WO2002088356A1

WO2002088356A1 - Novel g protein-coupled receptor protein and dna thereof

Info

Publication number: WO2002088356A1
Application number: PCT/JP2002/004200
Authority: WO
Inventors: Takeo Moriya; Takashi Ito; Yasushi Shintani; Nobuyuki Miyajima
Original assignee: Takeda Chemical Industries, Ltd.
Priority date: 2001-04-27
Filing date: 2002-04-26
Publication date: 2002-11-07

Abstract

A novel G protein-coupled receptor protein or its peptide fragments or salts thereof and polynucleotides (for example, DNA, RNA) encoding this receptor protein or its peptide fragments or derivatives thereof are usable: (1) in determining a ligand or an agonist; (2) in acquiring an antibody and an antiserum; (3) in constructing a recombinant receptor protein expression system; (4) in developing a receptor-bonded assay system and screening a candidate compound for a drug with the use of the expression system; (5) in performing drug design based on the comparison with a ligand or a receptor having a similar structure; (6) as a reagent for constructing a probe in gene diagnosis or a PCR primer; (7) in constructing a transgenic animal; or (8) as drugs such as genetic remedies.

Description

TECHNICAL FIELD The present invention relates to a novel G protein-coupled receptor protein derived from human testis, a salt thereof, and a 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 conjugated guanine nucleotide-binding protein (hereinafter sometimes abbreviated as G protein). Because they have a common structure with a transmembrane region, they are collectively referred to as G protein-coupled receptor Yuichi protein or 7-transmembrane receptor Yuichi protein (7TMR).

G protein-coupled receptor protein is present on the surface of each functional cell in living cells and organs, and is a target for molecules that regulate the function of those cells and organs, such as hormones, neurotransmitters, and biologically active substances. Plays a physiologically important role. The receptor transmits a signal into the cell through binding to a physiologically active substance, and this signal causes various reactions such as suppression of activation and activation of the cell.

Elucidating 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 protein, is important for various organisms. It will elucidate the functions of cells and organs and provide a very important means for drug development closely related to those functions.

For example, in various organs of a living body, physiological functions are regulated under the control of many hormones, hormone-like substances, neurotransmitters or bioactive substances. In particular, bioactive substances exist in various parts of the body, It regulates its physiological functions through Recept Yuichi protein. There are many unknown hormones, neurotransmitters, and other physiologically active substances in the living body, and the structure of their receptor proteins has not yet been reported. In addition, it is often unknown whether subtypes exist in known receptor overnight proteins.

Determining the relationship between substances that regulate complex functions in living organisms and their specific receptor proteins is a very important tool for drug development. In addition, in order to efficiently screen agonists and antagonists against receptor protein and develop pharmaceuticals, the function of the receptor protein gene expressed in the body should be elucidated and the It was necessary to use a suitable expression system.

In recent years, as a means of analyzing genes expressed in vivo, research on random analysis of the cDNA sequence has been actively conducted, and the cDNA fragment sequence obtained in this manner is expressed in an Expressed Sequence. Registered in the database as a 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 of a G protein-coupled receptor to a biologically active substance (ie, a ligand) or substances that bind to cause a signal transduction similar to that of a biologically active substance (ie, a ligand) are It has been used as a specific agonist or agonist at Recept Yuichi as a drug to regulate biological functions. Therefore, we have newly discovered a G protein-coupled receptor protein that is not only important in physiological expression in vivo but also a target for drug development, and its gene (eg, cDNA) Cloning is a very important tool for finding specific ligands ゃ, agonists and antagonists of novel G protein-coupled receptor proteins.

However, not all G protein-coupled receptors have been found, and at this time, unknown G protein-coupled receptors and the corresponding ligands have not been identified. There are a large number of fan receptors, and there is a keen need to search for new G protein-coupled receptors and elucidate their functions. The G protein-coupled receptor is useful for searching for a new physiologically active substance (that is, a ligand) using its signaling effect as an index, and for searching for an agonist or antagonist for the receptor. On the other hand, even if a physiological ligand is not found, an agonist or an anthony gonist against the receptor is prepared by analyzing the physiological action of the receptor from an inactivation experiment (knockout animal) of the receptor. It is also possible. These ligands, agonists or antagonists to the receptor can be expected to be used as preventive and / or therapeutic or diagnostic agents for diseases associated with dysfunction of G protein-coupled receptors.

Furthermore, the decrease or enhancement of the function of the receptor in the living body based on the gene mutation of the G protein-coupled receptor often causes some disease. In this case, not only administration of an antagonist 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 essential information for examining the presence or absence of a deletion or mutation in the gene, and the gene of the receptor is responsible for the disease involved in the dysfunction of the receptor. It can also be applied to prophylactic and / or therapeutic and diagnostic agents.

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 its partial peptide or a salt thereof, and a polynucleotide (DNA, RNA and a derivative thereof) encoding the G protein-coupled receptor protein or its partial peptide are prepared. Containing polynucleotide (DNA, RNA and derivatives thereof), a recombinant vector containing the polynucleotide, a transformant carrying the recombinant vector, the G protein-coupled receptor protein or a salt thereof. A production method, 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, and a G protein-coupled receptor protein Method for determining ligands for G protein, coupling of ligand to the G protein Type Recept with Yuichi Protein Screening method for compounds that change binding properties (angiagonists, agonists) or salts thereof, screening kits, ligands obtainable by using the screening methods or screening kits, and the G protein-coupled receptor Yuichi protein (Antagonist, agonist) or a salt thereof, and a compound that alters the binding between a ligand and the G protein-coupled receptor protein (antagonist, agonist) or the G protein It is intended to provide a drug or the like containing a compound that changes the expression level of a conjugated 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 human testis and analyzing its entire nucleotide sequence. . Then, when this base sequence was translated into an amino acid sequence, the first to seventh transmembrane regions were confirmed on the hydrophobicity plot, and the protein encoded by these cDNAs was conjugated to a seven-transmembrane G protein. Type receptor protein. The present inventors have further studied 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, which comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or 5;

(2) a G protein-conjugated receptor, which comprises the amino acid sequence represented by SEQ ID NO: 1 or 5, or a salt thereof;

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

(4) a polynucleotide comprising a polynucleotide having a nucleotide sequence encoding the G protein-coupled receptor protein according to (1),

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

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

(8) A transformant transformed with the recombinant vector according to (7).

(9) The G protein-coupled receptor according to (1), wherein the transformant according to (8) is cultured to produce the G protein-coupled receptor Yuichi protein according to (1). A method for producing a protein or a salt thereof,

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

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

(12) an antibody against the G protein-coupled receptor protein described in (1) above or the partial peptide described in (3) or a salt thereof,

(13) the antibody according to (12), which is a neutralizing antibody that inactivates signal transmission 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 according to (1), which can be obtained by using the G protein-coupled receptor according to (1) or the partial peptide according to (3) or a salt thereof. A ligand for a protein or a salt thereof,

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

(17) The G protein-coupled receptor protein described in (1) above, wherein the G protein-coupled receptor protein described in (1) above or the partial peptide described in (3) or a salt thereof is used. How to determine the ligand for that salt,

(18) A ligand characterized by using the G protein-coupled receptor protein described in (1) or the partial peptide described in (3) or a salt thereof, and a G protein-coupled receptor described in (1). A method for screening a compound or a salt thereof that alters the binding to a protein or a salt thereof,

(19) A ligand comprising the G protein-coupled receptor protein according to (1) or the partial peptide according to (3) or a salt thereof. And a kit for screening a compound or a salt thereof that alters the binding property to the G protein-coupled receptor protein or a salt thereof according to (1) above.

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

(21) The ligand obtainable by using the screening method described in (18) or the screening kit described in (19) above, and the G protein-coupled receptor protein or a salt thereof described in (1) above. 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 stringent conditions,

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

(24) The method for quantifying mRNA of a G protein-coupled receptor protein according to (1), wherein the polynucleotide according to (4) or a part thereof is used.

(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 changes the expression level of the G protein-coupled receptor Yuichi protein according to (1), which comprises using the quantification method according to (24);

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

(29) The above (1) which can be obtained by using the screening method described in the above (27). A compound or a salt thereof that alters the expression level of the described G protein-coupled receptor protein;

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

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

(33) The above-mentioned (17), wherein the test compound is brought into contact with the G protein-coupled receptor protein or the salt thereof described in the above (1) or the partial peptide or the salt thereof described in the above (3). The method of determining the described ligand,

(34) (i) contacting a ligand with a G protein-coupled receptor protein or a salt thereof described in (1) above or a partial peptide or a salt thereof described in (3) above, and (ii) ) A comparison is made between the case where the G protein-coupled receptor described in the above (1) or the salt thereof or the partial peptide described in the above (3) or the salt thereof is brought into contact with the ligand and the test compound. The screening method according to the above (18), wherein

(35) (i) contacting the labeled ligand with the G protein-coupled receptor protein or its salt as described in (1) above, or the partial peptide or its salt as described in (3) above; and (ii) When the labeled ligand and test compound are brought into contact with the G protein-coupled receptor protein described in (1) above or its salt or the partial peptide described in (3) or its salt, the labeled ligand described in (1) above The amount of binding to the G protein-coupled receptor protein or its salt or the partial peptide or its salt as described in (3) above is measured and compared with the ligand and the G protein as described in (1) above. Type receptor-a compound or its salt that alters the binding to the protein or its salt Cleaning method,

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

(37) (i) when the labeled ligand is brought into contact with the membrane fraction of cells containing the G protein-coupled receptor protein described in (1) above, and (ii) when the labeled ligand and test compound are The amount of the labeled ligand bound to the membrane fraction of the cell when the membrane is contacted with the membrane fraction of the cell containing the G protein-coupled receptor protein described in (1) above is measured and compared. A method for screening a compound or a salt thereof that alters the binding property between the characteristic ligand and the G protein-coupled receptor protein or a salt thereof according to the above (1);

(38) (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); (Ii) when the labeled ligand and the test compound are 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, Binding between the labeled ligand and the G protein-coupled receptor protein or its salt according to (1), wherein the amount of binding to the G protein-coupled receptor protein is measured and compared. A method of screening a compound or a salt thereof that changes the property,

(39) (i) A compound that activates the G protein-coupled receptor protein described in (1) or a salt thereof is contacted with a cell containing the G protein-coupled receptor protein described in (1). And (ii) a compound that activates the G protein-coupled receptor protein or its salt described in (1) above and a test compound, which contains the G protein-coupled receptor protein described in (1) above. A ligand characterized by measuring and comparing cell stimulating activity mediated by a G protein-coupled receptor protein when contacted with a cell and a G protein-coupled receptor protein described in (1) above. Or a method of screening for a compound or a salt thereof that changes the binding property to a salt thereof,

(40) A compound which activates the G protein-coupled receptor protein or a salt thereof described in (1) above, which is expressed in the cell membrane of the transformant by culturing the transformant according to (8). A compound that activates the G protein-coupled receptor protein or its salt described in (1) above and a test compound that is contacted with the protein-coupled receptor protein or the transformant described in (8). Measuring the cell-stimulating activity via the G protein-coupled receptor protein when the transformant is brought into contact with a G protein-coupled receptor protein expressed on the cell membrane of the transformant, and comparing the measured results. A compound that alters the binding property of the characteristic ligand to the G protein-coupled receptor protein or its salt according to (1), or The screening method of the salt,

(41) The compound that activates the G protein-coupled receptor protein described in (1) above is angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, Purin, vasopleucine, oxytocin, PACAP (e.g., PACAP27, PAC ΑΡ 38), secretin, glucagon, calcitonin, adrenomedullin, somatos, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intesty) Nal polypeptide), somatostin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene, pancreatastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine insulin Family (eg, CXC chemokines such as IL-8, GROa, GRO / 3, GROr, NAP-2, ENA-78, GCP-2, PF4, IP-10, Mig, PBS FZS DF-1 Subfamily; MCAFZMCP—1, MCP—2, MCP—3, MCP—4, eotaxin, RANTE S, MIP—1α, MIP—1 / 3, HCC—1, MIP-3α / LARC, MIP -3 3 / ELC, I-309, TARC, MI PF—l, MI PF-2 / eot ax in-2, MDC, DC—CC chemokine subfamily such as CK1 / P ARC, SLC; l C chemokine subfamily such as ympho tactin; fr ac talkine etc. CX3C chemokine subfamily, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA) or sphingosine 1-phosphate (39) Or the screening method according to (40) above,

(42) The binding between the ligand obtainable by the screening method according to any one of (34) to (41) and the G protein-coupled receptor protein or its salt according to (1). A compound to be changed or a salt thereof,

(43) The binding between the ligand obtainable by the screening method according to any of the above (34) to (41) and the G protein-coupled receptor protein or its salt according to the above (1). A medicament characterized by containing a compound to be changed or a salt thereof,

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

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

(46) The method according to (19), wherein the transformant according to (8) contains a G protein-coupled receptor protein expressed in the cell membrane of the transformant by culturing the transformant. Screening kit,

(47) The ligand, which can be obtained by using the screening kit according to any one of the above (44) to (46), and the G protein-coupled receptor Yuichi protein or a salt thereof according to the above (1) A compound that changes the binding property or a salt thereof,

(48) Binding between the ligand and the G protein-coupled receptor protein or the salt thereof according to (1), which can be obtained by using the screening kit according to any one of (44) to (46). A medicament comprising a compound or a salt thereof, (49) 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 according to (3) or a salt thereof. A method for quantifying a G protein-coupled receptor protein or a partial peptide or a salt thereof according to (3) above,

(50) Competition between the antibody described in (12) above and a test solution and the labeled G protein-coupled receptor protein described in (1) above or the partial peptide described in (3) above or a salt thereof And the above labeled antibody bound to the antibody

(1) The G protein-coupled receptor protein according to (1) in a test solution, wherein the ratio of the G protein-coupled receptor protein or the partial peptide or the salt thereof according to (3) is measured. Or the method for quantifying a partial peptide or a salt thereof according to (3) above,

(51) After reacting the test solution with the antibody of (12) insolubilized on the carrier and the labeled antibody of (12) simultaneously or continuously, the labeling agent on the insolubilized carrier is reacted. A method for quantifying the G protein-coupled receptor protein or the partial peptide or the salt thereof according to the above (1) in a test liquid, wherein the activity of the protein is measured.

