples of the genus Escherichia include, for example, Escherichia coli Escherichia coli K12 DH1 [Processing's of the 'National Academy of Ob-Sciences of the The Prosp. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research], (Nucleic Acids Research), 9, 309 (1981) 3, JA221 [Janal of Ob. .Biology (Journal of Molecular Biology), 120, 517 (1978)], HB101 [Journal of the 'Molecular' Biology, 1, 459 (1969)], C600 [Genetics, 39 , 40 (1954)], DH5a [Inoue, H., Nojima, Η. And Okayama, H., Gene, 96, 23-28 (1990)], DHIOB [Proceedings of the National Academy] Prob. Natl. Acad. Sci. USA, 87, 4645 Such as 4649 (1990)] is used.

 Examples of Bacillus spp. Include Bacillus subtilis MI114 [Gene, 24, 255 (1983) 3, 207-21 [Journal of Biochemistry, 95, 87] (1984)].

 Examples of yeast include Saccharomyces cerevisiae AH22, AH22R-, Sono-11A, DKD-5D, 20B-12; Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris and the like. Can be

Insect cells include, for example, when the virus is Ac NPV, a cell line derived from the larvae of night moth (Spodoptera frugiperda cell; S f cell), MG1 cell derived from the midgut of Trichoplusia ni, and egg derived from Trichoplusia ni egg High Five ™ cells, cells derived from Mamestra brassicae, cells derived from Estigmena acrea, etc. are used. PC monster hire 15

 32 When the virus is BmNP V, a silkworm-derived cell line (Bombyx mori N; BmN cell) or the like is used. Examples of the S: f cell include Sf9 cell (ATCC CRL1711) and Sf21 cell (Vaughn, JL et al., In Vivo, 13, 213-217 (1977)) and the like. Used.

 As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].

 Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), and dhfr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dhfr-) cell). ), Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, and human FL cells.

 Transformation of a genus Escherichia is described, for example, in Proc. Natl. Acad. Sci. USA, 69, Proc. Natl. Acad. Sci. USA. Vol., 2110 (1972) and Gene (17), 107 (1982).

 Transformation of a bacterium of the genus Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).

 Yeast transformation is described, for example, in Methods in Enzymology, Vol. 194, 182—187 (1991), Processings of the National Academy of Sciences. Natl. Acad. Sci. USA, 75, 1929 (1978).

 Transformation of insect cells or insects can be carried out, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).

Transformation of animal cells can be performed, for example, according to the method described in Cell Engineering Separate Volume 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). Can do it. Thus, a transformant transformed with the expression vector containing the DNA encoding the G protein-coupled receptor protein is obtained.

 Furthermore, the G protein-coupled receptor protein of the present invention can be produced by culturing the transformant in a medium suitable for the host.

 A liquid medium is preferable as a medium used when culturing a transformant whose host is a genus Escherichia or Bacillus. Such a medium preferably contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Here, as the carbon source, for example, glucose, dextrin, soluble starch, sucrose, etc .; As the nitrogen source, for example, ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal And inorganic or organic substances such as a nutrient extract; examples of the inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. Further, the medium may contain yeast extract, vitamins, growth promoting factors and the like. The pH of the medium is preferably about 5-8.

 As a medium used for culturing a transformant whose host is a bacterium belonging to the genus Escherichia, for example, M9 medium containing glucose and casamino acid [Mi Her, Journal of Experimen 'in' Molecular • Genetics (Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. For the purpose of making the promoter work efficiently, an agent such as 3 / 8-indolylacrylic acid may be added to the medium.

 When culturing a transformant whose host is a bacterium belonging to the genus Escherichia, the culturing is usually performed at about 15 to 43 ° C for about 3 to 24 hours. If necessary, ventilation or stirring may be performed.

When culturing a transformant whose host is a bacterium belonging to the genus Bacillus, the culturing is usually performed at about 30 to 40 ° C for about 6 to 24 hours. If necessary, ventilation or stirring may be performed. As a medium used for culturing a transformant in which the host is yeast, for example, Burkholder's minimum medium [Bostian, KL et al., "Procedures of the National Co., Ltd." Academy of Sciences Natl. Acad. Sci. USA, 77, 4505 (1980) J; SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. Zing's ブ ob ナ シ ョ ナ ル The National ォ Dec ォ ob · Sciences ブ ob ザ The ユ ー U 'S ェ (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984) ” . Preferably, the pH of the medium is about 5-8. The cultivation is usually performed at about 20 ° C to 35 ° C for about 24 to 72 hours. If necessary, ventilation or stirring may be performed.

 The culture medium used for culturing insect cells or transformants whose insect host is, for example, Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)) Examples thereof include those to which additives such as 10% serum are added as appropriate. The pH of the medium is preferably about 6.2 to 6.4. The culture is usually performed at about 27 ° C for about 3 to 5 days. If necessary, ventilation or stirring may be performed.

 Examples of a medium used for culturing a transformant whose animal host is an animal cell include, for example, a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)]; DMEM medium (Virology, 8, 396 (1959)); RPMI 1640 medium (Journal of the American Medical Association, 199, 519) (1967)), 199 medium [Proceeding of the Society for the Biological Medicine], 73, 1 (1950)] Can be Preferably, the pH of the medium is about 6-8. The cultivation is usually carried out at about 30 ° C to 40 for about 15 to 60 hours. If necessary, ventilation or stirring may be performed.

 As described above, the G protein-coupled receptor protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.

 The receptor protein of the present invention thus obtained can be separated and purified, for example, by the following method.

For example, after culturing the transformant, cells or cells are collected by a known method, suspended in an appropriate buffer, and sonicated, lysozyme and / or freeze-thawed. After the cells or cells are crushed, a crude extract of the receptor protein of the present invention can be obtained by centrifugation or filtration. Here, the buffer may contain a protein denaturant such as urine or guanidine hydrochloride, or a surfactant such as Triton X-100 ™.

 If the receptor protein is secreted into the culture solution during the culturing of the transformant, after the culture is completed, the supernatant is separated from the cells or cells by a known method, and the culture supernatant is collected. The receptor protein of the present invention can be separated.

 The receptor extract protein can be purified by subjecting the thus obtained crude extract or culture supernatant to known separation and purification methods. Here, known separation and purification methods include, for example, methods using solubility such as salting out and solvent precipitation; dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. A method mainly utilizing the difference in molecular weight; a method utilizing a difference in charge such as ion exchange chromatography; a method utilizing specific novelty such as affinity chromatography; and reverse-phase high-performance liquid chromatography. For example, a method using a difference in hydrophobicity such as an isoelectric point; a method using a difference in isoelectric points such as an isoelectric point method and a perturbation method are used. These methods can be appropriately combined.

 When the receptor protein obtained in this way is a free form, it can be converted to a salt by a known method or a method analogous thereto. When the receptor protein is a salt, it can be converted into a free form or another salt by a known method or a method analogous thereto.

 The receptor protein produced by the transformant can be arbitrarily modified or the polypeptide can be partially removed by allowing a suitable protein modifying enzyme to act on the receptor protein before or after purification. As the protein-modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.

 The activity of the receptor protein of the present invention or a salt thereof thus obtained can be determined by a binding experiment with a labeled ligand and a specific antibody.

It can be measured by means such as a size. An antibody against the receptor protein of the present invention or its partial peptide or a salt thereof may be an antibody capable of recognizing the receptor protein of the present invention or its partial peptide or a salt thereof, such as a polyclonal antibody or a monoclonal antibody. Any one may be used.

 An antibody against the receptor protein of the present invention or its partial peptide or a salt thereof (hereinafter sometimes abbreviated as the receptor protein of the present invention) may be obtained by using the receptor protein of the present invention as an antigen. It can be produced according to a known antibody or antiserum production method. Hereinafter, the method for producing an antibody against the receptor protein of the present invention will be described in detail.

 [Preparation of monoclonal antibody]

 (a) Preparation of monoclonal antibody-producing cells (hypridoma)

 The receptor protein or the like of the present invention is administered to a mammal at a site capable of producing an antibody upon administration, itself or together with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be used to enhance antibody production upon administration. Administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.

When producing monoclonal antibody-producing cells, a mammal immunized with the antigen, for example, an individual with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. By fusing the antibody-producing cells contained with the myeloma cells, a monoclonal antibody-producing hybridoma can be prepared. The measurement of the antibody titer in the antiserum can be performed, for example, by reacting the below-described labeled receptor protein or the like with the antiserum, and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Kayla and Milstein [Nature, 256, 495 (1975)]. When using a fusion accelerator Such a fusion promoter may be, for example, polyethylene daricol (PEG) or Sendai virus. Among them, PEG is preferred.

 Examples of myeloma cells include NS-1, P3U1, SP2 / 0 and the like, and P3U1 is preferably used.

 The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells is about 1: 1 to 20: 1. The fusion operation is carried out in the presence of PEG at a concentration of about 10 to 80% (preferably, PEG 1000 to> EG6000) at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 分 間 to 10 minutes. Can be implemented efficiently.

 Screening of the monoclonal antibody-producing hybridoma is performed according to a known method. Such methods include, for example, adding a hybridoma culture supernatant to a solid phase (eg, microplate) on which an antigen such as a receptor protein is adsorbed directly or together with a carrier, and then adding a radioactive substance or an enzyme. A method for detecting the monoclonal antibody bound to the solid phase by adding the anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice) A method in which a hybridoma culture supernatant is added to a solid phase to which an antibody or protein A is adsorbed, and a receptor protein or the like labeled with a radioactive substance, an enzyme, or the like is added to detect a monoclonal antibody bound to the solid phase. Is mentioned.

 Monoclonal antibodies can be selected by using a medium for selection and breeding according to a known method or a method analogous thereto. The selection of monoclonal antibodies is usually performed in animal cell culture media supplemented with HAT (hypoxanthine, aminopterin, thymidine).

As a medium for selection and breeding, any medium can be used as long as a hybridoma can grow, and specifically, RPMI containing 1 to 20%, preferably 10 to 20% fetal bovine serum. 1640 medium, GIT medium containing 1-10% fetal calf serum (Wako Pure Chemical Industries, Ltd.) or serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) . The culture temperature is usually 20 to 40 ° (:, preferably about 37 ° C.) The culture time is usually 5 days to 3 weeks, preferably 1 week Between ~ 2 weeks. The culture can be usually performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

 (b) Separation and purification of monoclonal antibodies

 Monoclonal antibodies can be separated and purified by known immunoglobulin separation and purification methods (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, adsorption / desorption using an ion exchanger (eg, DEAE), Centrifugation, gel filtration, antigen-binding solid phase or specific purification method in which only antibodies are collected using an active adsorbent such as protein A or protein G, and the bonds are dissociated to obtain antibodies.

 (Preparation of polyclonal antibody)

 The polyclonal antibody of the present invention can be produced by a known method or a method analogous thereto. The polyclonal antibody of the present invention can be used to immunize a mammal, for example, using an immunizing antigen (an antigen such as the receptor protein of the present invention) in the same manner as in the method for producing a monoclonal antibody described above. The antibody can be produced by collecting the antibody-containing substance against the receptor protein of the present invention and separating and purifying the antibody. The immunizing antigen (antigen such as receptor protein of the present invention) may be a complex with carrier protein.

 At this time, the type of the carrier protein and the mixing ratio of the carrier protein and the immunizing antigen are not particularly limited as long as the antibody can be efficiently produced against the immunizing antigen immunized by cross-linking the carrier. For example, a carrier protein such as serum serum albumin, serum cycloglobulin, keyhole, lysine hemocyanin, etc. may be mixed in a weight ratio of about 0.1 to 20, preferably about 1 to 5, with respect to 1 immunizing antigen. It is preferable to ring.

 The coupling between the immunizing antigen and the carrier protein can be performed using various condensing agents. Examples of the condensing agent include datalaldehyde, carposimid, maleimide active ester, and an active ester reagent containing a thiol group and a dithioviridyl group. ,

The immunizing antigen (including the complex of the immunizing antigen and the carrier protein) is supplied to the mammal at a site where antibody production is possible, together with the carrier itself or a diluent. Is administered. To increase antibody production during administration, complete:

Subjuvant or incomplete Freund's adjuvant may be used. Administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.

 The polyclonal antibody can be collected preferably from blood, such as blood, ascites, and the like of a mammal immunized by the above method.

 The polyclonal antibody titer in the antiserum can be measured by the same method as the measurement of the antibody titer in the serum described above. Separation and purification of the polyclonal antibody can be performed according to the above-mentioned known method of separating and purifying immunoglobulin.

The receptor encoding the receptor protein of the present invention or its salt, its partial peptide or its salt, and the receptor protein or its partial peptide are encoded by (1) a ligand for the G protein-coupled receptor protein of the present invention. (2) a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the G protein-coupled receptor protein of the present invention; (3) a gene diagnostic agent; and (4) a drug of the present invention. (5) A method for screening a compound that changes the expression level of a receptor protein or a partial peptide thereof, (5) prevention and / or prevention of various diseases containing a compound that changes the expression level of the receptor protein or a partial peptide thereof of the present invention. Therapeutic agent, (6) Quantitative method of ligand for G protein-coupled receptor protein of the present invention, (7) G protein poorly coupled receptor protein of the present invention and Riga (8) Compounds that alter the binding between the G protein-coupled receptor protein of the present invention and the ligand (agonists, antagonists) A prophylactic and / or therapeutic agent for various diseases, comprising: (9) quantification of the receptor protein of the present invention or its partial peptide or a salt thereof; (10) the receptor protein of the present invention in a cell membrane or its partial peptide (Ii) a method for screening and / or treating various diseases containing a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane; ) Neutralization of the receptor protein of the present invention or its partial peptide or a salt thereof with an antibody, (13) G of the present invention Used like for the production of non-human transformer diethyl nick animals with DN Alpha encoding white matter coupled receptions evening one protein Can be

 In particular, by using a receptor-binding assay system using the recombinant G protein-coupled receptor protein expression system of the present invention, the binding of a ligand to a G-protein-coupled receptor specific to a human mammal can be improved. The compound to be changed (eg, agonist, angyo gonist, etc.) can be screened, and the agonist or angyo gonist can be used as an agent for preventing or treating various diseases.

 The receptor protein or the partial peptide of the present invention or a salt thereof, the DNA encoding the receptor protein of the present invention or the partial peptide thereof (hereinafter, may be abbreviated as the DNA of the present invention), the receptor protein of the present invention, and the like. The use of the antibody (hereinafter, may be abbreviated as the antibody of the present invention) for the antibody is specifically described below.

 (1) Determination of ligand (agonist) for G protein-coupled receptor protein of the present invention

The receptor protein of the present invention or its salt or the partial peptide or its salt of the present invention can be used as a reagent for searching or determining a ligand (agonist) for the receptor protein of the present invention or its salt. Useful. That is, the present invention provides a method for determining a ligand for the receptor protein of the present invention, which comprises contacting the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof with a test compound. I will provide a. Test compounds include known ligands (for example, angiotensin, bombesin, cannapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasoprescin, oxytocin, PACAP (eg, PACAP 27, PACAP 38), secretin, glucagon, calcitonin, 7-drenomedullin, somatos, GHRH, CRF, ACTH, GRP, PTH, VIP (basoactive intestinal and related polypeptide), somatos, dopamine, motilin, Amylin, bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreatin, pro Staglandin, tropoxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GROa, GROj3, GRA, NAP-2, ENA-78, GCP-2, PF4, IP10, Mig CXC chemokine subfamily, such as PBSF / SDF-1, MCAF / MCP-1, MCP-2, MCP-13, MCP-4, eotaxin, RANTES, MIP-1H, MIP-1 / 3, HCC — 1, MIP-3a / LARC, MIP-3 ^ / ELC, 1—309, TARC, MI PF —1, MIPF-2 / eotaxin-2, MDC, DC-CK1 / PARC, 3 (such as (:( Chemokine subfamily; C chemokine subfamily such as lymphotactin; CX3 chemokine subfamily such as fractalkine etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), Sufingosine 1-monophosphate, etc.) In addition to zophosphatidylserine, sphingosyl phosphorylcholine, lysophosphatidylcholine, steroids, bile acids, isoprenoids, arachidonic acid metabolites, amines, amino acids, nucleotides, nucleosides, saturated or unsaturated fatty acids, for example, humans or mammals (eg, Tissue extract, cell culture supernatant, etc., for example, the tissue extract, cell culture supernatant, etc. of the receptor protein of the present invention. , And fractionating it while measuring cell stimulating activity, etc., to finally obtain a single ligand.

Specifically, the ligand determination method of the present invention uses the receptor protein of the present invention or its partial peptide or a salt thereof, or constructs a recombinant receptor-protein expression system, and Receptor binding using the ATS system is used to bind to the receptor protein of the present invention and to stimulate cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca "release, intracellular cAMP production, intracellular c A compound having an activity of promoting or inhibiting GMP production, inositol phosphate production, fluctuation of cell membrane potential, phosphorylation of intracellular protein, activation of c-fos, decrease of pH, etc. This is a method for determining peptide compounds, synthetic compounds, fermentation products, etc.) or salts thereof. In the ligand determination method of the present invention, when the receptor protein of the present invention or a partial peptide thereof is contacted with a test compound, for example, the amount of a test compound bound to the receptor protein or the partial peptide, It is characterized by measuring cell stimulating activity and the like.

 More specifically, the present invention provides

 (1) When a labeled test compound is brought into contact with the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof, the protein of the labeled test compound or a salt thereof, or the partial peptide is used. Or a method for determining a ligand for a receptor protein or a salt thereof according to the present invention, which comprises measuring the amount of binding to a salt thereof.

 (2) When a labeled test compound is brought into contact with a cell containing the receptor protein of the present invention or a membrane fraction of the cell, the amount of the labeled test compound bound to the cell or the membrane fraction is measured. A method for determining a ligand for the receptor Yuichi protein or a salt thereof according to the present invention,

 (3) When the labeled test compound is brought into contact with a receptor protein expressed on the cell membrane by culturing a transformant containing DNA encoding the receptor protein of the present invention, the receptor of the labeled test compound A method for determining a ligand for a receptor protein of the present invention, which comprises measuring the amount of binding to an evening protein or a salt thereof;

細胞 Cellular stimulating activity via the receptor protein when the test compound is brought into contact with cells containing the receptor protein of the present invention (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular c AMP generation, intracellular c GMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-1: activation of fos, reduction or activation of pH Activity, etc.), a method for determining a ligand for the receptor protein or a salt thereof of the present invention, and

細胞 Cell stimulation via receptor protein when a test compound is brought into contact with a receptor protein expressed on the cell membrane by culturing a transformant containing DNA encoding the receptor protein of the present invention. Activity (for example For example, arachidonic acid release, acetylcholine release, intracellular ^{Ca2 +} release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, and a method for determining a ligand for the receptor protein or a salt thereof of the present invention, which comprises measuring the activity of promoting or suppressing fos activation or pH reduction.

 In particular, it is preferable to carry out the tests 1 to 3 above after conducting the tests 1 to 3 above and confirming that the test compound binds to the receptor protein of the present invention. '

 First, any receptor protein used in the ligand determination method may be used as long as it contains the above-described receptor protein of the present invention or the partial peptide of the present invention. Receptacle protein is suitable.

 To produce the receptor protein of the present invention, the above-described expression method is used. It is preferable to express DNA encoding the receptor protein in mammalian cells or insect cells. As the DNA fragment encoding the protein portion of interest, cDNA is usually used, but is not necessarily limited thereto.For example, even if a gene fragment or synthetic DNA is used, Good. In order to introduce the DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, the DNA fragment should be transferred to a nucleus belonging to baculovirus using an insect as a host. Polyhedrin promoter of somatic disease virus (nuclear polyhedros is virus; NPV), promoter derived from SV40, promoter of retrovirus, metamouth thionine promoter, human heat shock promoter, cytomegalovirus promoter It is preferable to incorporate it downstream, such as the SR promoter. The quantity and quality of the expressed receptor can be determined by known methods, for example, as described in the literature [Nambi, P. et al., The 'Journal' of 'Biological' Chemistry (J. Biol. Cem.), 267, 19555- 19559, 1992].

Therefore, the receptor protein of the present invention or a partial peptide thereof or a salt thereof used in the ligand determination method of the present invention can be prepared according to a known method. The protein may be a purified receptor protein or its partial peptide or a salt thereof, or may be a cell containing the receptor protein or a cell membrane fraction thereof.

 When cells containing the receptor protein of the present invention are used in the ligand determination method of the present invention, the cells may be immobilized with daltaraldehyde, formalin, or the like. Immobilization can be performed according to a known method.

 The cell containing the receptor protein of the present invention means a host cell expressing the receptor protein of the present invention. Examples of the host cell include Escherichia coli, Bacillus subtilis, yeast, insect cells, and animal cells. Are used.

 The cell membrane fraction refers to a cell membrane-rich fraction obtained by crushing cells and then obtained by a known method. Methods for crushing cells include crushing cells with a Potter-Elvehj em homogenizer, crushing method using a single ring blender ゃ polytron (Kinematica), crushing method using ultrasonic waves, and French press. A crushing method in which cells are ejected from a fine nozzle while applying pressure, and the like, may be mentioned. The cell membrane thus obtained can be fractionated by a centrifugal fractionation method such as a differential centrifugation method or a density gradient centrifugation method. For example, the cell lysate is centrifuged at low speed (500-3000 rpm) for a short time (typically, about 1 minute to 10 minutes), and the supernatant is further centrifuged at high speed (15,000 to 30,000 rpm) for 30 minutes to 2 hours. By collecting the precipitate, a cell membrane fraction can be obtained. The cell membrane fraction contains a large amount of expressed receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.