(52) The above-mentioned agent which is a prophylactic and / or therapeutic agent for central disease, endocrine disease, metabolic disease, cancer, inflammatory disease, cardiovascular disease, respiratory disease, digestive system disease, immune system disease or infectious disease. 21) The pharmaceutical according to (31) or (32),

(53) Central disease, endocrine disease, metabolic disease, cancer characterized by administering to a mammal an effective amount of the compound according to (20), (29) or (30) or a salt thereof. , Inflammatory diseases, cardiovascular diseases, respiratory diseases, gastrointestinal diseases, immune system diseases or infectious disease prevention and / or treatment methods,

(54) To manufacture a preventive and / or therapeutic agent for a central disease, endocrine disease, metabolic disease, cancer, inflammatory disease, circulatory system disease, respiratory system disease, digestive system disease, immune system disease or infectious disease. The use of the compound according to the above (20), (29) or (30) or a salt thereof is provided. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a hydrophobicity plot of TGR 20-1.

FIG. 2 is a hydrophobicity plot of TGR20-2.

FIG. 3 is a diagram showing the amino acid sequence of TGR20-11 in one-letter code. FIG. 4 is a diagram showing the amino acid sequence of TGR20-12 in one-letter code. FIG. 5 is a diagram showing the distribution of TGR20 expression tissues. BEST MODE FOR CARRYING OUT THE INVENTION

The G protein-coupled receptor protein of the present invention (hereinafter sometimes abbreviated as receptor protein) may be the same as or similar to the amino acid sequence represented by SEQ ID NO: 1 or 5 (FIGS. 2 and 4). It is a receptor protein containing a substantially identical amino acid sequence.

The receptor protein of the present invention can be used, for example, in any cells (eg, spleen cells, nerves, etc.) of mammals (eg, humans, guinea pigs, rats, mice, rabbits, bushes, sheep, horses, monkeys, etc.). Cells, glial cells, knee / 3 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 , Mammary cells, hepatocytes or stromal cells, or their precursors, stem cells or cancer cells, etc.) or cells of the blood system (eg, leukocytes, erythrocytes), or those cells Any tissue present, for example, the brain, various parts of the brain (eg, olfactory bulb, nucleus pulposus, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus, cerebral cortex, medulla, cerebellum, cerebellum, occipital lobe, frontal lobe, Temporal lobe, putamen, caudate nucleus, brain stain, substantia nigra), spinal cord, pituitary, stomach, knee, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (Eg, large intestine, small intestine), proteins derived from blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testes, testes, ovaries, placenta, uterus, bones, joints, skeletal muscle, etc. Or a synthetic protein.

The amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 or 5 includes, for example, about 50% of the amino acid sequence represented by SEQ ID NO: 1 or 5 Or more, preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more. And the like.

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

Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 5 of the present invention include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 5 A protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 5 is preferred.

Examples of substantially the same activity include a ligand binding activity and a signal transduction activity. Substantially the same means that their activities are the same in nature. Therefore, the 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 20 times). However, the quantitative factors such as the degree of activity 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 activity can be measured according to a ligand determination method described later, ie, a screening method.

The receptor protein of the present invention includes: 1) one or more or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 or 5 (preferably, about 1 to 30, more preferably 1 to 1); An amino acid sequence in which about 0, more preferably several (1 to 5) amino acids have been deleted, (2) 1 or 2 or more (preferably 1 to 3) in the amino acid sequence represented by SEQ ID NO: 1 or 5 About 0 amino acids, more preferably about 1 to 10 amino acids, still more preferably several (1 to 5) amino acids; ③ an amino acid sequence represented by SEQ ID NO: 1 or 5 Or an amino acid sequence in which two or more (preferably about 1 to 30, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids have been substituted with other amino acids , Or タンパク質 Proteins containing an amino acid sequence obtained by combining them are also used. In the present specification, the receptor protein has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) 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 or 5, has a carboxyl group (1-COOH), a carboxylate (- COO—), amide (—CONH ₂ ) or ester (one COOR).

Here, as R in the ester, e.g., methyl, Echiru, n- propyl, alkyl groups such as isopropyl or n _ butyl, for example, ₃ cyclopentyl Le, cyclohexane, etc. cyclohexyl - ₈ cycloalkyl group, for example, phenyl, alpha - C ₆ _ ₁₂ Ariru groups such as naphthyl, for example, benzyl, phenylene Lou C Bok ₂ alkyl groups Moshikuwahi such phenethyl - C ₇ _ ₁₄ Ararukiru groups such as α- Nafuchiru C Bok ₂ alkyl group such as naphthylmethyl In addition, a bivaloyloxymethyl group commonly used as an oral ester is used.

When the receptor protein of the present invention has a carboxyl group (or carboxylate) at a position other than the C-terminal, 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 and the like are used.

Furthermore, the receptions evening one protein of the present invention, C Hi Ashiru groups in the protein described above, Amino group protecting groups Mechionin residues of N-terminal (e.g., formyl group, etc. which C ₂ _ ₆ Arukanoiru group of Asechiru ), Those whose N-terminal is cleaved in vivo and whose daltamyl group is pyroglutamine-oxidized, those on the side chains of amino acids in the molecule (eg, 1H, --SH, those amino group, imidazole group, indole group, etc. Guanijino group) is protected with a suitable protecting group (e.g., formyl group, etc. Ashiru groups such as C ₂ _ ₆ Arukanoiru group such Asechiru), or a sugar It also includes complex proteins such as so-called glycoproteins with linked chains.

Specific examples of the receptor protein of the present invention include, for example, a human testis-derived receptor protein TGR20 containing the amino acid sequence represented by SEQ ID NO: 1. 1, a human testis-derived receptor protein having an amino acid sequence represented by SEQ ID NO: 5, such as TGR20_2, is used.

The partial peptide of the receptor protein of the present invention (hereinafter, sometimes abbreviated as a 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 receptor binding activity may be used.

Specifically, as a partial peptide of the receptor Yuichi protein having the amino acid sequence represented by SEQ ID NO: 1 or 5, it is considered that it is an extracellular region (hydrophilic (Hydrophi 1ic) site) in the hydrophobicity plot analysis. The peptide containing the analyzed part. 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 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. Peptides having an amino acid sequence are preferred. A substantially identical amino acid sequence refers to an amino acid sequence of about 50% or more, preferably about 60% or more, more preferably about 70% or more, further preferably about 80% or more, and particularly preferably Represents an amino acid sequence having about 90% or more, most preferably about 95% or more homology.

Here, “substantially the same activity” has the same meaning as described above. The “substantially equivalent activity” can be measured in the same manner as described above.

In addition, the partial peptide of the present invention has one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence deleted. Or 1 or 2 or more (preferably, about 1 to 20; more preferably, about 1 to 10; more preferably, several (1 to 5)) amino acids are added to the amino acid sequence. Or 1 or 2 or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the amino acid sequence are replaced with other amino acids. It may be. The C-terminal of the partial peptide of the present invention may be any of a carboxyl group (one COOH), a carboxylate (—COO—), an amide (one C〇NH ₂ ) and an ester (one C◦OR). .

Further, similarly to the above-described receptor protein of the present invention, the partial peptide of the present invention has an N-terminal methionine residue in which the amino group of the methionine residue is protected by a protecting group, and is formed by cleavage of the N-terminal side in vivo. Examples include those in which Gin is pyroglutamine-oxidized, those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, and those in which a sugar chain is bonded, such as a so-called glycopeptide.

The C-terminal of the partial peptide of the present invention may be a carboxyl group (one COOH carboxylate (—C〇0-), an amide (one CO NH ₂ ), or an ester (—COOR).

Examples of the salt of the receptor protein or its partial peptide of the present invention include a physiologically acceptable salt with an acid or a base, and particularly preferably a physiologically acceptable acid addition salt. 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, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

The receptor protein of the present invention or a salt thereof can be produced from the above-mentioned mammalian cell or tissue by a known method for purifying the receptor protein, or encodes the receptor protein of the present invention described later. It can also be produced by culturing a transformant containing the DNA of interest. Also, the protein can be produced by the protein synthesis method described later or according to the method.

When producing from mammalian tissues or cells, homogenize the mammalian tissues or cells, extract with acids, etc., and combine the extract with chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.

For synthesis 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. You. Such resins include, for example, chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM Resin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ', 4'-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2', 4'-dimethoxyphenyl Two-way Fmoc aminoethyl) phenoxy resin. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods. At the end of the reaction, the protein is cleaved from the resin, and at the same time, various protecting groups are removed. Further, an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the target protein or its amide.

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 carbodiimides include DCC, N, N'-diisopropylcarbodiimide, N-ethylN '-(3-dimethylaminoprolyl) carbodiimide, and the like. For these activations, the protected amino acid may be added directly to the resin with a racemization inhibitor additive (eg, HOBt, HOOBt) or pre-protected as a symmetric anhydride or HOBt ester or HOOBt ester. The amino acid can be added to the resin after activation.

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-methylvinylidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol , Sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetate nitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof. Are 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 12 O: to 50. Activated key The amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, when 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, tertiary pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarponyl, C-Z, Br-Z, a Damantyloxycarponyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.

The lipoxyl group may be, for example, alkyl esterified (for example, methyl, ethyl, propyl, butyl, tert-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.). Or cyclic alkyl esterification), aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-methyl benzyl ester, benzhydryl esterification), phenacyl esterification, It can be protected by benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide and the like.

The hydroxyl group of serine can be protected, for example, by esterification or etherification. As a group suitable for the 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 benzyloxycarbonyl group and an ethoxycarbonyl group, and the like are used. Examples of groups suitable for etherification include a benzyl group, a tetrahydrovinyl group, a t-butyl group, and the like.

The protecting group of the phenolic hydroxyl group of tyrosine, for _{example, B zl, C l 2 -} B zl, 2- two Torobenjiru, B r- Z, such as tertiary butyl is used.

As the imidazole protecting group for histidine, for example, Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used. Examples of activated carboxyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4- Dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and ester with H〇Bt). As the activated amifu group of the raw material, for example, a corresponding phosphoric amide is used.

Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, or the like. Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia are also used. . The elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about 120 to about 40. In the acid treatment, for example, anisol, phenol, thioanisole, methacresol, paracresol, dimethyl Addition of cation capture agents such as sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, etc. is effective. In addition, the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tributophan is the above 1,2-ethanedithiol, 1,4. In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.

The protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.

As another method for obtaining an amide form of a protein, for example, first, after amidating and protecting one of the carboxyl groups of the carboxy terminal amino acid, a peptide (protein) chain is added to the amino group side to a desired chain length. After elongation, the protein except for the α-amino group protecting group at the N-terminus of the peptide chain and the protecting group at the C-terminal carbonyl group A protein from which only the protein has been removed is produced, and both proteins are condensed in a mixed solvent as described above. Details of the condensation reaction are the same 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 by using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.