The amount of receptions evening one protein of a cell and in their membrane fraction containing the receptor protein, per cell, preferably to 10 ⁸ molecules, more preferably to ¹⁰⁷ molecules. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which makes it possible not only to construct a highly sensitive screening system, but also to screen a large number of test compounds in the same lot.

In order to carry out the above-mentioned methods (1) to (3) for determining the ligand for the receptor protein or a salt thereof of the present invention, an appropriate receptor protein fraction and a labeled test compound are required. The receptor protein fraction is preferably a natural receptor protein fraction, or a recombinant receptor fraction having the same activity as the fraction. Here, “equivalent activity” means equivalent ligand binding activity, signal transduction action, and the like.

The labeled test compound, ^[3 H], ^[125 I], ^[14 C] or ^[35 S] angiotensin were identified respectively labels such as bombesin, Kan'nabi maytansinoid, cholecystokinin, glutamine, serotonin , Melatonin, new oral peptide Y, opioid, purine, vasopressin, oxitocin, ΡΑ CAP (eg, PACAP 27, PACAP 38), secretin, glucagon, calcitonin, 7-drenomediulin, somatostatin, GHRH, CRF, ACTH, GRP , PTH, VIP (Vasoactive Intestinal and Retained Polypeptides), Somatos-tin, Dopamine, Motilin, Amylin, Bradykinin, CGRP (Calcitonin Gene-Related Peptide), Leukotriene, Pancreatin, Prostaglandin, Trompoxane, Adenosine, 7-drenaline, chemokine family (eg IL-18, GRO, GRO3, GROA, NAP-2, ENA-78, GCP-2, PF4, IP10, Mig, PBSF / SDF One of the CXC chemokine subfamilies such as -l; MCAF / MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, MIP-1α, MIP-lj3, HCC-1 and MIP CC chemokine subfamily such as -3 / LARC, MIP-3β / ELC, I-309, TARC, MIPF-1 and MIPF-2 / eoiaxin-2, MDC, DC-CK1 / PARC, SLC; lymphotactin, etc. CX3 chemokine subfamily of fractalkine, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosine 1-lin Acid, lysophosphatidylserine, sphingosyl phosphoryl Choline, lysophosphatidylcholine, steroids, bile acids, isoprenoids, § Rakidon acid metabolites, amines, amino acids, nucleotides, nucleosides, etc. saturated or unsaturated fatty acids are preferred. Specifically, to carry out the method for determining a ligand for the receptor protein of the present invention or a salt thereof, first, cells or a membrane fraction of the cells containing the receptor protein of the present invention are suspended in an appropriate buffer. To prepare a sample of the reception. The buffer is not particularly limited as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer of ρΗ4 to 10 (preferably pH 6 to 8) or a tris-monohydrochloride buffer.

 Also, in order to reduce non-specific binding, various proteins such as surfactants such as CHAPS, Tween-80 ™ (Kao-Atlas), digitonin, dexcholate, and serum albumin and gelatin can be added to the buffer. Good. In addition, protein inhibitors such as SF, leptin, E-64 (manufactured by Peptide Research Institute), and pepstatin were added to the buffer to suppress the degradation of the receptor and ligand by the protease. You may.

Thus the receptions evening one specimen 0.01 to 10 ml thus obtained, a certain amount (5000 ~500000 c pm) of ^[3 H], ^[125 I], ^[14 C] labeled with a ^[35 S] Make the test compound coexist. On the other hand, in the control group, a large excess of unlabeled test compound is used in place of the labeled test compound in order to determine the amount of non-specific binding (NSB). The reaction between the receptor preparation and the test compound is carried out at about O ° C to 50, preferably about 4 ° C to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After the reaction, the reaction solution is filtered through a glass fiber filter paper, etc., and the glass fiber filter paper is washed with an appropriate amount of buffer (a buffer similar to a single receptor), and the radioactivity remaining on the glass fiber filter paper is measured by a liquid scintillation counter. Or, it is measured in the evening of the county. Using the obtained measurement values, the test compound having a count (B-NSB) of less than 0 cpm obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) exceeds the receptor protein of the present invention or its protein. It can be selected as a ligand for salt.

In the above (1) or (2), which determines the ligand for the receptor protein of the present invention or its salt, the cell stimulating activity (for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP Production, phosphoric acid production, cell membrane potential fluctuation, intracellular protein phosphorylation, c- The activity of promoting or suppressing the activation of fos, the decrease of pH, etc.) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured in a multiwell plate or the like. Prior to ligand determination, replace with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant. Then, the generated product is quantified according to each method. If the production of a substance that serves as an indicator of cell stimulating activity (for example, arachidonic acid) cannot be confirmed by the degrading enzyme contained in the cells, the assay may be performed by adding an inhibitor for the degrading enzyme. Good. In addition, activities such as inhibition of cAMP production can be detected as an activity of inhibiting production of cells whose basic production has been increased by forskolin or the like.

 The kit for determining a ligand that binds to the receptor protein of the present invention or a salt thereof includes a receptor protein of the present invention or a salt thereof, a partial peptide or a salt thereof of the present invention, a cell containing the receptor protein of the present invention, or It contains the membrane fraction of cells containing the receptor protein of the present invention.

 Examples of the kit for determining a ligand of the present invention include the following.

 1. Reagent for ligand determination

 ①Measurement buffer and washing buffer

 Hanks' Balanced Salt Solution (manufactured by Gibco) plus 0.05% serum albumin (manufactured by Sigma).

 Sterilize by filtration through a 0.45 m pore size filter and store at 4 ° C, or prepare at use.

 ② G protein conjugated receptor Yuichi protein preparation

CHO cells expressing the receptor protein of the present invention, 12-well plates and passaged 5 10 ⁵ holes, 37, 5% C0 _2, with 95% ai r 2 days followed by culturing.

③ Labeled test compound

Commercially available ^[3 H], C ¹²⁵ I], [ "C], ^[35 S] labeled compounds or the like, was or those labeled by an appropriate method Store the solution in an aqueous solution at 4 ° C or -20 ° C, and dilute to 1 M with the measurement buffer before use. For test compounds that are poorly soluble in water, dissolve in dimethylformamide, DMSO, methanol, etc.

 ④Unlabeled test compound

 Prepare the same labeled compound at a concentration 100-1000 times higher.

 2. Measurement method

 (1) Wash the CH〇 cells expressing the receptor protein of the present invention cultured on a 12-well tissue culture plate twice with 1 ml of the measurement buffer, and then add 490 / l of the measurement buffer to each well. .

 ② Add 5/1 of labeled test compound and react at room temperature for 1 hour. To determine the amount of non-specific binding, add 5 a 1 of unlabeled test compound.

 ③ Remove the reaction solution and wash 3 times with 1 ml of washing buffer. The labeled test compound bound to the cells is dissolved in 0.2 N NaOH-1% SDS, and mixed with 4 ml of liquid scintillator A (Wako Pure Chemical Industries).

 放射 Measure radioactivity using a liquid scintillation counter (Beckman Coulter, Inc.).

Examples of the ligand capable of binding to the receptor protein of the present invention or a salt thereof include substances specifically present in the hypothalamus, cerebral cortex, colon cancer, lung cancer, cardiac placenta, lung, and the like. Specifically, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, pudding, vasoprescin, oxitocin, PACAP (e.g., PACAP 27, PACAP 38), secretin, Glucagon, calcitonin, adrenomedullin, somatos quintin, GHRH, CRF, ACTH, GRP, PTH, VIP (basoactive intestinal and lit polypeptide), somatos quintin, dopamine, motilin, amylin, bradykinin, CGRP (Calcitonin Gene Relay Peptide, leukotriene, pancreatin, prostaglandin, tropoxane, adenosine, adrenaline, gemokine superfamily (eg, IL-18, GROo !, GRROjS, GROa, NA CXC chemokine subfamily such as P-2, ENA-78, GCP-2, PF4, IP10, Mig, PBSF / SDF-1; MCAF / MCP-1, MCP-2, MCP-13, MCP-4, eotaxin, RANTES, MIP-1H, MIP-1jS, HCC-1, MIP-3 «/ LAR MIP-3i3 / ELC, 1-309, TARC, MIPF-1 and MIPF-2 / eotaxin-2 , MD.C, CC-CK1 / PARC, SLC, etc .; CC chemokine subfamily; lyinphotactin, etc .; CX3 chemokine subfamily, such as iractalkine; endothelin, enterogastrin, histamine, etc. Neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosin monophosphate, lysophosphatidylserine, sphingosylphosphorylcholine, lysophosphatidylcholine, steroids, bile acids, isoprenoids, araki Metabolites of donic acid, amines, amino acids, nucleotides, nucleosides, saturated or unsaturated fatty acids, and the like. (2) A preventive and / or therapeutic agent for a disease associated with dysfunction of the G protein-coupled receptor protein of the present invention

 In the above method (1), if the ligand for the receptor protein of the present invention is revealed, the receptor protein of the present invention or the DNA encoding the receptor protein of the present invention can be used in accordance with the action of the ligand. It can be used as a medicament such as a prophylactic and / or therapeutic agent for diseases associated with receptor protein dysfunction.

 For example, in the case where there is a patient who cannot expect the physiological action of the ligand because the receptor protein of the present invention is deficient or reduced in the living body (dysfunction of the receptor protein), (1) the receptor protein of the present invention It may be administered to a patient to supplement the amount of the receptor protein, or (i) administering the receptor protein of the present invention to the patient.

Or (ii) introducing the DNA encoding the receptor protein of the present invention into target cells and expressing the cells, and then transferring the cells to the patient. Increase the amount of receptor protein in the patient's body, such as by transplanting it into Can be That is, DNA encoding the receptor protein of the present invention is useful as an agent for preventing and / or treating a disease associated with dysfunction of the safe and low-toxic receptor protein of the present invention.

 The receptor protein of the present invention is a novel seven-transmembrane receptor protein having approximately 27% homology at the amino acid sequence level with 5HT1D (serotonin receptor).

 The receptor protein of the present invention or the DNA encoding the receptor protein may be a central disease (eg, depression, Alzheimer's disease, dementia, eating disorder, etc.), an internal sulcus disease (eg, hypertension, gonad dysfunction) , Thyroid dysfunction, pituitary dysfunction, etc.), metabolic diseases (eg, diabetes, lipid metabolism disorders, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder) It is used as a medicament useful for prevention, Z or treatment of cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., heart disease (eg, angina, myocardial infarction, etc.).

 When the receptor protein of the present invention is used as a medicine, it can be formulated according to a method commonly used in the technical field of formulation.

 On the other hand, when the DNA encoding the receptor protein of the present invention is used as a medicine, the DNA of the present invention may be used as it is, or a retrovirus vector, an adenovirus vector, an adenovirus-associated virus vector, or the like. After insertion into an appropriate vector, a pharmaceutical preparation can be prepared in the same manner as the receptor protein of the present invention. The DNA of the present invention can be administered as it is, or together with an auxiliary agent for promoting uptake, using a gene gun or a catheter such as a hydrogel catheter.

Examples of the dosage form of the drug include oral preparations such as sugar-coated tablets, capsules, elixirs, and micropulcers, and parenteral preparations such as injections, if necessary. These preparations include, for example, (1) a receptor protein of the present invention or (2) DNA encoding the receptor protein, a preparation additive, for example, a physiologically acceptable carrier, a flavoring agent, an excipient, a vehicle. It can be manufactured by mixing with preservatives, stabilizers, binders and the like. The content of the receptor protein or DNA of the receptor of the present invention in these preparations is determined by the following formula of the present invention. 2 04215

 51 Separately determined taking into account the dose of protein or DNA.

 For example, additives used in the manufacture of disintegrants and capsules include, for example, binders such as gelatin, cornstarch, tragacanth, and arabic gum; shaping agents such as crystalline cellulose; cornstarch, gelatin, alginic acid, and the like. Swelling agents; lubricating agents such as magnesium stearate; sweeteners such as sucrose, lactose or sachets; and phosphorus and other sweeteners (j; flavoring agents such as peppermint, cocoa oil or cherry), and the like. In addition to additives, liquid carriers such as oils and fats may be further contained.

 Examples of the additives used in the production of injections include aqueous vehicles such as water for injection and saline, and oily vehicles such as vegetable oils such as sesame oil, coconut oil, and soybean oil. When an aqueous vehicle is used, isotonic agents such as butdu sugar, D-sorbitol, D-mannitol, and sodium chloride; alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), A solubilizing agent such as a zwitterionic surfactant (eg, polysorbate 80 ™, HCO-50) may be used. When an oil vehicle is used, a solubilizing agent such as benzyl benzoate or benzyl alcohol may be used. The injection solution is usually filled in a suitable ampoule.

 Examples of the formulation additives include a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, prochloride hydrochloride, etc.), a stabilizer (eg, human serum albumin) , Polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.

 Since the preparations thus obtained have low toxicity, they are safe for humans and mammals (for example, rats, mice, rabbits, sheep, sheep, bush, horses, cats, dogs, monkeys, etc.). Can be administered.

The dosage of the receptor protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method and the like. In the case of oral administration, for example, about 0.1 per day for a cancer patient (body weight 60 kg). 100100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the receptor of the present invention The dose of the protein is, for example, usually about 0.01 to 30 mg, preferably about 0.1 to 20 mg per day for a cancer patient (body weight 60 kg) in the case of intravenous injection. Preferably about 0.1 to 10 mg.

 The dosage of the DNA of the present invention varies depending on the administration subject, the target organ, the condition, the administration method, and the like. In the case of oral administration, for example, about 0.1 to 10 mg / day for a cancer patient (body weight 60 kg). 100 mg, preferably about 1.0 to 50 mg, more preferably thread 1. (! To 20 mg. In the case of parenteral administration, the dose of the DNA of the present invention is, for example, in the case of intravenous injection of an injection. For example, it is usually about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day for a cancer patient (assuming a body weight of 60 kg).

(3) Genetic diagnostics

 The DNA of the present invention can be used as a probe to produce the receptor protein of the present invention in humans or mammals (eg, rat, mouse, rabbit, rabbit, sheep, bush, rabbit, cat, dog, monkey, etc.). Abnormality (genetic abnormality) of DNA or mRNA encoding the partial peptide can be detected, for example, damage, mutation or reduced expression of the DNA or mRNA, or DNA or mRNA of the DNA or mRNA. It is useful as a diagnostic agent for genes such as increased or overexpressed genes. '

 The above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the well-known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Processing's Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, pp. 2766-2770 (1989)) Can be implemented.

(4) A method for screening a compound that changes the expression level of the receptor protein or a partial peptide thereof of the present invention

By using the DNA of the present invention as a probe, the receptor of the present invention can be used. It can be used for screening a compound that changes the expression level of one protein or its partial peptide.

 That is, the present invention relates to, for example, (i) non-human mammal blood, ② specific organs, ③ tissues or cells isolated from organs, or (ii) transformants of the present invention. Provided is a method for screening a compound that changes the expression level of the receptor protein or its partial peptide of the present invention by measuring the mRNA amount of the receptor protein or its partial peptide.

 The measurement of the mRNA amount of the receptor protein of the present invention or its partial peptide is specifically performed as follows.

 (0 normal or disease model non-human mammals (eg, mouse, rat, rabbit, sheep, pigeon, pig, cat, cat, dog, monkey, etc., more specifically, dementia rat, obese mouse, atherosclerotic rabbit, Drugs (eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.) After a certain period of time, blood or a specific organ (eg, brain, lung, colon, etc.), or tissue or cells isolated from the organ is obtained.

 The mRNA of the receptor protein of the present invention or its partial peptide contained in the obtained cells can be obtained, for example, by extracting mRNA from cells or the like by a conventional method, and using a method such as TaqMan PCR. And can be analyzed by performing Northern blotting by known means.

 (ii) A transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method, and the mRNA of the receptor protein of the present invention or the partial peptide thereof contained in the transformant is prepared. Can be quantified and analyzed in the same manner.

 Screening for a compound that changes the expression level of the receptor protein or its partial peptide of the present invention comprises:

(i) A given time before giving a drug or physical stress to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour Before ~ 6 hours before) or after a certain time (30 Minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or the test compound is administered simultaneously with the drug or physical stress, and after a certain time (30 minutes) After 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the cells is quantified and analyzed. Can be done by

 (ii) Transformant When culturing according to a conventional method, the test compound is mixed with the medium, and after culturing for a certain period of time (1 to 7 days, preferably 1 to 3 days, more preferably 2 to 3 days) After that, the amount can be determined by quantifying and analyzing the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the transformant.

 The compound obtained by using the screening method of the present invention or a salt thereof is a compound having an action of changing the expression level of the receptor protein or a partial peptide thereof of the present invention. Specifically, (a) the receptor of the present invention By increasing the expression level of Yuichi protein or its partial peptide, cell stimulating activity via G protein-coupled receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca "release, intracellular CAMP Production, intracellular c-GMP production, inositol monophosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. (B) reducing the expression level of the receptor protein of the present invention or a partial peptide thereof to attenuate the cell stimulating activity. Compound der Ru.

 Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound.

 The compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein of the present invention.

The compound that attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein or the like of the present invention. The compound or a salt thereof obtained by using the screening method of the present invention can be made into various preparations in the same manner as in the case of the above-mentioned receptor protein of the present invention.

 The preparations obtained in this way are safe and low toxic, so they can be used, for example, in human mammals (for example, rats, mice, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). Can be administered.

 The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.In the case of oral administration, for example, about 0,0 per day for a cancer patient (body weight 60 kg). It is 1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the dose of the compound or a salt thereof is usually, for example, about 0.01% per day for a cancer patient (60 kg body weight) when intravenously injected. 3030 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg. (5) A preventive and / or therapeutic agent for various diseases containing a compound that alters the expression level of the receptor protein or its partial peptide of the present invention

 As described above, the receptor protein of the present invention is considered to play some important role in vivo, such as central function. Therefore, the compound that changes the expression level of the receptor protein of the present invention or its partial peptide can be used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention.

 Here, the disease associated with dysfunction of the receptor protein of the present invention includes, for example, central diseases (eg, depression, Alzheimer's disease, dementia, eating disorders, etc.), endocrine diseases (eg, hypertension, gonad dysfunction, Thyroid dysfunction, pituitary dysfunction, etc.), metabolic disorders (eg, diabetes, dyslipidemia, 'hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer) , Breast cancer, cervical cancer, colon cancer, rectal cancer, etc., and heart diseases (eg, angina pectoris, myocardial infarction, etc.).

The compound is useful for preventing diseases associated with dysfunction of the receptor protein of the present invention. 02 04215

 When used as 56 and Z or a therapeutic agent, the compound can be made into various preparations in the same manner as in the case of the receptor protein of the present invention described above.

 The preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and mammals (for example, rats, mice, puppies, higgs, bushes, cats, cats, dogs, monkeys, etc.). Can be administered.

 The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.In the case of oral administration, for example, about 0,0 per day for a cancer patient (body weight 60 kg). It is 1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the dose of the compound or a salt thereof may be, for example, in the case of intravenous injection, usually about 0.01% per day for a cancer patient (body weight 60 kg). 3030 mg, preferably about 0.1-20 mg, more preferably about 0.1-10 mg.

(6) Method for quantifying ligand for G protein-coupled receptor protein of the present invention Since the receptor protein of the present invention has a binding property to a ligand, the concentration of the ligand in the living body can be quantified with high sensitivity. can do.

 The quantification method of the present invention can be carried out, for example, by combining with the 'competition method'. That is, the ligand concentration in the sample can be measured by bringing the sample into contact with the receptor protein of the present invention or the like. Specifically, for example, the quantification method of the present invention can be carried out according to the method described in (1) or (2) below or a method analogous thereto.

 (1) Hiro Irie "Radio No Tsutsui" (Kodansha, published in 1974)

(2) Hiro Irie, “Radio Immunoassay” (Kodansha, published in 1979) (7) Compounds that alter the binding between the G protein-coupled receptor Yuichi protein of the present invention and a ligand (agonists, Screening Method for Antagonists, etc.) By using the receptor protein or the like of the present invention or constructing an expression system for a recombinant receptor protein or the like, and using a receptor-binding Atsei system using the expression system, the ligand and the present invention can be used. Changes the binding to the receptor protein of the invention Picture 2/04215

 This includes screening for compounds (eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof efficiently.

Such compounds, (a) G-protein coupled receptions evening through an cell-stimulating activity (e.g., Arakidon acid release, acetylcholine release, intracellular C a ²⁴ Yu away, intracellular c AM P product, cells Compounds that have an activity of promoting or inhibiting intracellular cGMP production, inositol monophosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, activation of c-fos, pH reduction, etc. (B) a compound not having the cell stimulating activity (so-called antagonist to the receptor protein of the present invention); (c) a ligand and a G protein-coupled receptor of the present invention. And (d) a compound that decreases the binding strength between the ligand and the G protein-coupled receptor protein of the present invention. (The compound (a) is preferably screened by the above-described ligand determination method).

 That is, the present invention relates to (υ) the case where the receptor protein of the present invention or its partial peptide or a salt thereof is contacted with a ligand; and (ii) the receptor protein of the present invention or its partial peptide or a salt thereof. And a ligand and a test compound, and comparing the ligand with a receptor protein of the present invention or a partial peptide thereof or a salt thereof, or a salt thereof, A screening method is provided.

 The screening method of the present invention is characterized in that, in the cases (i) and (ii), for example, the amount of a ligand bound to the receptor protein or the like, the cell stimulating activity, and the like are measured and compared.