To obtain an ester of a protein, for example, after condensing the α_ carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the ester of the desired protein can be obtained in the same manner as the protein amide. Can be obtained.

The partial peptide of the protein of the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptidase. As a method for synthesizing a peptide, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion, and if the product has a protecting group, removing the protecting group. . Known methods for condensation and elimination of protecting groups include, for example, the methods described in the following ① to ⑤.

(DM. Bodanszky and M.A. Ondet ti, Peptide Synthes is, Interscience Publ ishers, 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, Biochemistry Laboratory Course 1, Protein Chemistry IV, 205, (1977)

治 Supervised by Haruaki Yajima, Development of Continuing Drugs Volume 14 Peptide Synthesis Hirokawa Shoten

After the reaction, the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. The partial peptide obtained by the above method is a free form In some cases, it can be converted to an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted to a free form by a known method.

The polynucleotide encoding the receptor protein of the present invention may be any polynucleotide as long as it contains the above-described nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention. Is also good. The polynucleotide is a DNA encoding the receptor protein of the present invention, RNA such as mRNA, and 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. If single stranded, it may be the sense strand (ie, the coding strand) or the antisense strand (ie, the non-coding strand).

Using the polynucleotide encoding the receptor protein of the present invention, for example, a method described in the known experimental medicine special edition "New PCR and its application" 15 (7), 1997 or a method analogous thereto Thus, the mRNA of the receptor protein of the present invention can be quantified.

The DNA encoding the receptor protein of the present invention may be any of genomic DNA, a genomic DNA library, the above-described cDNA derived from cells and tissues, the above-described cDNA library derived from cells and tissues, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells and tissues described above.

Specifically, the DNA encoding the receptor protein of the present invention is, for example, a DNA containing the base sequence represented by SEQ ID NO: 2 or 6, or the base represented by SEQ ID NO: 2 or 6. It has DNA that hybridizes under high stringent conditions to DNA having a sequence, and has substantially the same activity (eg, ligand binding activity, signal transduction activity, etc.) as the receptor protein of the present invention. Any DNA may be used as long as it encodes a receptor protein having the DNA. It is highly stringent with DNA having the nucleotide sequence represented by SEQ ID NO: 2. The DNA that hybridizes under the conditions is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with the base sequence represented by SEQ ID NO: 2. For example, a DNA containing a nucleotide sequence having homology of

Examples of the DNA that hybridizes with the DNA having the nucleotide sequence of SEQ ID NO: 6 under high stringent conditions include, for example, about 70% or more, preferably about 70% or more of the nucleotide sequence of SEQ ID NO: 6 A DNA containing a nucleotide sequence having a homology of 80% or more, more preferably about 90% or more, and most preferably about 95% or more is used.

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

The high stringent conditions include, for example, a sodium concentration of about 19 to 40 mM, preferably about 19 to 2 OmM, and a temperature of about 50 to 70, preferably about 60 to 65 :. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 is most preferable.

More specifically, as the DNA encoding the receptor protein TGR 20-1 containing the amino acid sequence represented by SEQ ID NO: 1, a DNA containing the base sequence represented by SEQ ID NO: 2, As the DNA encoding the receptor protein TGR 201-11 having the amino acid sequence represented by SEQ ID NO: 5, a DNA having the base sequence represented by SEQ ID NO: 6 and the like are used.

A part of the nucleotide sequence of the DNA encoding the receptor protein of the present invention, or a polynucleotide containing a part of the nucleotide sequence complementary to the DNA, is a polynucleotide encoding the following partial peptide of the present invention. Not only does it encompass DNA, but also RNA.

According to the present invention, G protein-coupled receptor protein protein replication or Designs and synthesizes antisense polynucleotides (nucleic acids) that can inhibit expression based on cloned or determined DNA sequence information of the DNA encoding the G protein-coupled receptor protein. Can. Such a polynucleotide (nucleic acid) can hybridize to RNA of a G protein-coupled receptor protein gene and can inhibit the synthesis or function of the RNA, or can inhibit the synthesis or function of G protein-coupled receptor protein. G protein-coupled receptor protein gene expression can be regulated and controlled through interaction with quality-related RNA. Polynucleotides that are complementary to a selected sequence of G protein-coupled receptor protein-related RNA and that can specifically hybridize with G protein-coupled receptor protein-associated RNA are It is useful for regulating and controlling the expression of G protein-coupled receptor protein gene in vitro and in vitro, and is also useful for treating or diagnosing diseases and the like. The term "corresponding" means having homology or being complementary to a particular sequence of nucleotides, base sequences or nucleic acids, including genes. “Corresponding” between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) in the instructions derived from the nucleotide (nucleic acid) sequence or its complement. ing. 5'-end hairpin loop, 5'-end 6—base-ba-repeat, 5'-end untranslated region, 5'-end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation termination Codons, 3 'untranslated region, 3' palindrome region, and 3 'hairpin loop may be selected as preferred regions of interest, but any region within the G protein coupled conjugate receptor protein Can be selected as

The relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region or the relationship between the target nucleic acid and a polynucleotide that can hybridize with the target can be said to be “antisense”. Antisense polynucleotides include polynucleotides containing 2-dexoxy-D-ribose, polynucleotides containing D-ribose, and other types of polynucleotides that are N-glycosides of purine or pyrimidine bases. Nucleotide or non-nucleotide backbone Other polymers that have a specific bond (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers that contain special bonds (provided that the polymer is a base polymer such as that found in DNA or RNA). Pairing (contains a nucleotide having a configuration permitting attachment of a base)). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can further comprise unmodified polynucleotides (or unmodified oligonucleotides). Nucleotides), as well as those with known modifications, eg, labeled, capped, methylated, or one or more natural nucleotides replaced with analogs, as known in the art , Modified with an intramolecular nucleotide, such as those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphorothioate) , Such as proteins (nucleases, nucleases, inhibitors, etc.) Has side-chain groups such as syn, antibody, signal peptide, poly-L-lysine, etc. and sugars (eg, monosaccharides), and has an inter-current compound (eg, acridine, psoralen, etc.) , Containing chelating compounds (eg, metals, radioactive metals, boron, oxidizable metals, etc.), those containing alkylating agents, those with modified bonds (eg, Nucleic acid). Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functionalities such as ethers, amines, etc. It may be converted to a group.

The antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA, or a modified nucleic acid (RNA, DNA). Specific examples of modified nucleic acids include sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides. And those that are resistant to degradation of oligonucleoside amides, but are not limited thereto. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to have a greater affinity for the target sense strand, and to be more toxic if it is toxic. Make sense nucleic acid less toxic.

Thus, many modifications are known in the art, for example, J. Kawakami et al., Phami Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed. , Ant isense Research and Applicat ions, CRC Press, 1993.

The antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes or microspheres, may be applied by gene therapy, It could be given in additional form. Thus, additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes or increase nucleic acid uptake. For example, hydrophobic substances such as phospholipid and cholesterol) can be mentioned. Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). Such a substance can be attached to the 3 'end or 5' end of a nucleic acid, and can be attached via a base, a sugar, or an intramolecular nucleoside bond. Other groups include cap groups specifically arranged at the 3 'end or the 5' end of nucleic acids, which prevent degradation by nucleases such as exonuclease and RNAse. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene dalicol.

The antisense nucleic acid inhibitory activity is 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 the G protein-coupled receptor protein. be able to. The cells may be prepared by various methods known in the art. Applicable to

The DNA encoding the partial peptide of the present invention may be any DNA containing the above-described nucleotide sequence encoding the partial peptide of the present invention. Also, genomic DNA, genomic DNA Any of a library, a cDNA derived from the above-described cells and tissues, a cDNA library derived from the above-described cells and tissues, and a synthetic DNA may be used. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using an 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) DNA having a partial base sequence of DNA having the base sequence represented by SEQ ID NO: 2 or 6, or (2) sequence It has a DNA that hybridizes under high stringent conditions with DNA having the nucleotide sequence represented by No. 2 or 6, and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, For example, DNA having a partial base sequence of DNA encoding a receptor protein having a signal transduction effect having a signal signaling function is used.

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, most preferably at least about 95% or more is used. Examples of the DNA that hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 6 under high stringent conditions include, for example, about 70% or more, preferably about 80% or more of the nucleotide sequence represented by SEQ ID NO: 6 DNA having a nucleotide sequence having homology of about 90% or more, most preferably about 95% or more is used.

Cloning of the DNA completely encoding the receptor protein of the present invention or a partial peptide thereof (hereinafter, sometimes abbreviated as the receptor protein of the present invention) includes the receptor protein of the present invention. Bases of DNA to encode Either amplify by PCR using a synthetic DNA primer having a partial base sequence of the sequence or incorporate DNA incorporated into an appropriate vector into part or all of the receptor protein of the present invention. Selection can be performed by hybridization with a DNA fragment to be coded or labeled with a synthetic DNA. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, the procedure can be performed according to the method described in the attached instruction manual.

The DNA base sequence can be converted using PCR or a known kit such as Mutan ^™ -super Express Km (Takara Shuzo Co., Ltd.) or Mutan ™ -K (Takara Shuzo Co., Ltd.) using the ODA-LAPCR method or the Gapped method. It can be carried out according to a known method such as the duplex method or the Kunkel method 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 by digesting with a restriction enzyme or adding a linker if desired. The DNA may have ATG as a translation initiation codon at its 5 'end, and TAA, at the end of translation, or at its 3' end at the 3 'end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter.

The expression vector of the receptor protein of the present invention may be prepared, for example, by (a) cutting out a DNA fragment of interest from DNA encoding the receptor protein of the present invention, and (mouth) converting the DNA fragment into an appropriate expression vector. It can be produced by ligating downstream of the promoter.

Examples of the vector include E. coli-derived plasmids (eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13), and Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194). ), Yeast-derived plasmids (eg, pSH19, pSH15), bacteriophage such as λ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., as well as ρAlll, pXTl, pRc / CMV, pRc / RSV, pc DNA I / Neo, etc. are used. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. For example, when animal cells are used as host, SRa Promoter, SV40 promoter, LTR motor, CMV Promoter, HSV-TK promoter and the like can be mentioned. Of these, it is preferable to use the CMV promoter, the SR α promoter, and the like. When the host is Eshierihia genus bacterium, trp promoter one, lac flop port motor, re cA promoter evening one, AP _L promoter and foremost, etc. l pp promoter Isseki one is, when the host is Bacillus, When the host is yeast, such as SP〇1 promoter, SP〇2 promoter, pen P promoter, etc., PH05 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.

In addition to the above, an expression vector containing, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, and an SV40 replication origin (hereinafter sometimes abbreviated as SV40 ori) may be used. Can be used. Examples of selectable markers include a dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], an ampicillin resistance gene (hereinafter sometimes abbreviated as Amp), Neomycin resistance gene (hereinafter sometimes abbreviated as Neo, G418 resistance), etc. In particular, when the dh fr gene is used as a selection marker using CHO (dh fr ") cells, the target gene Can also be selected with a thymidine-free medium.

If necessary, a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention. When the host is a genus Escherichia, the PhoA-signal sequence, a 0-A signal sequence, etc., when the host is a Bacillus genus, an α-amylase signal sequence, a subtilisin, a signal sequence, etc. If the host is an animal cell, such as MF, signal sequence, SUC2, signal sequence, etc. Etc. can be used respectively.

DNA encoding the receptor protein of the present invention thus constructed A transformant can be produced using a vector containing

As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.

Specific examples of the genus Escherichia include Escherichia coli K12, DH1 [Processing's of the 'National Academy of Sciences of the United States (Proc. Natl. Acad. Sci. USA), Vol. 60, 160 (1968)], JM103 [Nucleic Acids Research, (Nucleic Acids Research), Vol. 9, 309 (198 1)], J J221 [Journalo Journal of Molecular Biology], 120, 517 (1978)], HB 101 [Journal of Molecular Biology, 41, 459 (1969)], C 600 [ Genetics

(Genetics), 39, 440 (1954)], DH5a [Inoue, H., Nojima, H. and Okayama, H., Gene, 96, 23-28 (1990)], DH10B [ Proc.'S of 'The' National 'Academy.' 'Sciences' of the USA (Proc. Natl. Acad. Sci. USA), Vol. 87, 4645-4649 (1 990)] Can be.

Examples of Bacillus bacteria include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95 Vol. 87 (1 984)].

Examples of yeast include, for example, Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC 1913, NCYC2036 , Pichia pastoris, etc. are used.

As insect cells, for example, when the virus is AcNPV, a cell line derived from a larva of Spodoptera (Spodoptera frugiperda cell; Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, High Five ™ derived from egg of Trichoplusia ni Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. Wooly When the virus is BmNP V, a cell line derived from silkworm (Bombyxmori N; BmN cell) or the like is used. Examples of the Sf cell include Sf9 cell (ATCC CRL1711) and Sf21 cell (Vaughn, Jugh et al., In Vivo, 13, 213-217, (1977)) Are used.

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

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

In order to transform a microorganism belonging to the genus Escherichia, see, for example, Processings of the National Academy of Sciences, Proc. Natl. Acad. Sci. USA, 69. Vol., 211 (1972) and Gene, Vol. 17, 107 (1982).

Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 11 (1799).

To transform yeast, see, for example, Methods in Enzymology, 194, 182–187 (1991), Processing's of the National Academy of Sciences. Natl. Acad. Sci. USA, Vol. 75, 1929 (1978).

Insect cells or insects can be transformed, for example, by the method described in Bio / Technology, 6, 47-55 (1988).

Transformation of animal cells is described, for example, in Cell Engineering Separate Volume 8, New Cell Engineering Experimental Protocol. Can be performed according to the method described in Thus, the DN encoding the G protein-coupled receptor Yuichi protein

A transformant transformed with the expression vector containing A is obtained.

When culturing a transformant whose host is a bacterium belonging to the genus Escherichia or Bacillus, a liquid medium is suitable as a medium to be used for culturing, and a carbon source necessary for the growth of the transformant is contained therein. , Nitrogen sources, inorganic substances and the like. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn chip liquor, peptone, potato zein, meat extract, soybean meal, and potato extract. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.

Examples of a medium for culturing the genus Escherichia include, for example, an M9 medium containing glucose and casamino acids (Miller, Journal of Experiments, Molecular Genetics, Journal of Experiments in Molecular Genetics). ), 431-433, Cold Spring Harbor Laboratory, New York 19

72] is preferred. Here, if necessary, a drug such as 3 / 3-indolyl acrylic acid can be added to make the promotion work efficiently. When the host is a bacterium belonging to the genus Escherichia, cultivation is usually performed at about 15 to 43 for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.

When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 for about 6 to 24 hours. If necessary, aeration and stirring can be applied.

When culturing a transformant whose host is yeast, for example,

—Burkholder Minimal Medium [Bostian, KL et al., “Proc. Natl. Acad. Sci. USA ), 77, 4505 (1980)] or an SD medium containing 0.5% casamino acid [Bitter, GA et al., "Processings of the National Academy of Sciences" Natl. Acad. Sci. USA, 81, 5330 (1984)] The pH of the medium is preferably adjusted to about 5 to 8. Culture is usually performed at about 2 Ot: Perform at about 35 for about 24-72 hours, adding aeration and agitation as needed.

When culturing an insect cell or a transformant whose host is an insect, the medium used is 10% serum immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). And the like to which additives such as the above are appropriately added are used. Preferably, the ρΗ of the culture medium is adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 for about 3 to 5 days, and aeration and agitation are added as necessary.

When culturing a transformant in which the host is an animal cell, the medium may be, 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 (1992), 519 (1 967)] and 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] Used. Preferably, the pH is about 6-8. Culture is usually performed at about 30t: ~ 40 for about 15-60 hours, and aeration and agitation are added as necessary.

As described above, the G protein-coupled receptor overnight 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 can be separated and purified from the culture by, for example, the following method.

When extracting the receptor protein of the present invention from cultured cells or cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or After the cells or cells are destroyed by freeze-thawing or the like, a method of obtaining a crude extract of the receptor protein by centrifugation or filtration is used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 ™. If the receptor protein is secreted into the culture solution, after the culture is completed, the supernatant is separated from the cells or cells by a known method, and the supernatant is collected.

Receptor contained in the culture supernatant or extract obtained in this way The protein can be purified by appropriately combining known separation and purification methods. These known separation and purification methods mainly include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, and the like. Methods that use differences in charge, methods that use charge differences such as ion-exchange chromatography, methods that use specific novelty such as affinity chromatography, and methods that use reverse-phase high-performance liquid chromatography. Methods using the difference in hydrophobicity, methods using the difference in isoelectric point such as isoelectric focusing, and the like are used.

When the receptor protein thus obtained is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when it is obtained in a salt, a known method Alternatively, it can be converted into a free form or another salt by a method analogous thereto.

Receptor protein produced by the recombinant can be optionally modified or partially removed by treating it with an appropriate protein modifying enzyme before or after purification. it can. 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 produced can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.

An antibody against the receptor protein of the present invention or its partial peptide or a salt thereof may be a polyclonal antibody as long as it can recognize the receptor protein of the present invention or its partial peptide or a salt thereof. Any of an antibody and a monoclonal antibody may be used.

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

[Preparation of monoclonal antibody] (a) Preparation of monoclonal antibody-producing cells

The receptor protein or the like of the present invention is administered to a mammal at a site capable of producing an antibody by administration, itself or together with a carrier or a diluent. Upon administration, Complete Freund's adjuvant / Incomplete Freund's adjuvant may be administered to enhance antibody production ability. The 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, goats, and mice and rats are preferably used.

When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with an antigen, for example, an individual with an antibody titer from a mouse, and collect the spleen or lymph node 2 to 5 days after the final immunization By fusing antibody-producing cells contained therein with myeloma cells, monoclonal antibody-producing hybridomas can be prepared. The antibody titer in the antiserum can be measured, for example, by reacting the antiserum with a labeled receptor protein or the like described later, and then measuring the activity of a labeling agent bound to the antibody. The fusion operation can be carried out according to a known method, for example, the method of Koehler and Mills [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.

Examples of myeloma cells include NS-1, P3U1, SP2Z0, and the like, with P3U1 being preferred. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and the PEG (preferably, PEG1000 to PEG6000) power is about 10 to 80%. Cell fusion can be carried out efficiently by incubating at about 20 to 40, preferably about 30 to 37 for about 1 to 10 minutes.

Various methods can be used to screen the monoclonal antibody-producing hybridomas. For example, the hybridomas may be attached to a solid phase (eg, a microplate) on which an antigen such as receptor protein is adsorbed directly or together with a carrier. The culture supernatant is added, and then an anti-immunoglobulin antibody (labeled for cell fusion) If the cells to be treated are mice, an anti-mouse immunoglobulin antibody is used.) Or a method for detecting the monoclonal antibody bound to the solid phase by adding tin A, A method of adding a hybridoma culture supernatant, adding a receptor protein or the like labeled with a radioactive substance, an enzyme, or the like, and detecting a monoclonal antibody bound to a solid phase can be used.

The selection of the monoclonal antibody can be carried out according to a known method or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added. As a selection and breeding medium, any medium can be used as long as it can grow a hybridoma. For example, RPMI 1640 medium containing 1 to 20%, preferably 10 to 20% fetal bovine serum, GIT medium containing 1 to 10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) Alternatively, a serum-free medium for hybridoma culture (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The cultivation temperature is usually from 20 to 40, preferably about 37. The culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks. The culture can be usually performed under 5% carbon dioxide. The antibody titer of the culture supernatant of the eight hybridomas can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

(b) Purification of monoclonal antibodies

Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies. [Examples: salting out, alcohol precipitation, isoelectric focusing, electrophoresis, ion exchangers (ex. , DEAE), ultracentrifugation, gel filtration, antigen-binding solid phase or specific antibody obtained by collecting only the antibody with an active adsorbent such as protein A or protein G, and dissociating the bond to obtain the antibody. Purification method].

(Preparation of polyclonal antibody)

The polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as the receptor protein of the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the method for producing a monoclonal antibody described above. Recept Yuichi By collecting antibody-containing substances against proteins, etc., and separating and purifying the antibodies. Can be manufactured.

Regarding the complex of immunizing antigen and carrier-1 protein used to immunize mammals, the type of carrier-1 protein and the mixing ratio between carrier and hapten depend on the efficiency of antibody against hapten immunized by cross-linking with carrier. If possible, any material may be cross-linked at any ratio.For example, serum serum albumin, thyroglobulin, keyhole limpet, hemocyanin, etc. may be used in a weight ratio of hapten. On the other hand, a method of coupling at a rate of about 0.1 to 20, preferably about 1 to 5 is used.

Further, various condensing agents can be used for force coupling between the hapten and the carrier. For example, an active ester reagent containing a daltaraldehyde, a carbodiimide, a maleimide active ester, a thiol group or a dithioviridyl group is used.

The condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible. Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration. The 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 from blood, ascites, etc., preferably from blood, of the mammal immunized by the above method.

The measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner 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 same immunoglobulin separation and purification method as the above-described separation and purification of the monoclonal antibody.

The receptor protein of the present invention or its salt, its partial peptide or its salt, and the DNA encoding the receptor protein or its partial peptide are: (1) a ligand (agonist) 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, (4) a receptor protein of the present invention or A method for screening a compound that changes the expression level of the partial peptide, (5) prevention of various diseases containing a compound that changes the expression level of the receptor protein of the present invention or the partial peptide thereof, and prevention of Z or Is a therapeutic agent; (6) a method for quantifying a ligand for the G protein-coupled receptor protein of the present invention; and (7) a compound that changes the binding property of the ligand to the G protein-coupled receptor protein of the present invention. (8) Compounds that alter the binding between the G protein-coupled receptor Yuichi protein and the ligand of the present invention (agonists, angel gonists) A prophylactic and / or therapeutic agent for various diseases comprising: (9) quantification of the receptor protein of the present invention or a partial peptide thereof or a salt thereof; (10) a receptor protein of the present invention in a cell membrane or a portion thereof A method for screening a compound that changes the amount of a peptide, (11) a receptor protein of the present invention or a partial peptide thereof in a cell membrane. A prophylactic and / or therapeutic agent for various diseases containing a compound that alters the amount of (12) neutralization by an antibody against the receptor protein of the present invention or its partial peptide or a salt thereof, (13) the present invention Can be used to create a non-human animal having a DNA encoding the G protein-coupled receptor protein.

In particular, by using the receptor-binding Atsei system using the recombinant G protein-coupled receptor protein expression system of the present invention, the ligand binding property to a mammal-specific G protein-coupled receptor is changed. Compounds (eg, agonist, angelic gonist, etc.) can be screened, and the agonist or angelic gonist can be used as an agent for preventing and / or treating various diseases.

A receptor protein or a partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as a receptor protein of the present invention, etc.), a DNA encoding a receptor protein of the present invention or a partial peptide thereof (hereinafter, referred to as a peptide). The use of an antibody against the receptor protein of the present invention or the like (hereinafter sometimes abbreviated as the antibody of the present invention) 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 overnight or its salt or the partial peptide of the present invention Alternatively, the salt thereof is useful as a reagent for searching for or determining a ligand (agonist) for the receptor protein of the present invention or a salt thereof.

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. Provide a way. Test compounds include known ligands (for example, angiotensin, bombesin, canapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasoprescin, oxotocin, ΡACAP (eg, PACAP 2 7, PACAP 3 8), secretin, glucagon, calcitonin, adrenomedullin, somatos, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Rerated Polypeptide), somatos Tin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene, pancreatastatin, prostaglandin, tropoxane, adenosine, adrenaline, chemokine superfamilyー (eg, IL-8, GROa, GR〇 / 3, GROr, NAP—2, ENA—78, GCP-2, PF4, IP-10, Mig, PBS F / SDF— 1 CXC chemokine subfamily such as; MCAF / MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, MIP-1 and MIP-1 / 3, HCC-1 and MIP-3 α / LARC, MI P-3 / 3 / ELC, I-309, TARC, MIPF-1, MIP F-2 / eotaxi n-2, MDC, DC-CK1 / P ARC, CC such as SLC Chemokine subfamily; C chemokine subfamily such as 1 ymp hotactin; CX3 C chemokine subfamily such as fracta 1 kine, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polyp. In addition to suidoid, galanin, lysophosphatidic acid (LPA), sphingosine monomonophosphate, etc., for example, mammalian Organization Distillate, such as cell culture supernatant is used. For example, the tissue extract, the cell culture supernatant, etc. are added to the receptor protein of the present invention, and the cell stimulating activity and the like are measured. Fractionation, and finally a single ligand can be obtained.