 More specifically, the present invention provides

(1) When the labeled ligand is brought into contact with the receptor protein of the present invention or the like, and when the labeled ligand or test compound is brought into contact with the receptor protein or the like of the present invention, the receptor of the labeled ligand is used. Ligand characterized by measuring and comparing the amount of binding to protein etc. and receptor protein of the present invention 4215

 58 a method of screening for a compound or a salt thereof that alters the binding property to

(2) When a labeled ligand is brought into contact with a cell containing the receptor protein or the like of the present invention or a membrane fraction of the cell, or when a labeled ligand and a test compound are brought into contact with a cell containing the receptor protein or the like of the present invention or Threads of the labeled ligand to the cell or the membrane fraction when contacted with the membrane fraction of the cell; the amount of the mixture is measured and compared; the ligand and the receptor protein of the present invention, etc. Screening method for a compound or a salt thereof that changes the binding property with

(3) When the labeled ligand is brought into contact with the receptor protein expressed on the cell membrane by culturing the transformant containing the DNA of the present invention, the labeled ligand and the test compound are converted to the DNA of the present invention. Measuring the amount of labeled ligand bound to the receptor protein or the like when the transformant containing the protein is brought into contact with the receptor protein or the like of the present invention expressed on the cell membrane by culturing, and comparing. A method for screening a compound or a salt thereof that alters the binding property between the characteristic ligand and the receptor protein of the present invention,

(4) A compound that activates the receptor protein or the like of the present invention (eg, a ligand for the receptor protein or the like of the present invention) is brought into contact with a cell containing the receptor protein or the like of the present invention; Yuichii When a compound that activates a protein or the like and a test compound are brought into contact with cells containing the receptor protein or the like of the present invention, cell-stimulating activity via the receptor (for example, arachidonic acid release, acetylcholine release, Intracellular ^{Ca 2+} release, intracellular CAMP generation, intracellular cGMP generation, inositol phosphate production, fluctuation of cell membrane potential, intracellular protein phosphorylation, activation of c-f0s, pH The activity of promoting or suppressing the decrease in the activity of a ligand is measured and compared, and a compound that changes the binding property between the ligand and the receptor protein of the present invention or the like is characterized. Screening method for a substance or a salt thereof, and

化合物 A compound that activates the receptor protein of the present invention (eg, a ligand for the receptor protein of the present invention) was expressed on the cell membrane by culturing a transformant containing the DNA of the present invention. Receptor Yuichi protein of the present invention 02 04215

 The receptor of the present invention expressed on the cell membrane by culturing a transformant containing the DNA of the present invention with a compound that activates the receptor protein or the like of the present invention and a test compound. Receptor-mediated cell stimulating activity upon contact with Yuichi protein etc. (eg, arachidonic acid release, acetylcholine release, intracellular Ca "release, intracellular cAMP generation, intracellular cGMP Production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, and activities that promote or suppress pH reduction, etc.) It is intended to provide a method for screening a compound or a salt thereof that changes the binding property between a characteristic ligand and the receptor protein of the present invention.

 Prior to obtaining the receptor protein or the like of the present invention, when screening for a G protein-coupled receptor agonist or an angiogonist, first, a cell, tissue or cell membrane containing a G protein-coupled receptor receptor protein such as a rat is used. A candidate compound is obtained using the fraction (primary screening), and then a test is performed to confirm whether the candidate compound actually inhibits the binding of human G protein-coupled receptor protein to ligand (Secondary screening) was required. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins are also mixed, so it is difficult to actually directly screen an agonist or an antagonist for the target receptor protein. Was.

 However, for example, the use of the mouse-derived receptor protein of the present invention eliminates the need for primary screening, and allows efficient screening of compounds that inhibit the binding between ligand and G-protein-coupling receptor protein. Can be. Furthermore, whether the screened compound is an agonist or an antagonist can be easily evaluated.

 The specific description of the screening method of the present invention is as follows.

First, the receptor protein of the present invention used in the screening method of the present invention may be any as long as it contains the above-described receptor protein of the present invention. Cell membrane fractions of mammalian organs containing proteins and the like are preferred. However, especially human organs are not available. Because it is extremely difficult, a mouse-derived receptor protein expressed in large amounts using a recombinant or the like is suitable for screening.

 The above method is used to produce the receptor protein of the present invention and the like, but it is preferable to express the DNA of the present invention in mammalian cells and insect cells. As the DNA fragment encoding the protein portion of interest, cDNA is used, but it is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. In order to introduce the DNA fragment encoding the receptor protein of the present invention into host animal cells and to express them efficiently, the DNA fragment must be a nucleopolyhedron belonging to a baculovirus using an insect as a host. Disease virus (nul polyhedros is virus; NPV) polyhedrin promoter, promoter derived from SV40, retrovirus promoter overnight, metamouth thionine promoter, human heat shock promoter, cytomegalo It is preferable to incorporate it downstream such as a virus promoter and an SR promoter. Inspection of the amount and quality of the expressed receptor can be carried out by a known method, for example, as described in the literature [Namb i, P. et al., The Journal of Biological Biology Chemistry, vol. , Pp. 19555-19559, 1992]. The “receptor protein or the like of the present invention” used in the screening method of the present invention may be a receptor protein or the like purified according to a known method, a cell containing the receptor protein or the like, a cell membrane containing the receptor protein or the like. Any of fractions may be used.

 When cells containing the receptor protein or the like of the present invention are used in the screening method of the present invention, the cells may be immobilized with daltaraldehyde, formalin, or the like. Immobilization can be performed according to a known method. As the “cells containing the receptor protein or the like of the present invention” and the “cell membrane fraction”, those described in the method for determining a ligand of the present invention are used.

The amount of the receptor protein in the cell or membrane fraction containing the receptor protein or the like is preferably 10 ³ to 10 molecules, more preferably 10 ⁵ to 10 ⁷ molecules per cell. It is. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction, which not only enables the construction of a highly sensitive screening system, but also enables screening of a large number of test compounds in the same lot. .

 In order to carry out the above (1) to (3) for screening for a compound that alters the binding between the ligand and the receptor protein of the present invention, for example, an appropriate receptor protein fraction and a labeled ligand are required. is there.

 The receptor protein fraction is preferably a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto. Here, “equivalent activity” refers to equivalent ligand binding activity, signal transduction activity and the like.

As the labeled ligand, a labeled ligand, a labeled ligand analog compound, or the like is used. For example, ligands labeled with [ ³ H], [ ¹²⁵ I], [ ¹⁴ C], [ ³⁵ S] and the like are used.

 Specifically, to screen for a compound that alters the binding between the ligand and the receptor protein of the present invention, etc., first, a cell or a membrane fraction of the cell containing the receptor protein of the present invention is used. Prepare a receptor protein sample by suspending it in an appropriate buffer. The buffer is not particularly limited as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a tris-HCl buffer.

 In addition, a surfactant such as CHAPS, Tween-80 ™ (Kao-Atlas), digitonin, or dexcholate may be added to the buffer for the purpose of reducing non-specific binding. In addition, a protease inhibitor such as PMSF, leptin, E-644 (manufactured by Peptide Research Institute), or peptide suptin may be added to the buffer for the purpose of suppressing the degradation of the receptor and ligand by the protease. .

A fixed amount (5000-500000 cpm) of the labeled ligand was added to 0.01 to 10 ml of the Recept Yuichi protein preparation thus obtained, and ⁴ to ^ ^-1 at the same time. M test compounds are allowed to coexist. On the other hand, in the control group, a large excess of unlabeled ligand was used instead of labeled ligand to determine the amount of non-specific binding (NSB). Coexist. The reaction between the receptor protein preparation and the test compound or labeled ligand is performed at about 0 ° C to 50 ° C, preferably about 4 ° C to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 hours. It takes from a minute to three hours. After the reaction, the reaction solution is filtered through a glass fiber filter paper, etc., and the glass fiber filter paper is washed with an appropriate amount of buffer (a buffer similar to the receptor protein sample). Then, the radioactivity remaining on the glass fiber filter paper is measured by a liquid scintillation counter. Measure with one or T-counter. Using the obtained measurement value, when the count (B. One NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B.) when there is no antagonist is 100%, the specific binding amount ( For example, a test compound having a B-NSB) of 50% or less can be selected as a candidate substance capable of competitive inhibition.

In the above (1) or (2), which screens for a compound that alters the binding between the ligand and the receptor protein of the present invention, the cell stimulating activity (for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, cell Activity or suppression that promotes intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. Activity) can be measured by known methods or using a commercially available measurement kit.

 Specifically, first, cells containing the 'receptor protein of the present invention and the like are cultured on a multi-well plate or the like. Before performing screening, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, and then extract cells or collect supernatant. Then, the generated product is quantified according to each method. If the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult due to the presence of a degrading enzyme contained in cells, the assay may be performed by adding an inhibitor against the degrading enzyme. Good. In addition, activities such as inhibition of CAMP production can be detected as production inhibitory effects on cells whose basic production has been increased by forskolin or the like.

In order to perform screening by measuring cell stimulating activity, cells expressing an appropriate receptor protein are required. Expressing the receptor protein of the present invention, etc. The cell line is preferably a cell line having the natural receptor protein of the present invention or the like, or a cell line expressing the above-mentioned recombinant receptor protein or the like.

 As test compounds, for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds are novel compounds. Or a known compound.

 A screening kit for a compound or a salt thereof that alters the binding property of the ligand to the receptor protein of the present invention or the like comprises a cell containing the receptor protein of the present invention, the receptor protein of the present invention, or And those containing the membrane fraction of cells containing the receptor protein of the present invention.

 Examples of the screening kit of the present invention include the following.

1. Screening reagent

 ①Measurement buffer and washing buffer

 Hanks' Balanced Salt Solution (manufactured by Gibco) plus 0.05% serum serum albumin (manufactured by Sigma).

 The solution may be sterilized by filtration through a 0.45 m filter and stored at 4 ° C, or may be prepared at use.

 ② G protein-coupled receptor preparation

The receptions evening CHO cells expressing an protein of the present invention, passaged 5 10 ⁵ cells on a 12-well plate, 37 ° (:, which was cultured for 2 days in _{5% C0 2, 95% ai} r .

③ Labeled ligand

Commercially available ^[3 H], ^[I25 I], ^[14 C], ^[35 S] those states of labeled ligand solution and stored at 4 ° C or single 20 ° C, etc., measurement buffer at use Dilute to 1 M with the solution.

 ④Ligand standard solution

 Dissolve the ligand in PBS containing 0.1% で serum albumin (manufactured by Sigma) to 1 mM, and store at 20 ° C.

 2. Measurement method

① Expression of the receptor protein of the present invention cultured on a 12-well tissue culture plate C After washing the HO cells twice with 1 ml of the measurement buffer, add 490 l of the measurement buffer to each well.

② After adding 10- ³ to ^10-1Q test compound solution 5 1 M, the labeled ligand 5 1 was added to react at room temperature for one hour. To determine the amount of nonspecific binding, add 5 l of 1 ({ ³ } ligand) instead of the test compound.

③ Remove the reaction solution and wash 3 times with 1 ml of washing buffer. The labeled ligand bound to the cells is dissolved in 0.2 N NaOH-1% SDS, and mixed with 4 ml of liquid scintillator overnight A (manufactured by Wako Pure Chemical Industries).