Specifically, the ligand determination method of the present invention uses the receptor protein of the present invention or a partial peptide thereof or a salt thereof, or constructs an expression system for a recombinant receptor protein, and By using the receptor binding system using the receptor system, it is possible to bind to the receptor protein of the present invention and to stimulate cell stimulation (for example, release of arachidonic acid, release of acetylcholine, release of intracellular Ca ^2+, release of intracellular c AMP generation, intracellular c GMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity to promote or suppress pH reduction, etc.) This is a method for determining a compound (eg, peptide, protein, non-peptide compound, synthetic compound, fermentation product, etc.) or a salt thereof.

In the ligand determination method of the present invention, when the test compound is brought into contact with the receptor protein of the present invention or its partial peptide, for example, the amount of the 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 the labeled test compound is brought into contact with the receptor protein of the present invention or its salt or the partial peptide of the present invention or its salt, the protein of the labeled test compound or its salt, 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 partial peptide or 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 a protein or a salt thereof according to the present invention,

(3) When the labeled test compound is brought into contact with the receptor protein expressed on the cell membrane by culturing a transformant containing the DNA encoding the receptor protein of the present invention, the labeled test compound The receptor according to the present invention, wherein the amount of binding to the receptor protein or a salt thereof is measured. Yuichiichi Method for determining ligand for protein,

細胞 Cell stimulating activity via receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ^{2+) when} a test compound is brought into contact with cells containing the receptor protein of the present invention. Activity or suppression that promotes release, intracellular CAMP generation, intracellular cGMP generation, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease in pH, etc. And a method for determining a ligand for the receptor protein of the present invention or a salt thereof, and

を When the test compound is brought into contact with the receptor protein expressed on the cell membrane by culturing a transformant containing DNA encoding the receptor protein of the present invention, the receptor protein is Cell stimulating activity (e.g., arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular CAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein Phosphorylation, activation of c-fos, activity of suppressing or reducing pH, etc.), and determination of ligands for the receptor protein or its salt according to the present invention. Provide a way.

In particular, it is preferable to carry out the above tests 1 to 3 after conducting the tests 1 to 3 above and confirming that the test compound binds to the receptor protein of the present invention. First, the receptor protein used in the ligand determination method may be any protein as long as it contains the above-described receptor protein of the present invention or the partial peptide of the present invention. A protein expressed in a large amount at night is suitable.

To produce the receptor protein of the present invention, the above-described expression method is used. It is preferable that the DNA encoding the receptor protein is expressed in mammalian cells or insect cells. Complementary DNA is usually used as the DNA fragment encoding the protein portion of interest, but is not necessarily limited to this. 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 introduced into an insect host and used as a baculovirus. Polyhedros is virus (NPV) polyhedrin promoter, SV40-derived promoter, retroviral promoter — Yuichi, meta-mouth thionein promoter, human heat shock It is preferable to incorporate the gene into the downstream of a promoter, cytomegalovirus promoter, SRα promoter and the like. Examination of the amount and quality of the expressed receptor can be performed by a known method. For example, the method is performed according to the method described in the literature [Nambi, P. et al., The Journal of Biological 'Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992]. Can be.

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

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

The cells containing the receptor protein of the present invention include host cells expressing the receptor protein of the present invention. The host cells include Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like. Is used.

The cell membrane fraction refers to a cell membrane-rich fraction obtained by disrupting cells and then obtained by a known method. Cell crushing methods include a method of crushing cells with a Potter-Elvehj em-type homogenizer, Warinda Blender ゃ Polytron.

(Kinematica), crushing by ultrasonic waves, crushing by ejecting cells from a narrow nozzle while applying pressure with a French press, and the like. For cell membrane fractionation, centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used. For example, the cell lysate is centrifuged at a low speed (500 rpm to 300 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further spun at a higher speed (150 rpm to 300 rpm). The mixture is centrifuged at 0,000 rpm for 30 minutes to 2 hours, and the resulting precipitate is used as the membrane fraction. In the membrane fraction, the expressed receptor protein and cell-derived proteins were included. It is rich in membrane components such as phospholipids and membrane proteins.

The amount of receptor protein in the cells containing the receptor protein and the membrane fraction thereof is preferably 10 ³ to 10 ⁸ molecules per cell, more preferably 10 ⁵ to 10 ⁷ molecules per cell. Is preferred. 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 the measurement of a large number of samples in the same lot. become.

In order to carry out the above-mentioned methods (1) to (3) for determining the ligand for the receptor protein or its salt of the present invention, an appropriate receptor protein fraction and a labeled test compound are required.

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

Labeled test compounds include angiotensin, bombesin, canapinoid, cholecystokinin, dalyumin, serotonin, melatonin, labeled with [], [ ¹²⁵ I], ["c", [S], etc. Neuropeptide Y, opioids, purines, vasopressin, oxatocin, PACAP (e.g., PACAP 27, PACAP 38), secretin, glucagon, calcitonin, adrenomedullin, somato sutin, GHRH, CRF, ACTH, GRP, PTH, VIP ( Basoactive Intestinal and Retained Polypeptide), Somatos Retin, Dopamine, Motilin, Amylin, Bradykinin, CGRP (Calcitonin Gene Reited Peptide), Leukotriene, Pancreastatin, Prostaglandin, Thromboxan, Adenosine, Adrenaline , Chemokines family (eg, IL-8, GROa, GRO / 3, GROr, NAP-2, ENA-78, GCP-2, PF4, IP_10, Mig, PBSF / SDF-1 etc. Of the CXC chemokine subfamily; MCAFZMCP-1, MCP-2, MCP-3, MCP-4, eot ax in, RANTES, MIP-1 and MlP-1β, HCC-1 and ΜIΜ-3 α / LARC, Μ I Ρ-3 β / ELC, I-309, TARC, MI PF— 1, MI PF— 2 / eot ax in-2, MDC, CC-chemokine subfamily such as DC-CK 1 / PARC, SLC; C chemokine subfamily such as 1 ym photactin; CX3 C chemokine subfamily such as fracta 1 kine etc.), endothelin, enterogastrin, histamine, neuro Tensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), and sphingosine 1-phosphate are preferred.

Specifically, to carry out the method for determining a ligand for the receptor protein of the present invention or a salt thereof, a cell containing the receptor protein of the present invention or a membrane fraction of the cell is first suitable for the method for determination. Prepare a standard sample of the receptor by suspending in a buffer. The buffer may be any buffer such as a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8) or a buffer of tris-hydrochloric acid, which does not inhibit the binding between the ligand and the receptor protein. In order to reduce non-specific binding, various proteins such as detergents such as CHAP S, Tween-80 ™ (Kao Ichi Atlas Co., Ltd.), digitonin and dexcholate, and serum albumin and gelatin are buffered. Can also be added. Furthermore, a protease inhibitor such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and peptide suptin can be added for the purpose of suppressing the degradation of the receptor and the ligand by the protease. To 0.0 lm. 1 to 10 ml of the receptions evening over solution, a certain amount (50 00 c pm~500000 c pm) of ^[3 H], ^[125 1], [ "C] labeled with a ^[35 S] test Prepare a reaction tube containing a large excess of unlabeled test compound to determine the amount of non-specific binding (NSB) Reaction is about 0T: ~ 50, preferably about 4 and ~ 37 After the reaction, the reaction is filtered through a glass fiber filter paper, washed with an appropriate amount of the same buffer, and the radioactivity remaining in the glass fiber filter paper is removed. Measure with a liquid scintillation counter 1 or an counter 1. A test compound having a count (B-NSB), which is obtained by subtracting the non-specific binding amount (N SB) from the total binding amount (B), exceeding 0 cpm is used as a receptor according to the present invention. Select as a ligand for one protein or its salt can do.

The above for determining a ligand for the receptor protein of the present invention or a salt thereof In order to carry out the methods (1) to (4), cell stimulating activity via the receptor protein (for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, cell Or the activity of promoting or inhibiting intracellular C GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc.) It can be measured using a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured in a multiwell plate or the like. Before performing ligand determination, 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 remove supernatant. Collect and quantify the product produced according to each method. When the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult to be assayed by a degrading enzyme contained in a cell, an inhibitor for the degrading enzyme may be added to perform the assay. In addition, activities such as suppression of cAMP production can be detected for production suppression of cells whose basal production has been increased with forskolin or the like.

The kit for determining a ligand binding to the receptor protein of the present invention or a salt thereof contains the receptor protein of the present invention or a salt thereof, the partial peptide or a salt thereof of the present invention, and the receptor protein of the present invention. It contains a cell or a membrane fraction of a cell 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 serum albumin (manufactured by Sigma).

Sterilize by filtration through a 0.45 m pore size filter and store at 4 or prepare it before use.

② G protein protein conjugated receptor Yuichi protein preparation

The C HO cells expressing the receptor protein of the present invention, 1 2-well plates were passaged with 5 X 1 0 ⁵ cells / well, at 37, 2 days of culture at 5% C_〇 _2, 9 5% air did thing.

③ Labeled test compound

Commercially available compounds labeled with [], [ ¹²⁵¹ ], [ ¹⁴ c], [ ³⁵ s], or those labeled by an appropriate method

Store the solution in an aqueous solution at 4 or -2 Ot: and dilute to 1 M with the measuring buffer before use. Test compounds that are poorly soluble in water should be dissolved in dimethylformamide, DMSO, methanol, etc.

④Unlabeled test compound

The same as the labeled compound is prepared at a concentration 100 to 1000 times higher.

2. Measurement method

(1) Wash CHO 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 add 490 / ^ 1 measurement buffer to each well. Add.

2) Add 5/1 labeled test compound and react at room temperature for 1 hour. To determine the amount of non-specific binding, add 51 unlabeled test compounds.

(3) Remove the reaction solution and wash three times with lm1 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 overnight A (manufactured by Wako Pure Chemical Industries).

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

Examples of the ligand capable of binding to the receptor protein or a salt thereof of the present invention include substances specifically present in organs such as blood, brain, large intestine, spleen, spleen, and ovary, and the like. Specifically, angiotensin, bombesin, cana pinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasoprescin, oxaxin, PACAP (e.g., PACAP 27, PACAP 38) , Secretin, glucagon, calcitonin, adrenomedullin, somatos, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Reputed Polypeptide), somatos, dopamine, motilin, Amylin, Radikinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine family (eg, IL-8, GRO, GROj3, GROr, NAP) -2, ENA-78, GCP-2, PF4, IP-10, Mig, CXC chemokine subfamily, including PBSF / SDF-1; MCAF / MCP-1, MCP-2, MCP-3, MCP- 4, eot ax in, RANTES, MIP— 1 H, MIP-1 / 3, HCC-1, MI P-3 a / LA RC, MI P-3 β / ELC, 1—309, TARC, MI PF— 1 , MI PF-2 / eotaxi n-2, CC chemokine subfamily such as MDC, DC-CK1 / PARC, SLC; 1 C chemokine subfamily such as ymp hotactin; CX 3 C chemokine subfamily such as fracta 1 kine Etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide Galanin, lysophosphatidic acid (LPA), including Sufuingoshin 1 monophosphate is used.

(2) A preventive and / or therapeutic agent for a disease associated with dysfunction of the G protein-coupled receptor Yuichi protein of the present invention

In the above method (1), if the ligand for the receptor protein of the present invention is identified, the receptor protein of the present invention or the DNA encoding the receptor protein can be prepared according to the action of the ligand. It can be used as a medicament such as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention.

For example, when there is a patient who cannot expect the physiological action of the ligand because the receptor protein of the present invention is reduced in the living body (the deficiency of the receptor protein), Or (2) administering to the patient the DNA encoding the receptor protein of the present invention and expressing it, or (mouth) subject cells. After inserting and expressing the DNA encoding the receptor protein of the present invention into the patient, the cells are transplanted into the patient, etc., to increase the amount of the receptor protein in the patient's body, and the action of the ligand. Can be fully exhibited. That is, the 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 has about 39% homology at the amino acid sequence level to KIAA0758 [DNA Res., 5 (5), 277-286 (1998)] which is a G protein-coupled receptor protein. It is a novel seven-transmembrane receptor protein that is recognized.

The DNA encoding the receptor protein or the receptor protein of the present invention may be a central disease (eg, Alzheimer's disease, dementia, eating disorder, depression, epilepsy, etc.), an endocrine disease (eg, Addison's disease, Cushing's syndrome, brown cells) Species, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic diseases (eg, diabetes, dyslipidemia, diabetic complications, obesity, gout, Cataract, hyperlipidemia, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), inflammatory disease ( For example, allergies, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc., cardiovascular diseases (eg, hypertension, cardiac hypertrophy, stenosis) Heart disease, arteriosclerosis, myocardial infarction, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, chronic lymphocytic leukemia, Chronic myelogenous leukemia, malignant melanoma, multiple myeloma, etc.), respiratory diseases (eg, cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombus, pulmonary embolism, etc.), gastrointestinal diseases (eg, Gastric ulcer, duodenal ulcer, gastritis, reflux esophagitis, knee inflammation, etc., immune system disease (eg, autoimmune disease, etc.), infectious disease (eg, immune dysfunction, 'pneumonia, cytomegal virus, influenza virus) , Virus infection such as herpes virus, rickettsial infection, bacterial infection, etc.).

When the receptor protein of the present invention is used as the above-mentioned prophylactic and / or therapeutic agent, it can be formulated according to conventional means.

On the other hand, when the DNA encoding the receptor protein of the present invention (hereinafter sometimes abbreviated as the DNA of the present invention) is used as the above-described prophylactic and / or therapeutic agent, the DNA of the present invention may be used alone or as a retrovirus. Vector, adenovirus vector

: To a suitable vector such as a virus vector After insertion, it can be carried out according to conventional means. The DNA of the present invention can be administered as it is or together with an adjuvant for promoting uptake, using a gene gun or a catheter such as Hyde-mouth gel power catheter.

For example, (1) DNA encoding the receptor protein of the present invention or (2) the receptor protein may be, if necessary, orally as tablets, capsules, elixirs, microcapsules, etc., coated with sugar or water or water. It can be used parenterally in the form of an injectable solution such as a sterile solution with a pharmaceutically acceptable liquid or a suspension. For example, (1) the receptor protein of the present invention or (2) DNA encoding the receptor protein is generally used together with known physiologically acceptable carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like. Recognized formulation It can be manufactured by mixing in the unit dosage form required for practice. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry. When the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can. As an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. Agents, such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene daricol), non-ionic surfactants (eg, polysorbate 80 ™, HCO-50) . As the oily liquid, for example, sesame oil, soybean oil, and the like are used. Good.

Examples of the prophylactic and / or therapeutic agent include a buffer (for example, a phosphate buffer, a sodium acetate buffer), a soothing agent (for example, benzalkonidum chloride, proactive hydrochloride, etc.), a stabilizer (for example, , Human serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection solution is usually filled in a suitable ampoule.

Because the preparations obtained in this way are safe and have low toxicity, they can be used, for example, in mammals (eg, humans, rats, mice, egrets, sheep, bush, foxes, cats, dogs, monkeys, etc.). Can be administered.

The dose of the receptor protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like. However, in the case of oral administration, in general, for example, in a cancer patient (as 60 kg), It is about 0.1 mg to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. For example, in the case of injection, it is usually used for cancer patients (as 6 O kg). In this case, it is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg by intravenous injection. . In the case of other animals, the amount converted per 60 kg can be administered.

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. However, in the case of oral administration, in general, for example, in a cancer patient (as 6 O kg), one dose is required. About 0.1 mg to 100 mg per day, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. For example, in the case of an injection, it is usually used, for example, for cancer patients (as 6 O kg). It is convenient to administer about 0.01 to 3 Omg, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 1 Omg by intravenous injection. is there. In the case of other animals, the dose can be administered in terms of 60 kg. (3) Gene diagnostic agent

The DNA of the present invention can be used as a probe to produce the receptor protein of the present invention in mammals (eg, human, rat, mouse, egret, sheep, bush, horse, cat, dog, monkey, etc.). Since abnormalities (DNA abnormalities) in DNA or mRNA encoding the partial peptide can be detected, for example, damage, mutation, or decreased expression of the DNA or mRNA, and increased or excessive expression of the DNA or mRNA, etc. It is useful as a gene diagnostic agent for.

The above-described genetic diagnosis using the DNA of the present invention includes, for example, the well-known Northern Hybridization and the PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proc. 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 its partial peptide of the present invention

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

That is, the present invention relates to, for example, (i) non-human mammals: (1) blood, (2) specific organs, (3) tissues or cells isolated from the organs, or (ii) the receptor of the present invention contained in a transformant or the like. The present invention provides 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 one protein or its partial peptide.

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

(i) Normal or disease model non-human mammals (for example, mice, rats, rabbits, higgs, bushus, horses, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerosisゥ A heron, a tumor-bearing mouse, etc.), a drug (eg, an anti-dementia drug, a blood pressure lowering drug, an anti-cancer drug, an anti-obesity drug, etc.) or physical stress (eg, After a certain period of time, such as infiltration stress, electric shock, light and dark, low temperature, etc., blood or specific organs (eg, brain, liver, spleen, large intestine, knee, ovary, etc.) or isolated from organs Obtained tissue or cells.

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

(ii) A transformant expressing the receptor protein of the present invention or its partial peptide is prepared according to the above method, and the mRNA of the receptor protein of the present invention or its partial peptide 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 fixed time before drug or physical stress is given to a normal or disease model non-human mammal (30 minutes to 24 hours before, preferably 30 minutes before)

~ 12 hours before, more preferably 1 hour to 6 hours ago or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours After) or the test compound is administered simultaneously with the drug or physical stress, and after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours later), by quantifying and analyzing the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the cells,

(ii) When culturing the transformant according to a conventional method, the test compound is mixed in a medium, and after culturing for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days) (After day), 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 or a salt thereof obtained by using the screening method of the present invention has an action of changing the expression level of the receptor protein of the present invention or a partial peptide thereof. Specifically, (a) increasing the expression level of the receptor protein of the present invention or its partial peptide to thereby increase the cell stimulating activity through G protein-coupled receptor (eg, arachidonic acid Release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos , A compound that enhances the activity of promoting or suppressing pH reduction, etc.) (Mouth) By reducing the expression level of the receptor protein of the present invention or its partial peptide, the cell stimulating activity is reduced. Is a compound that attenuates

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

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 or the like of the present invention.

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

When a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned drug containing the receptor protein of the present invention.

The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like. About 0.1 to 100 mg / day, preferably about 1.0 to 50 mg / day, more preferably about 1.0 to 20 mg / day. In the case of parenteral administration, the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc. Is one It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(5) A preventive and / or therapeutic agent for various diseases containing a compound that changes 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 alters the expression level of the receptor protein or its partial peptide of the present invention can be used as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention. .

Here, examples of the disease associated with dysfunction of the receptor protein of the present invention include central diseases (eg, Alzheimer's disease, dementia, eating disorders, depression, epilepsy, etc.), endocrine diseases (eg, Addison's disease, Cushing's syndrome, pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic disorders (eg, diabetes, dyslipidemia, diabetic complications) , Obesity, gout, cataract, hyperlipidemia, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.) , Inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.), cardiovascular diseases (eg, hypertension, cardiac hypertrophy) Angina, arteriosclerosis, myocardial infarction, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, chronic lymphocytic leukemia) , Chronic myelogenous leukemia, malignant melanoma, multiple myeloma, etc.), respiratory diseases (eg, cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis, pulmonary embolism, etc.), gastrointestinal diseases (eg, , Gastric ulcer, duodenal ulcer, gastritis, reflux esophagitis, knee inflammation, etc., immune system diseases (eg, autoimmune diseases, etc.), infectious diseases (eg, immune dysfunction, pneumonia, cytomegal virus, influenza virus) Virus infections such as herpes virus, rickettsial infections, bacterial infections, 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, it can be formulated according to a conventional method. it can.

For example, the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or another pharmaceutically acceptable liquid. It can be used parenterally or in the form of injections such as suspensions. For example, the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry. When the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can. Examples of the aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like. Agents, such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, polysorbate 80 ™, HCO-50) . As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.

Examples of the prophylactic and / or therapeutic agent include a buffer (for example, a phosphate buffer and a sodium acetate buffer), a soothing agent (for example, benzalkonidum chloride, proactive hydrochloride, etc.), a stabilizer (for example, , Human serum albumin, polyethylene glyco And preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection solution is usually filled in a suitable ampoule.

The dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like. However, in the case of oral administration, for example, in a cancer patient (as 6 O kg), one dose is generally used. About 0.1 to per day: L0 Omg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, etc., for example, usually in the form of an injection, for example, a cancer patient (as 6 O kg) In this case, it is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg, by intravenous injection. In the case of other animals, the amount converted per 60 kg can be administered.

(6) The ligand of the present invention for the G protein-coupled receptor protein The receptor protein of the present invention has a binding property to the ligand. Can be.

The quantification method of the present invention can be used, for example, in combination with a competition method. That is, the ligand concentration in the specimen can be measured by bringing the specimen into contact with the receptor protein of the present invention or the like. Specifically, for example, it can be used according to the method described in the following (1) or (2) or a method analogous thereto.

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

②Irie Hiroshi, edited "Radio Imno Tsutsui" (Kodansha, published in 1979)

(7) Screening method for compounds (eg, agonists, antagonists, etc.) that alter the binding between the G protein-coupled receptor protein of the present invention and a ligand The ligand and the receptor of the present invention can be obtained by using the receptor protein of the present invention or by constructing an expression system for a recombinant receptor protein or the like and using a receptor-binding assay system using the expression system. A compound that changes the binding property to a protein or the like (eg, a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, etc.) or a salt thereof can be efficiently screened. Such compounds include (ii) cell stimulating activity via G protein-coupled receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular c A compound that has the activity of promoting or inhibiting GMP production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, reduction of pH, etc. (Agonist against receptor protein of the present invention), (mouth) Compound not having the cell stimulating activity (so-called agonist against receptor protein of the present invention), (8) Ligand and G protein of the present invention A compound that enhances the binding force to the conjugated receptor protein, or (2) the ligand and the G protein-coupled receptor protein of the present invention. Compound that reduces the binding strength and the like are included (the compound of the above (I) is not preferred to be screened by the ligand determination methods described above).

That is, the present invention relates to (i) the case where the receptor protein of the present invention or its partial peptide or a salt thereof is brought into contact with a ligand, and (Π) the case where the receptor protein of the present invention or its partial peptide or a salt thereof is present. And a compound or a salt thereof that alters the binding property between the ligand and the receptor protein of the present invention or a partial peptide thereof or a salt thereof, which is compared with a case where the ligand and the test compound are brought into contact with each other. 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 and 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, and when the labeled ligand and the test compound are brought into contact with the receptor protein, etc. of the present invention. The amount of binding of the labeled ligand to the receptor protein or the like in the case of the binding is measured and compared. Screening method,

(2) When the labeled ligand is brought into contact with a cell containing the receptor protein of the present invention or the membrane fraction of the cell, and when the labeled ligand and the test compound contain the receptor protein of the present invention or the like. And measuring the amount of the labeled ligand bound to the cell or the membrane fraction when contacting the cell or the membrane fraction of the cell with the ligand of the present invention. A method of screening for a compound or a salt thereof that alters the binding to protein or the like,

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

場合 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; Cell stimulating activity via receptor receptor (eg, arachidonic acid release) when a compound activating the receptor protein of the present invention and a test compound are brought into contact with cells containing the receptor protein of the present invention. , Acetylcholine release, intracellular ^{Ca 2+} release, intracellular ^cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation The activity of promoting or suppressing pH reduction, etc.), and comparing the ligand with the receptor protein of the present invention. For screening a compound or a salt thereof that changes the binding property to

を A compound that activates the receptor protein or the like of the present invention (for example, a ligand for the receptor protein or the like of the present invention) is treated with a trait containing the DNA of the present invention. When the transformant is cultured and the receptor is brought into contact with the receptor protein or the like of the present invention expressed on the cell membrane, the compound of the present invention that activates the receptor protein or the like and the test compound are the DNA of the present invention. Cell stimulatory activity through receptor X (eg, arachidonic acid release, acetilco Phosphorus release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, pH A compound that changes the binding property between the ligand and the receptor protein of the present invention, etc. Other provides a method of screening a salt thereof.

Prior to obtaining the receptor protein of the present invention, when screening for a protein-coupled receptor agonist or an antagonist, first, a cell containing the protein-coupled receptor protein such as a rat, A candidate compound is obtained using the tissue or its cell membrane fraction (primary screening), and then the candidate compound actually inhibits the binding of human G protein-coupled receptor protein to ligand A test (secondary screening) was needed to confirm whether or not this was the case. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins will also be present. Therefore, it has been difficult to actually screen for an agonist or an antagonist of the desired receptor protein.