 放射 Measure radioactivity using a liquid scintillation counter (manufactured by Beckman Coulter), and determine Percent Maximum Binding (PMB) by the following formula.

 PMB = [(B-NSB) / (B. One NSB)] X100

 PMB: Percent Maximum Binding

 B: Value when the sample is added

 NSB: Non-specific Binding

 B. : Maximum binding amount

The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding property between a ligand and the receptor protein of the present invention. a) Cell stimulating activity via G protein-coupled receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation , A compound having an activity of promoting or inhibiting intracellular phosphorylation, activation of c-fos, lowering of pH, etc. (a so-called agonist against the receptor protein of the present invention), ( b) the compound having no cell-stimulating activity (so-called antagonist to the receptor protein of the present invention); (c) the ligand and the compound of the present invention. A compound that enhances the binding force to the G protein-coupled receptor protein, or (d) a compound that decreases the binding force between the ligand and the G protein-coupled receptor protein of the present invention.

Such compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, Fermentation products and the like may be mentioned, and these compounds may be novel compounds or known compounds.

 Since the agonist against the receptor protein or the like of the present invention has the same activity as the physiological activity of the ligand for the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug according to the ligand activity.

 Since the antagonist to the receptor protein or the like of the present invention can suppress the physiological activity of the ligand to the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug for suppressing the ligand activity.

 The compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention is useful as a safe and low-toxic drug for enhancing the physiological activity of the ligand for the receptor protein of the present invention or the like. It is.

 A compound that decreases the binding force between the ligand and the G protein-coupled receptor protein of the present invention is useful as a safe and low-toxic drug for reducing the physiological activity of the ligand for the receptor protein or the like of the present invention. is there.

 The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention can be made into various preparations in the same manner as in the case of the receptor protein of the present invention described above.

 The preparations obtained in this way are safe and low toxic, so they can be administered, for example, to humans and mammals (eg, rats, puppies, higgs, bush, puppies, cats, dogs, sals, etc.). can do.

 The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.In the case of oral administration, for example, about 0 mg / day for a cancer patient (body weight 60 kg). 1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the dose of the compound or a salt thereof is usually, for example, about 0.01 in a cancer patient (body weight 60 kg) per day when an injection is intravenously injected. -30 mg, preferably about 0.1-20111, more preferably about 0.1-10111.

(8) Alters the binding between the G protein-coupled receptor protein of the present invention and a ligand. And Z or therapeutic agents for various diseases containing compounds to be converted (agonist, angyu gonist)

 As described above, the receptor protein of the present invention is considered to play some important roles in vivo, such as central function, circulatory function, digestive function, and cardiac function. Therefore, a compound that alters the binding property between the receptor protein of the present invention and the ligand (agonist, angonist) or a ligand for the receptor protein of the present invention is dysfunctional of the receptor protein of the present invention. It can be used as a prophylactic and / or therapeutic agent for diseases related to the disease.

 Here, diseases associated with dysfunction of the receptor protein of the present invention include, for example, central illness (eg, depression, Alzheimer's disease, dementia, eating disorder, etc.) and endocrine diseases (eg, hypertension, gonad dysfunction, thyroid gland) Dysfunction, pituitary dysfunction, etc.), metabolic diseases (eg, diabetes, dyslipidemia, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer) , Cervical cancer, colon cancer, rectal cancer, etc.), and heart diseases (eg, angina pectoris, myocardial infarction, etc.).

 When the compound or ligand is used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, the compound or ligand may be used as the above-described receptor protein of the present invention. Various preparations can be prepared in the same manner as described above.

 Further, the above-mentioned prophylactic / therapeutic agent can be used in combination with an appropriate drug, for example, as a DDS preparation specifically targeting an organ or tissue in which the receptor protein of the present invention is highly expressed.

 The preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and mammals (for example, rats, mice, puppies, higgs, bushes, cats, cats, dogs, monkeys, etc.). Can be administered.

The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.In the case of oral administration, for example, about 0,0 per day for a cancer patient (body weight 60 kg). It is 1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the compound or its salt The dose is, for example, usually about 0.01 to 30 mg, preferably about 0.1 to 30 mg per day for a cancer patient (body weight 60 kg) when an injection is injected intravenously. 20 mg, more preferably about C-10 mg. (9) Determination of the receptor protein of the present invention or its partial peptide or its salt

 Since the antibody of the present invention can specifically recognize the receptor protein and the like of the present invention, it is used for quantification of the receptor protein of the present invention in a test solution, particularly for quantification by a sandwich immunoassay. can do. That is, the present invention, for example,

 (i) reacting the antibody of the present invention with a test solution and a labeled receptor protein in a competitive manner, and measuring the ratio of the labeled receptor protein bound to the antibody in the test solution. A method for quantifying the receptor protein of the present invention,

 (ii) Simultaneously or continuously reacting the test solution with the antibody of the present invention insoluble on the carrier and the labeled antibody of the present invention, and then measuring the activity of the labeling agent on the insoluble carrier. The present invention provides a method for quantifying the receptor protein of the present invention in a test solution, characterized in that:

 In the above (ii), it is preferable that one antibody is an antibody that recognizes the N-terminal of the receptor protein or the like of the present invention, and the other antibody is an antibody that reacts with the C-terminal of the receptor protein or the like of the present invention. .

In addition to measuring the receptor protein of the present invention using the monoclonal antibody against the receptor protein of the present invention (hereinafter sometimes abbreviated as the monoclonal antibody of the present invention), the detection by tissue staining or the like can be performed. You can do it. For these purposes, the antibody molecule itself may be used, or F (at) ′) ₂ , Fab ′, or Fab fraction of the antibody molecule may be used. The assay method using an antibody against the receptor protein or the like of the present invention is not particularly limited, and the amount of the antibody, antigen or antibody-antigen complex corresponding to the antigen amount (for example, the amount of receptor protein) in the test solution Any method can be used as long as it is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. A method for measuring the displacement may be used. As the measuring method, for example, nephelometry, a competitive method, an immunometric method and a sandwich method are preferably used, but the sandwich method described later is particularly preferable in terms of sensitivity and specificity.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ^I4 C] and the like are used. As the above-mentioned enzyme, a stable enzyme having a large specific activity is preferable. For example, / 3-galactosidase,] 3-darcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiosinate and the like are used. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin and the like are used. Further, a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.

 In the case of insolubilizing an antigen or an antibody, a method using physical adsorption or a chemical bond usually used for insolubilizing or immobilizing a protein or an enzyme is used. Examples of the carrier used for physical adsorption include insoluble polysaccharides such as agarose, dextran, and cellulose; synthetic resins such as polystyrene, polyacrylamide, and silicon; and glass.

 In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction). By measuring the activity of the agent, the amount of the receptor protein of the present invention in the test solution can be determined. The primary reaction and the secondary reaction may be performed in reverse order, may be performed simultaneously, or may be performed with a time delay. Here, the same labeling agent as described above is used. Insolubilization is performed in the same manner as described above.

 In the immunoassay using the sandwich method, the antibody used for the solid phase antibody or the labeling antibody does not necessarily need to be one type, and a mixture of two or more types of antibodies is used for the purpose of improving the measurement sensitivity, etc. You may.

In the method for measuring the receptor protein and the like by the sandwich method of the present invention, As the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different binding site such as a receptor protein is preferably used. That is, the antibodies used in the primary reaction and the secondary reaction are, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is other than the C-terminal For example, it is preferable to recognize the N end.

 The monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry. In the competitive method, the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, and then the unreacted labeled antigen, (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation) The amount of any of B, F and the label is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as the antibody, B / F separation is performed using a polyethylene glycol, a liquid phase method using a second antibody against the above antibody, etc .; and an immobilized antibody is used as the first antibody, or As the first antibody, a soluble antibody is used, and an immobilization method using an immobilized antibody as the second antibody is used. In the immunometric method, an antigen in a test solution and a solid-phased antigen are subjected to a competitive reaction with a fixed amount of a labeled antibody, and then the solid phase and the liquid phase are separated, or Then, the unreacted labeled antibody is allowed to bind to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution. .

 In nephelometry, the amount of insoluble sediment resulting from an antigen-antibody reaction in a gel or in a solution is measured. When the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry using laser scattering is preferably used.

 In applying these individual immunological assay methods to the assay method of the present invention, no special conditions, operations, and the like need to be set. The measurement system for the receptor protein of the present invention or a salt thereof may be constructed by adding ordinary technical considerations of those skilled in the art to ordinary conditions and procedures in each method. For such technical considerations, it is possible to refer to reviews and written documents. Specifically, Hiroshi Irie

(Kodansha, published in 1974), edited by Hiroshi Irie Five

 70 Immunoassay "(Kodansha, published in 1979), Eiji Ishikawa et al.," Enzyme Immunoassay "(Medical Publishing, published in 1978), Eiji Ishikawa et al.," Enzyme Immunoassay "(2nd edition) (Medical) Shoin, published in 1982), Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition) (Medical Publishing, published in 1987), “Methods in ENZYMOLOGY” J Vol. 70 ( 73 (Immunochemical Techniques (Part B)), ibid.Vol. 74 (Immunochemical Techniques (Part 0), ibid.Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)) Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Ant ibodies and General Immunoassay Methods)) Vol. 121 (Immunochemical Techniques (Part I: Hybridonia Technolgy and Monoclonal Ant ibodies)) Press Company).

 As described above, the receptor protein of the present invention or a salt thereof can be quantified with high sensitivity by using the antibody of the present invention.

 Furthermore, by quantifying the receptor protein of the present invention or a salt thereof in a living body using the antibody of the present invention, it is possible to diagnose various diseases associated with dysfunction of the receptor protein of the present invention. it can.

 Further, the antibody of the present invention can be used for specifically detecting the receptor protein of the present invention or the like present in a subject such as a body fluid or a tissue. Further, preparation of an antibody column used for purifying the receptor protein and the like of the present invention, detection of the receptor protein of the present invention in each fraction at the time of purification, and receptor of the present invention in test cells It can be used for analyzing the behavior of Yuichi proteins. (10) A method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in a cell membrane

Since the antibody of the present invention can specifically recognize the receptor protein of the present invention or its partial peptide or a salt thereof, a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane Skull of Can be used for reading.

 That is, the present invention, for example,

 (i) After destruction of (1) blood, (2) specific organ, and (3) tissue or cells isolated from the non-human mammal, the cell membrane fraction is isolated, and the receptor of the present invention contained in the cell membrane fraction is destroyed. A method for screening a compound that changes the amount of the receptor protein or its partial peptide of the present invention in the cell membrane by quantifying the protein or its partial peptide,

 (ii) After disrupting a transformant or the like that expresses the receptor protein of the present invention or its partial peptide, the cell membrane fraction is isolated, and the receptor protein of the present invention contained in the cell membrane fraction or its partial peptide A method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by quantifying

 (iii) The degree of staining of the receptor protein on the cell surface by using immunostaining after sectioning (1) blood, (2) specific organs, (3) tissues or cells isolated from the organs of a non-human mammal. A method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane by quantifying

(iv) Quantifying the degree of staining of the receptor protein on the cell surface by using an immunostaining method after sectioning a transformant or the like that expresses the receptor protein of the present invention or a partial peptide thereof. Thus, there is provided a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane. '

 The quantitative determination of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically performed as follows.