However, for example, the use of the human receptor protein of the present invention eliminates the need for primary screening, and makes it possible to efficiently screen for a compound that inhibits the binding of a ligand to a G protein-coupled receptor protein. it can. Further, it is possible to easily evaluate whether the screened compound is an agonist or an antagonist.

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

First, as the receptor protein of the present invention used in the screening method of the present invention, any protein may be used as long as it contains the above-described receptor protein of the present invention. Cell membrane fractions of mammalian organs containing the receptor protein and the like of the present invention are suitable. However, especially human-derived organs are extremely available Because of its difficulty, it is suitable to use, for example, a human-derived receptor protein expressed in large amounts by using a recombinant, etc. for screening.

The above method is used to produce the receptor protein of the present invention, etc., but it is preferable to carry out the expression by expressing the DNA of the present invention in mammalian cells or insect cells. A complementary DNA is used for the encoding DNA fragment, but is not necessarily limited to this. For example, a gene fragment or a synthetic DNA may be used. In order to introduce a 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 nuclear polyhedrosis belonging to a baculovirus using an insect as a host. Virus (nuclear polyhedros is virus; NPV) polyhedrin promoter, SV40-derived promoter, retroviral promoter, meta-mouth thionine promoter, human heat shock promoter, cytomegalovirus promoter, It is preferably incorporated downstream, such as the SR a promoter. Examination of the amount and quality of the expressed receptor can be performed by a known method. For example, it can be carried out according to the method described in the literature [Nambi, P. et al., The 'Journal of Biological Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992]. it can.

Therefore, in the screening method of the present invention, the protein containing the receptor protein of the present invention may be a receptor protein purified according to a known method or the like. A cell containing a protein or the like may be used, or a membrane fraction of a cell containing the receptor protein or the like may be used. In the screening method of the present invention, when a cell containing the receptor protein of the present invention or the like is used, the cell may be immobilized with daltaraldehyde, formalin, or the like. The immobilization method can be performed according to a known method.

Cells containing the receptor protein of the present invention and the like refer to host cells expressing the receptor protein and the like. Examples of the host cells include Escherichia coli, Bacillus subtilis, yeast, insect cells, and animals. Cells and the like are preferred.

The cell membrane fraction refers to a cell membrane-rich fraction obtained by disrupting cells and then obtained by a known method. Methods for crushing cells include crushing cells with a Potter-Elvehj em-type homogenizer, Waring Blender ゃ Polytron. (Kinematica), crushing by ultrasonic waves, crushing by ejecting cells from a narrow nozzle while applying pressure with a French press or the like. For cell membrane fractionation, centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used. For example, the cell lysate is centrifuged at low speed (500 rpm to 300 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged (150 rpm to 1000 rpm). The mixture is centrifuged at 300 rpm for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction. The membrane fraction is rich in the expressed receptor protein and other membrane components such as cell-derived phospholipids and membrane proteins.

The amount of receptions evening one protein of a cell or membrane fraction containing the receptor protein or the like, 1 0 ³ ~ per cell: is preferably in the range of L 0 ⁸ molecule, is 1 0 ^5-1 0 ⁷ molecules Is preferred. The higher the expression level, the higher the ligand binding activity (specific activity) per membrane fraction. Become.

In order to carry out the above steps (1) to (3), which screen for a compound that changes the binding property between the ligand and the receptor protein of the present invention, for example, an appropriate receptor protein fraction and a labeled ligand are required. It is.

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

As the labeled ligand, a labeled ligand, a labeled ligand analog compound and the like are used. For example, ligands labeled with [ ³ H], C ¹²⁵ 1], [ 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 or the like of the present invention, first, a cell or a membrane fraction of the cell containing the receptor or the like of the present invention is subjected to screening. Prepare the receptor protein preparation by resuspending it in a buffer suitable for screening. The buffer may be a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8), or a buffer that does not inhibit the binding of a ligand such as Tris-HCl buffer to the receptor protein. Any may be used. Surfactants such as CHAPS, Tween-80 ™ (Kao-Atlas), digitonin, and dexcholate can also be added to the buffer to reduce non-specific binding. In addition, protease inhibitors such as PMS F, Leptin, E-64 (manufactured by Peptide Research Laboratories), and Peptidin can be added for the purpose of suppressing receptor and ligand degradation by proteases. . To 0.0 lm. 1 to 10 m l of the receptions evening first solution, presence of a certain amount (5000 c pm~ 500000 c pm) was added the labeled ligand concurrently 1 (T ⁴ M~1 (T ^ID test compound M Prepare a reaction tube containing a large excess of unlabeled ligand to determine the amount of non-specific binding (NSB) The reaction is about 0 to 50, preferably about 4 to 37, After about 20 minutes to 24 hours, preferably about 30 minutes to 3 hours After the reaction, the reaction solution is filtered through a glass fiber filter paper and washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured using a liquid scintillation counter or When the count (B _fl —NSB) obtained by subtracting the non-specific binding amount (NSB) from the count (B _D ) when there is no antagonist is 100%, the specific binding amount (B -NSB), for example, a test compound with 50% or less It can be selected as a candidate substance capable of.

In order to carry out the above methods (1) to (4) for screening a compound that changes the binding property between the ligand and the receptor protein of the present invention, for example, a cell stimulating activity mediated by the receptor protein (eg, arachidone) Acid release, acetylcholine release, intracellular Ca2 ⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, pH Activity that promotes or suppresses reduction, etc.) can be measured by a known method 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 conducting screening, replace the cells 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, extract cells or collect the supernatant. The products produced are quantified according to the respective method. The production of substances (eg, arachidonic acid) that serve as indicators of cell stimulating activity, Therefore, when the assay is difficult, the assay may be performed by adding an inhibitor against the degrading enzyme. In addition, an activity such as inhibition of cAMP production can be detected as an activity of inhibiting production of cells whose basal 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. Examples of cells expressing the receptor protein of the present invention include cell lines having the natural receptor protein of the present invention and cell lines expressing the recombinant receptor protein of the present invention. desirable.

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 a ligand to the receptor protein or the like of the present invention includes a cell containing the receptor protein of the present invention or the like, the protein of the present invention or the like. Or a cell membrane fraction 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

Filtration can be performed by filtration through a 0.45 / zm filter, and stored at 4 or may be prepared at the time of use.

② G protein conjugated receptor evening sample

The receptions evening CHO cells expressing an protein of the present invention, 1 2-well plates and passaged 5 X 1 0 ⁵ or holes, 3 7, 5% C 0 2, and cultured for 2 days in 9 5% air thing.

③ Labeled ligand

Commercially available ^[3 H], ^[125 I], ^[14 C], and stored at 4 or in one 2 0 those states of the ligand solution labeled with a ^[35 S], a buffer for the measurement Dilute to 1 M with.

④Ligand standard solution

Dissolve the ligand in PBS containing 0.1% ゥ serum albumin (Sigma) so as to obtain ImM, and store the solution at 1-20.

2. Measurement method

(1) Wash CH2 cells expressing the receptor protein of the present invention cultured on a 12-well tissue culture plate twice with a measurement buffer (lm1), and add 4901 measurement buffer to each well.

② 10- ³ ~10- 1 ⁽⁾ after M test compound solution was added 5 / Z 1, the labeled ligand 5 1 was added to react at room temperature for one hour. A supplementary 5 ^ 1 ligands of 10- ³ M in place of the test compound to determine the amount of non-specific binding.

(3) Remove the reaction solution and wash three times with lm1 of washing buffer. The labeled ligand bound to the cells is dissolved in 0.2N NaOH-1% SDS, and mixed with 4 ml of liquid scintillating liquid A (manufactured by Wako Pure Chemical Industries).

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

PMB = [(B-NS B) / (B ₀ — NSB)] X 100

PMB: Percent Maximum Binding

B: Value when the sample is added

NSB: Non-specific Binding

. : Fx large mouth D '

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 the ligand and the receptor protein of the present invention. ) Cell stimulating activity via G protein-coupled receptor (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, cell (A so-called agonist for the receptor protein of the present invention) having an activity of promoting or inhibiting the phosphorylation of internal proteins, activation of c-fos, reduction of pH, etc. A compound having no cell-stimulating activity (a so-called angonist for the receptor protein of the present invention); Or (2) a compound that decreases the binding force between the ligand and the G protein-coupled receptor protein of the present invention.

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. is there.

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

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

The 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.

When the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional method. For example, tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as in the above-described medicine containing the receptor protein of the present invention.

The dose of the compound or its salt depends on the subject of administration, target organ, symptoms, administration method, etc. In the case of oral administration, in general, for example, in a cancer patient (assuming 60 kg), about 0. 0 to 10 mg / day, preferably about 1.0 to 5 mg / day 0 mg, more preferably about 1.0-20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like. In this case, it is convenient to administer about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. It is. In the case of other animals, the dose can be administered in terms of 60 kg.

(8) A prophylactic and / or therapeutic agent for various diseases containing a compound (agonist, angonist) that changes the binding property between the G protein-coupled receptor protein and the ligand 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, circulatory function, and digestive function. Therefore, a compound (agonist, angiogonist) that changes the binding property between the receptor protein and the ligand of the present invention and the ligand for the receptor protein of the present invention are dysfunctional of the receptor protein of the present invention. It can be used as a prophylactic and / or therapeutic agent for diseases related to.

Here, examples of the disease associated with the dysfunction of the receptor protein of the present invention include central diseases (eg, Alzheimer's disease, dementia, eating disorders, depression, epilepsy, etc.), endocrine diseases (eg, Addison's disease, Cushing's syndrome, pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic diseases (eg, diabetes, dyslipidemia, diabetic complications) , Obesity, gout, cataract, hyperlipidemia, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.) , Inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.), cardiovascular diseases (eg, hypertension, cardiac hypertrophy) Angina, arteriosclerosis, myocardial infarction, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, chronic lymphocytic leukemia) , Chronic myelogenous leukemia, malignant melanoma, multiple myeloma, etc.), respiratory diseases (eg, cold syndrome, asthma, pneumonia, lung) Hypertension, pulmonary thrombosis, pulmonary embolism, etc., digestive system diseases (eg, gastric ulcer, duodenal ulcer, gastritis, reflux esophagitis, 塍 inflammation, etc.), immune system diseases (eg, autoimmune diseases, etc.), infection Disease (eg, immune dysfunction, pneumonia, viral infections such as cytomegal virus, influenza virus, herpes virus, rickettsial infection, bacterial infection, etc.).

When the compound or ligand is used as a prophylactic and / or therapeutic agent for a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to conventional means.

For example, the compound or ligand may be orally administered as a sugar-coated tablet, tablet, elixir, microcapsule, or the like, if necessary, or mixed with water or another pharmaceutically acceptable liquid. It can be used parenterally in the form of injections, such as sterile solutions or suspensions. For example, the compound may be formulated in a unit dosage form required for generally accepted pharmaceutical practice with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like. It can be manufactured by mixing. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry. When the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can. Examples of the aqueous liquid for injection include physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like. Agent, for example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene Cole) and nonionic surfactants (eg, Polysorbate 80 ™, HCO-50). As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.

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

The preparations obtained in this way are safe and have low toxicity, so they can be used in mammals (eg, humans, rats, mice, puppies, sheep, pigs, 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, symptom, administration method, and the like. About 0.1 to: L 0 Omg, preferably about 1.0 to 5 Omg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like. In this case, it is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg, by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(9) Quantification 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 of the present invention and the like, the quantification of the receptor protein and the like of the present invention in a test solution, particularly sandwich It can be used for quantification by Tsuchi immunoassay. That is, the present invention provides, 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; A method for quantifying the receptor protein of the present invention in a test solution,

(ii) Measuring the activity of the labeling agent on the insolubilized carrier after simultaneously or continuously reacting the test solution with the antibody of the present invention and the labeled antibody of the present invention insolubilized on the carrier. A method for quantifying the receptor protein of the present invention in a test solution is provided.

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

The receptor protein and the like of the present invention can be measured using a monoclonal antibody against the receptor protein and the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and detection by tissue staining and the like can be performed. You can also. For these purposes, the antibody molecule itself may be used, or F (ab ') ₂ , Fab', or Fab fraction of the antibody molecule may be used. The measurement method using an antibody against the receptor protein of the present invention is not particularly limited, and may be an antibody, an antigen or an antibody corresponding to the amount of an antigen (for example, the amount of the receptor protein) in a test solution. Any method that detects the amount of one antigen complex by chemical or physical means and calculates this from a standard curve prepared using a standard solution containing a known amount of the antigen can be used. You may. For example, nephelometry, a competitive method, an immunometric method and a sandwich method are suitably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity.