(i) Normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc .; more specifically, demented rats, obese mice, arteriosclerotic rabbits) Drugs (eg, anti-dementia drugs, antihypertensive drugs, drugs, anti-obesity drugs, etc.) or physical strikes After a certain period of time (eg, immersion stress, electric shock, light / dark, low temperature, etc.), blood, or a specific organ (eg, brain, lung, colon), or tissue isolated from the organ, Alternatively, obtain cells. The obtained organ, tissue, or cell is suspended in, for example, an appropriate buffer (eg, Tris-HCl buffer, phosphate buffer, Hase buffer, etc.), and the organ, tissue, or cell is broken. Then, a cell membrane fraction is obtained by using a surfactant (eg, Triton X_100 ™, Tween-20 ™, etc.), and further using a ^ method such as centrifugation, filtration, or column fractionation.

 The cell membrane fraction refers to a cell membrane-rich fraction obtained by crushing cells and obtained by a known method. Cell crushing methods include crushing cells with a Potter-Elvehj em-type homogenizer, crushing method using a Warlinda blender or a polytron (Kinematica); sonication crushing method; French press A crushing method in which cells are ejected from a thin nozzle while pressurizing with, for example, is mentioned. The cell membrane thus obtained can be fractionated by a centrifugal fractionation method such as a differential centrifugation method or a density gradient centrifugation method. For example, the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1-10 minutes), and the supernatant is further centrifuged at a high speed (15000-30000 rpm) for 30 minutes to 2 hours. By collecting the precipitate, a cell membrane fraction can be obtained. The cell membrane fraction contains a large amount of expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.

 The receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, Western blot analysis, or the like.

 Such a sandwich immunoassay can be performed in the same manner as described above, and western blot analysis can be performed by known means.

 (ii) Producing a transformant expressing the receptor protein of the present invention or its partial peptide according to the above method, and quantifying the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction. Can be.

The amount of the receptor protein of the present invention or its partial peptide in the cell membrane Screening for compounds to be altered

 (i) A given time before giving a drug or physical stress to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour Before or after 6 hours) or after a certain time (after 30 minutes to 3 days, preferably after 1 hour to 2 days, more preferably after 1 hour to 24 hours), or simultaneously with the drug or physical stress. After a certain period of time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours) after administration, the receptor protein of the present invention or a portion thereof in the cell membrane (Ii) When the transformant is cultured according to a conventional method, the test compound is mixed in the medium, and after culturing for a certain period of time (after 1 day to 7 days, preferably Is one day later ~ 3 After more favorable Mashiku after 2 days to 3 days), or the receptor protein of the present invention on the cell membrane can be carried out by quantifying the amount of the part base peptide.

 Confirmation of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically carried out as follows.

 (iii) Normal or disease model non-human mammals (for example, mice, rats, rabbits, sheep, higgs, bush, birds, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, arteriosclerosis rabbits) Drugs (eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.) And after a certain period of time, obtain blood or a specific organ (eg, heart, placenta, lung, etc.), or tissue or cells isolated from the organ. The obtained organs, tissues or cells are cut into tissue sections according to a conventional method, and immunostained using the antibody of the present invention. By quantifying the degree of staining of the receptor protein on the cell surface, and confirming the protein on the cell membrane, the receptor protein of the present invention or its partial peptide on the cell membrane can be quantitatively or qualitatively determined. You can check the quantity.

(iv) It can also be confirmed by taking the same means using a transformant or the like that expresses the receptor protein of the present invention or its partial peptide. The compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the amount of the receptor protein of the present invention or its partial peptide in a cell membrane, and specifically, (a ) By increasing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, the cell stimulating activity via G protein-coupled receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release) Activates or suppresses intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease in pH, etc. (B) reducing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. Is a compound that decrease the cell stimulating activity.

 Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound.

 The compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein of the present invention.

 The compound that attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein or the like of the present invention.

 When a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, the compound or a salt thereof may be prepared into various preparations in the same manner as in the case of the receptor protein of the present invention described above. Can be.

 The preparations obtained in this way are safe and have low toxicity, so they can be administered to humans and mammals (for example, rats, mice, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc.). can do.

The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.In the case of oral administration, for example, about 0,0 per day for a cancer patient (body weight 60 kg). It is 1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the dose of the compound or a salt thereof is usually, for example, in the case of intravenous injection, for example, a cancer patient (body 4215

 The amount is about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day based on 75 kg (60 kg).

(11) A preventive and / or therapeutic agent for various diseases containing a compound that alters the amount of the receptor protein or its partial peptide of the present invention in the cell membrane. The receptor protein of the present invention is as described above, for example, heart or heart. It is considered to play some important role in vivo, such as function. Therefore, a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be used as a preventive and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention.

 Here, diseases associated with dysfunction of the receptor protein of the present invention include, for example, central illness (eg, depression, Alzheimer's disease, dementia, eating disorder, etc.) and endocrine diseases (eg, hypertension, gonad dysfunction, thyroid gland) Dysfunction, pituitary dysfunction, etc.), metabolic diseases (eg, diabetes, dyslipidemia, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer) , Cervical cancer, colon cancer, rectal cancer, etc.), heart disease (eg, angina pectoris, myocardial infarction, etc.).

When the compound is used as a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, the compound may be a compound of the present invention described above. / Various preparations can be prepared in the same manner as in the case of the septa-protein.

 The preparations obtained in this way are safe and have low toxicity, so they can be administered to humans and mammals (for example, rats, mice, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc.). can do.

The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.In the case of oral administration, for example, about 0,0 per day for a cancer patient (body weight 60 kg). It is 1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the dose of the compound or a salt thereof is usually, for example, about 0.01% per day for a cancer patient (60 kg body weight) when an injection is intravenously injected. ~ 30 mg, preferably about 0.1-20 mg, more preferably about 0.1 to 10 mg.

(12) Neutralization by an antibody against the receptor protein of the present invention, its partial peptide, or a salt thereof

 The neutralizing activity of the antibody against the receptor protein of the present invention or its partial peptide or a salt thereof against the receptor protein or the like means the activity of inactivating the signal transduction function involving the receptor protein. I do. Therefore, the antibody has a neutralizing activity due to signal transduction associated with the receptor protein, for example, cell stimulating activity via the receptor protein (for example, arachidonic acid release, acetylcholine release, intracellular C release). a Release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuation of cell membrane potential, phosphorylation of intracellular protein, c-1: activation of fos, reduction of pH, etc. Activity or inhibitory activity). Therefore, the antibody of the present invention having a neutralizing activity can be used for prevention and / or treatment of diseases caused by overexpression of the receptor protein.

(13) Creation of a transgenic animal having a DNA encoding the G protein-coupled receptor protein of the present invention

 Using the DNA of the present invention, transgenic animals expressing the receptor protein and the like of the present invention can be produced. Examples of animals include mammals (for example, rats, mice, rabbits, sheep, sheep, pigs, rabbits, cats, dogs, monkeys, etc.) (hereinafter sometimes abbreviated as animals). Mice, egrets and the like are preferred.

When introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of being expressed in animal cells. For example, when introducing the DNA of the present invention derived from a egret, a gene construct linked to the downstream of various promoters capable of expressing the DNA of the present invention derived from an animal having high homology with the DNA in animal cells, for example, The present invention is characterized by microinjection into fertilized eggs of egrets. 4215

 77 DNA-introduced animals that produce high levels of protein and other proteins can be produced. As the promoter, for example, a virus-derived promoter, a ubiquitous expression promoter such as metamouth thionein and the like are used, and a promoter of a gene specifically expressed in the heart is preferable.

 Introduction of the DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target animal. The presence of the receptor protein or the like of the present invention in the germinal cells of the produced animal after the introduction of the DNA indicates that all the offspring of the produced animal have the receptor protein and the like of the present invention in all of the germinal and somatic cells. means. The progeny of this type of animal that has inherited the gene has the receptor protein of the present invention in all of its germ cells and somatic cells. The DNA-introduced animal of the present invention can be bred in a normal breeding environment as a DNA-bearing animal after confirming that the gene is stably maintained by crossing. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the introduced gene on both homologous chromosomes are obtained, and by crossing the male and female animals, all the offspring can be obtained. It can be propagated to carry the DNA.

 Since the animal into which the DNA of the present invention has been introduced expresses the receptor protein of the present invention at a high level, it is useful as an agonist or an animal for screening an antagonist against the receptor protein of the present invention.

The DNA-introduced animal of the present invention can also be used as a cell source for tissue culture. For example, by directly analyzing the DNA or RNA in the tissue of the DNA-introduced mouse of the present invention, or by analyzing the tissue in which the receptor-protein of the present invention expressed by a gene is present, the receptor protein of the present invention can be obtained. Etc. can be analyzed. Cells of a tissue having the receptor protein or the like of the present invention are cultured by standard tissue culture techniques, and these are used to study the function of cells from tissues that are generally difficult to culture such as those derived from brain or peripheral tissues. be able to. Also, by using the cells, for example, it is possible to select a drug that enhances the function of various tissues. In addition, if there is a cell line that highly expresses the receptor protein of the present invention, the receptor protein of the present invention is isolated and purified therefrom. 4215

 It is also possible to do

(14) A medicine containing an antisense polynucleotide (nucleic acid) '' can complementarily bind to the polynucleotide (eg, DNA) of the present invention and suppress the expression of the polynucleotide (eg, DNA). The antisense polynucleotide of the present invention has low toxicity and can suppress the function of the protein of the present invention or the polynucleotide of the present invention (eg, DNA) in a living body. It can be used as a prophylactic and / or therapeutic agent for diseases associated with dysfunction of receptor protein.

 Here, the disease associated with the dysfunction of the receptor protein of the present invention includes, for example, central illness (eg, depression, Alzheimer's disease, dementia, eating disorder, etc.), endocrine disease (eg, hypertension, gonad dysfunction, thyroid gland) Dysfunction, pituitary dysfunction, etc.), metabolic diseases (eg, diabetes, dyslipidemia, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer) , Cervical cancer, colon cancer, rectal cancer, etc.), and heart diseases (eg, angina pectoris, myocardial infarction, etc.).

 When the antisense polynucleotide is used as the therapeutic or prophylactic agent, the antisense polynucleotide may be formulated in the same manner as in the case of the DNA encoding the receptor protein of the present invention described above. it can. The product obtained in this way has low toxicity and is orally or parenterally administered to humans or mammals (eg, rats, puppies, higgs, bush, puppies, cats, dogs, monkeys, etc.). Can be administered in a controlled manner.

 The antisense polynucleotide can be administered as it is or together with a physiologically acceptable carrier such as an auxiliary for promoting ingestion, using a gene gun or a catheter such as a hydrogel gel catheter.

The dosage of the antisense polynucleotide varies depending on the target disease, the administration subject, the administration route, and the like. For example, for the purpose of treating cancer, the antisense polynucleotide of the present invention is administered to an organ (eg, liver, lung, heart, When administered topically to the kidney (eg kidney), the dose is about 0.1 to 100 mg per day for an adult (body weight 60 kg). Furthermore, the antisense polynucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence or expression of the DNA of the present invention in tissues or cells. In the present specification and the drawings, when bases, amino acids, and the like are indicated by abbreviations, the indications are based on the abbreviations by IUPAC-IUB Communication on Biochemical Nomenclature or abbreviations commonly used in the art. An example is shown below. When amino acids may have optical isomers, the L-form is indicated unless otherwise specified.

 Gly-Glycine

 Ala-Alanin

 Val: Valine

 Leu: Leucine

 l ie-Ishiguchi Ishi,

 Ser: Serine

 Thr: Sure Saino

 Cys: Cystine

 Met: Methionin

 Glu: Glutamic acid

 Asp: Aspartic acid

 Lys • 1 ton

 Arg: Arginine

 His: histidine

 Phe: feniralanin

 Tyr: Tyrosine

 Trp: Tributofuan

 Pro: Proline

 Asn: Asparagine

Gin: Glutamine pGlu: pyroglutamic acid

 Me: methyl group

 Et: ethyl group

 Bu: butyl group

 Ph: phenyl group

 TC.: Thiazolidine-1 (R) mono- lipoxamide group

Further, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols. '

 To s: ρ-toluenesulfonyl

CHO: Formyl

B z 1

C 1 ₂ B z 1: 2, 6

Bom

Z: benzyloxycarponyl

C 1-Z:

B rZ:

B oc t-butoxycarbonyl

DNP dinitrophenol

T r t Trityl

Bum t—butoxymethyl

Fmo c: N-9-fluorenylmethoxycarbonyl

HOB t: 1-hydroxybenztriazole

HOOB t: 3, 4-dihydro-3-hydroxy-4-oxo-1

 1, 2, 3—Benzotriazine

 HONB: 1-Hydroxy-5-norpolene-2,3-dicarboximide.

DCC The sequence numbers in the sequence listing in the present specification indicate the following sequences. SEQ ID NO: 1

 1 shows the amino acid sequence of a mouse-derived novel G protein-coupled receptor protein TGR38 of the present invention.

 SEQ ID NO: 2

 1 shows the nucleotide sequence of cDNA encoding the novel mouse-derived G protein-coupled receptor protein TGR38 of the present invention.

 SEQ ID NO: 3

 1 shows the nucleotide sequence of primer 1 used in the PCR reaction in Example 1 below. '

 SEQ ID NO: 4

 The nucleotide sequence of Primer 1 used in the PCR reaction in Example 1 below was set to SEQ ID NO: 5

 1 shows the nucleotide sequence of the 5 ′ untranslated region of the DNA encoding the novel mouse-derived G protein-coupled receptor protein TGR38 of the present invention.

 SEQ ID NO: 6

 1 shows the nucleotide sequence of the 3 ′ untranslated region of the DNA encoding the novel mouse-derived G protein-coupled receptor protein TGR38 of the present invention. The transformant, Escherichia coli T0P10 / pCR2.1-TGR38, obtained in Example 1 below has been used since April 19, 2001 at 1-1-1 Higashi, Tsukuba City, Ibaraki Pref. The 6th (zip code 305-8566) Independent National Institute of Advanced Industrial Science and Technology (AIST) Patent Depositary Depositary No. F ERM BP-7554 from April 11, 2001, Yodogawa, Osaka, Osaka It has been deposited with the Foundation for Fermentation (IFO), 2-17-85, Jusanhoncho, Ward (postal code 532-8686) under the deposit number IF〇16614. Example

Hereinafter, the present invention will be described in more detail with reference to Examples. Is not limited. The genetic engineering method using Escherichia coli should be performed according to the method described in Molecular cloning.

Example 1 (Cloning of cDNA encoding G protein-coupled receptor Yuichi protein from mouse brain and determination of nucleotide sequence)

 Using mouse brain cDNA (CL0NTECH) as type I, PCR was carried out using two primers, Primer 1 (SEQ ID NO: 3) and Primer 1 (SEQ ID NO: 4). In the PCR reaction, the above cDNA 1, Advantage-GC2 Polymerase Mix (CLONTECH) l xl, Primer 1 (SEQ ID NO: 3) and Primer 2 (SEQ ID NO: 4) 0.5 M each, dNTPs 200 iM, and Advantage-GC2 Po A reaction solution 50 JL61 consisting of 10 l of the buffer attached to the merase Mix and GC Melt 51 was used. In the PCR reaction, 95 ° C for 1 minute, 95 ° C for 30 seconds, 68 ° C for 2 minutes, 5 cycles, 95 ° C for 30 seconds, 66 ° C for 30 seconds, The cycle of the reaction at 68 ° C for 2 minutes was repeated 5 times, the cycle of the reaction at 95 ° C for 30 seconds, 30 seconds at 64 ° C, and the reaction for 68 minutes at 68 ° was repeated 30 times, and the reaction was finally performed at 68 ° C for 7 minutes.

 The PCR reaction product was subcloned into the plasmid vector pCR2.1 (Invitrogen) according to the method described in the T0P0-TA cloning kit (Invitrogen). The resulting plasmid vector was introduced into E. coli T0P10, and added to LB agar medium containing ampicillin to select a clone having cDNA. As a result of analyzing the sequences of various clones, a cMA sequence (SEQ ID NO: 2) encoding a novel G protein-coupled receptor protein was obtained.

 The novel G protein-combined receptor protein containing an amino acid sequence obtained by translating the cDNA sequence was designated as TGR38. Further, the transformant was named Escherichia coli TOP10 / pCR2.1-TGR38. Industrial applicability

The G protein-coupled receptor protein of the present invention or a partial peptide thereof or a salt thereof, a polynucleotide encoding the receptor protein or a partial peptide thereof (eg, DNA, RNA and derivatives thereof) are: (2) Acquisition of antibodies and antisera, (3) Construction of a recombinant receptor protein expression system, (4) Development of a receptor-binding atsushi system using the same expression system and drug candidate compounds Screening, ド ラ ッ グ drug design based on comparison with structurally similar ligands and receptors, プ ロ probes in genetic diagnosis, 試 薬 reagents for creating PCR primers, ⑦ transgenics. It can be used for animal production or as a drug such as a gene prevention and treatment agent.

Claims

The scope of the claims 1. A G protein-coupled receptor protein or a salt thereof, which comprises an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1.  2. A G protein-coupled receptor protein or a salt thereof, comprising the amino acid sequence represented by SEQ ID NO: 1.  3. A partial peptide of the G protein-coupled receptor protein according to claim 1, or a salt thereof. 4. A polynucleotide containing a polynucleotide encoding the G protein-coupled receptor protein according to claim 1.  5. The polynucleotide according to claim 4, which is DNA.  6. The polynucleotide according to claim 5, which has the nucleotide sequence represented by SEQ ID NO: 2. 7. A recombinant vector containing the polynucleotide according to claim 4. 8. A transformant transformed with the recombinant vector according to claim 7. 9. The transformant according to claim 8 is cultured to produce the G protein-coupled receptor protein according to claim 1, or the G protein-coupling receptor protein according to claim 1 or a protein thereof. Method for producing salt. 10. A pharmaceutical comprising the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof.  11. A pharmaceutical comprising the polynucleotide according to claim 4.  12. An antibody against the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof. 13. The antibody according to claim 12, which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to claim 1.  1. A diagnostic agent comprising the antibody according to claim 12. 15. The claim 1, which can be obtained by using the G protein-coupled receptor protein according to claim 1 or the partial peptide or the salt thereof according to claim 3. Ligand for the G protein-coupled receptor Yuichi protein or salt thereof.  16. A medicament comprising the ligand according to claim 15.  17. The G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof, wherein the G protein-coupled receptor protein according to claim 1 or a salt thereof is used. Decision method. 18. The G protein-coupled receptor protein according to claim 1 or a salt thereof, wherein the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof is used. For screening a compound or a salt thereof that alters the binding property between a compound and a ligand.  19. The G protein-coupled receptor protein or a salt thereof according to claim 1, which contains the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof. A kit for screening a compound or a salt thereof that changes the binding property to a ligand. 20. A ligand obtainable by using the screening method according to claim 18 or the screening kit according to claim 19; and the G protein-coupled receptor protein according to claim 1 or the same. A compound or a salt thereof that changes the binding property to a salt.  21. Binding of the ligand obtainable by using the screening method according to claim 18 or the screening kit according to claim 19 to the G protein-coupled receptor protein according to claim 1 or a salt thereof A medicament comprising a compound that changes sex or a salt thereof. '  22. The polynucleotide of claim 4 and highly stringent conditions 23. A polynucleotide comprising a nucleotide sequence complementary to the polynucleotide according to claim 4 or a part thereof.  24. The method for quantifying mRNA of a G protein-coupled receptor protein according to claim 1, wherein the polynucleotide according to claim 4 or a part thereof is used. 25. The G according to claim 1, wherein the antibody according to claim 12 is used. A method for quantifying a protein-coupled receptor protein.  26. The method for diagnosing a disease associated with the function of a G protein-coupled receptor according to claim 1, wherein the quantification method according to claim 24 or 25 is used.  27. The method for screening a compound or a salt thereof, which changes the expression level of a G protein-coupled receptor protein according to claim 1, wherein the method according to claim 24 is used.  28. Screening of the compound or a salt thereof that changes the amount of the G protein-coupled receptor protein according to claim 1 in a cell membrane, characterized by using the quantification method according to claim 25. Method.  29. A compound or a salt thereof, which alters the expression level of the G protein-coupled receptor protein according to claim 1, which can be obtained by using the screening method according to claim 27.  30. A compound or a salt thereof that alters the amount of the G protein-coupled receptor protein of claim 1 in a cell membrane obtainable by using the screening method of claim 28.  31. A medicament comprising a compound that changes the expression level of the G protein-coupled receptor protein according to claim 1, which can be obtained by using the screening method according to claim 27, or a salt thereof.  32. A medicament comprising a compound that changes the amount of the G protein-coupled receptor protein of claim 1 or a salt thereof in a cell membrane obtainable by using the screening method of claim 28.  33. The medicament according to any one of claims 21 to 31, which is a preventive or therapeutic agent for central diseases, endocrine diseases, metabolic diseases, cancer or heart diseases. 3 4. A mammal, comprising administering to the mammal an effective amount of the compound or a salt thereof according to any one of claims 20, 29, or 30. Prevention and treatment of central diseases, endocrine diseases, metabolic diseases, cancer or heart diseases in Japan. 3 5. Prophylactic and therapeutic agents for central illness, endocrine disease, metabolic disease, cancer or heart disease Use of the compound or a salt thereof according to any one of claims 20 to 29 or 30 for the manufacture.