As a labeling agent used in a measuring 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], [ ¹⁴ 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-dalcosidase, alkaline phosphatase, peroxidase, malic acid 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.

For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing or immobilizing a protein or enzyme may be used. As the carrier, for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.

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 the reverse order, may be performed simultaneously, or may be performed at staggered times. The labeling agent and the method of insolubilization can be based on those described above.

In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one kind, and a mixture of two or more kinds of antibodies is used for the purpose of improving measurement sensitivity and the like. May be used.

In the method for measuring receptor protein and the like by the sandwich method of the present invention, the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is preferably an antibody having different binding sites such as receptor proteins. . That is, when the antibody used in the primary reaction and the secondary reaction is, for example, the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is preferably the C-terminal. For example, an antibody that recognizes other than the N-terminal part is used. ·

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 competition method, the antigen in the test solution and the labeled antigen were allowed to react competitively with the antibody. The unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody (BZF separation), and the amount of either B or F is measured, and the amount of antigen in the test solution is determined. Quantify. In this reaction method, a soluble antibody is used as the antibody, BZF separation is carried out using a polyethylene glycol, a liquid phase method using a second antibody against the above antibody, or a solid phase antibody is used as the first antibody. An immobilization method using a soluble antibody as the first antibody and using an immobilized antibody as the second antibody is used.

In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in either phase is measured to determine the amount of antigen in the test solution.

In nephelometry, the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing laser scattering is preferably used.

In applying each of these immunological assay methods to the assay method of the present invention, no special conditions, operations, and the like need to be set. What is necessary is just to construct the measuring system of the receptor protein or its salt of the present invention by adding ordinary technical considerations of those skilled in the art to ordinary conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews and written books. [For example, Hiroshi Irie “Radiom Noassy” (Kodansha, published in Showa 49), Hiroshi Irie “Radio Noizi” (Munoassy) (Kodansha, published in Showa 54), "Enzyme immunoassay" edited by Eiji Ishikawa et al. (Medical Shoin, published in 1953), "Enzyme immunoassay" edited by Eiji Ishikawa et al. (Second edition) (Issue of Medical Sciences, published in 1977), Eiji Ishikawa et al. “Enzyme Immunoassay” (3rd edition) (Issue of Medical Sciences, published in 1962), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Tec niaues (Part A))> Ibid.Vol. 73 (Immunochemical Techniaues (Part B)), Ibid.Vol. 74 (Immunochemical TechniQues (Part C)), Ibid.Vol.84 (Ichemical nochemical Techniaues (Part D:. Se lected Immunoassays)) ¾ ibid Vol 92 (Immunochemical Techniaues (Part E: Monoclonal Ant ibod ies and General Immunoassay Methods)), ibid., Vol. 121 (Immunochemical Tec niQues (Part I: Hybridoma Technology and Monoclonal Ant ibodies)) (all published by Academic Press).

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

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.

Further, the antibody of the present invention can be used for specifically detecting the receptor protein of the present invention present in a subject such as a body fluid or a tissue. Further, preparation of an antibody column used for purifying the receptor protein of the present invention and the like, detection of the receptor protein of the present invention in each fraction at the time of purification, and the present invention in a test cell It can be used for analyzing the behavior of the receptor protein.

(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, the receptor protein of the present invention or its partial peptide in the cell membrane can be recognized. Can be used for screening a compound that changes the amount of the compound.

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, a cell membrane fraction is isolated, and the receptor of the present invention contained in the cell membrane fraction is isolated. A method for screening a compound that changes the amount of the receptor protein or its partial peptide of the present invention in a cell membrane by quantifying the protein or its partial peptide,

(11) After disrupting the transformant expressing the receptor protein or its partial peptide of the present invention, the cell membrane fraction is isolated, and the receptor protein of the present invention contained in the cell membrane fraction is isolated. Alternatively, the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is changed by quantifying the partial peptide thereof. Compound screening method,

(iii) Sections of (1) blood, (2) specific organs, and (3) tissues or cells isolated from the organs of non-human mammals, and then using immunostaining to obtain the receptor protein on the cell surface The present invention provides 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 by confirming the protein on the cell membrane by quantifying the degree of staining of the protein.

(iv) After transforming the receptor protein of the present invention or a transformant that expresses a partial peptide thereof, etc., into a section, immunostaining is used to obtain the receptor protein at the cell surface layer. Provided is 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 by confirming the protein on the cell membrane by quantifying the degree of staining of the cell. The quantification 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, sheep, bush, birds, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteries, etc.) Drugs (eg, anti-dementia drugs, antihypertensive 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, liver, spleen, large intestine, knee, ovary, etc.), or a tissue or cell isolated from the organ is obtained. The obtained organ, tissue or cell is suspended in, for example, an appropriate buffer (for example, Tris-HCl buffer, phosphate buffer, Hess buffer, etc.) to destroy the organ, tissue or cell Then, a cell membrane fraction is obtained by using a surfactant (eg, Triton XI 00 ™, Twin 20 ™, etc.), and further using a method such as centrifugation, filtration, or column fractionation.

(Kinematica), crushing by ultrasonic waves, crushing by ejecting cells from a narrow nozzle while applying pressure with a French press or the like. Fine Fractionation by centrifugal force, such as differential centrifugation and density gradient centrifugation, is mainly used for fractionation of the cell membrane. For example, the cell lysate is centrifuged at a low speed (500 rpm to 300 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further spun at a higher speed (150 rpm to 300 rpm). The mixture is centrifuged at 0,000 rpm for 30 minutes to 2 hours, and the resulting precipitate is used as the membrane fraction. The membrane fraction is rich in 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 the Western plot 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.

Screening for a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is performed by:

(i) A given time before drug or physical stress is applied to a normal or disease model non-human mammal (30 minutes to 24 hours before, preferably 30 minutes to 12 hours before, Preferably 1 hour to 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 drug or physical The test compound is administered at the same time as the target stress, and after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the cell membrane By quantifying the amount of the receptor protein of the present invention or a partial peptide thereof,

(ii) When culturing the transformant according to a conventional method, the test compound is mixed in a medium, and after culturing for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days) (After day), by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane.

The receptor protein of the present invention contained in a cell membrane fraction or a partial peptide thereof The confirmation of the code is specifically performed as follows.

(iii) Normal or disease model non-human mammals (eg, mice, rats, rabbits, sheep, sheep, bush, birds, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, arteries, etc.). Drugs (eg, anti-dementia drugs, antihypertensive 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 specific organs (eg, brain, liver, spleen, large intestine, kidney, ovary, etc.), or tissues or cells isolated from the organs are obtained. The obtained organs, tissues or cells are cut into tissue sections according to a conventional method, and immunostaining is performed using the antibody of the present invention. By confirming the protein on the cell membrane by quantifying the degree of staining of the receptor protein on the cell surface, quantitatively or qualitatively, the receptor protein of the present invention or its receptor protein on the cell membrane can be obtained. The amount of the partial peptide can be confirmed.

(iV) It can also be confirmed by taking the same means using a transformant expressing the receptor protein of the present invention or its partial peptide, or the like.

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 amount of the receptor protein of the present invention or a partial peptide thereof in a cell membrane. 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 C a2 ⁺ release, intracellular ^cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, intracellular protein phosphorylation, activation of c-fos, decrease in pH, etc. (Mouth) the receptor protein of the present invention in a cell membrane or a partial peptide thereof Is a compound that reduces the cell stimulating activity by reducing the amount of

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 or the like.

The dose of the compound or a salt thereof varies depending on the administration subject, target organ, condition, administration method, and the like. However, in the case of oral administration, for example, in a cancer patient (assuming 60 kg), one dose is required. About 0.1 to per day: L0 O mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. For example, in the case of injection, it is usually, for example, a cancer patient (as 6 O kg) About 0.01 to 3 O mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 O mg per day. It is. In the case of other animals, the amount converted per 60 kg can be administered.

(11) A preventive and / or therapeutic agent for 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.The receptor protein of the present invention is, for example, as described above. It is thought to play some important role in vivo, such as central functions. 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 an agent for preventing and / or treating 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, Alzheimer's disease, dementia, eating disorder, depression, epilepsy, etc.), endocrine disease (eg, Addison's disease, Cushing's syndrome, brown cell type, primary aldosteronism, menopause, endometriosis , Dysgonadism, thyroid dysfunction, pituitary dysfunction, etc.), metabolic diseases (eg, diabetes, lipid metabolism, diabetic complications, obesity, gout, cataract, hyperlipidemia, etc.), cancer (eg, non-small) Cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, hepatitis, Retinopathy, cystitis, pneumonia, etc.), cardiovascular diseases (eg, hypertension, cardiac hypertrophy, angina, arteriosclerosis, myocardial infarction, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, Prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, malignant melanoma, multiple myeloma, etc.), respiratory disease (for example, Common cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis / pulmonary embolism, etc. Autoimmune diseases, etc.), infectious diseases (eg, immune dysfunction, pneumonia, viral infections such as cytomegal virus, influenza virus, herpes virus, rickettsial infections, bacterial infections, 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, it can be formulated according to conventional means.

For example, the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or another pharmaceutically acceptable liquid. It can be used parenterally or in the form of injections such as suspensions. For example, the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by the following. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.

Examples of additives that can be mixed with tablets, capsules, etc. include binders such as gelatin, corn starch, tragacanth, gum arabic, and crystalline cells. Excipients such as loin, leavening agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, peppermint, coconut oil or cherry. Such flavoring agents are used. When the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can. As an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used. Agents, such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, polysorbate 80 ™, HCO-50) . As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.

Because the preparations obtained in this way are safe and low toxic, they can be used, for example, in mammals (eg, humans, 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, in a patient with cancer (6 O kg), the About 0.1 to per day: L 0 O mg, preferably about 1.0 to 5 O mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. For example, in the form of an injection, for example, in a cancer patient (as 60 kg), about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg per day It is convenient to administer the degree by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

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

The neutralizing activity of an 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 that it inactivates a signal transduction function involving the receptor protein. Means activity. Therefore, when the antibody has neutralizing activity, signal transduction associated with the receptor protein, for example, cell stimulating activity via the receptor protein (for example, arachidonic acid release, acetylcholine release, intracellular Ca ^{2 +} Activation or suppression that promotes release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, lowering of pH, etc. Activity, etc.) can be inactivated. Therefore, it can be used for prevention and Z or treatment of diseases caused by overexpression of the receptor protein.

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

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

In transferring the DNA of the present invention to 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 transferring the DNA of the present invention derived from a egret, a DNA construct of the present invention, which is highly homologous to the DNA and is linked to the downstream of various promoters capable of expressing the DNA of the present invention in animal cells, may be used, for example. , To heron fertilized egg By microinjection, a DNA-transferred animal that highly produces the receptor protein of the present invention can be produced. As the promoter, for example, a promoter derived from a virus or a ubiquitous expression promoter such as meta-mouth thionein can be used, but an NGF gene promoter specifically expressed in the brain and a phenolase gene promoter are preferably used. .

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 DNA transfer means that all the offspring of the produced animal have the receptor protein of the present invention in all of its germ cells 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.

After confirming that the DNA-transferred animal of the present invention stably retains the gene by breeding, it can be reared in a normal breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all the offspring will have the DNA Breeding to have Since the animal into which the DNA of the present invention has been transferred has a high level of expression of the receptor protein of the present invention, etc., it is used for screening of an agonist or an angiosperm against the receptor protein of the present invention. Useful as animals.

The DNA-transferred 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-transferred 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 present invention can be used. It can analyze the quality of protein at the reception and evening. Cells of a tissue having the receptor protein of the present invention and the like are cultured by standard tissue culture techniques, and the functions of cells from tissues that are generally difficult to culture such as those derived from the brain and peripheral tissues are used by these. Can study. In addition, by using the cells, for example, a drug that enhances the function of various tissues can be selected. Moreover, if there is a high expression cell line, it is possible to isolate and purify the receptor protein of the present invention and the like from there. In the present specification and drawings, bases, amino acids, and the like are indicated by abbreviations based on the abbreviations by the IUPAC-I UB Commission on Biochemical Nomenclature or commonly used abbreviations in the art, and examples thereof are described below. When there is an optical isomer with respect to the amino acid, the L-form is indicated unless otherwise specified.

DNA deoxylipo nucleic acid

c DNA complementary deoxyribonucleic acid

A adenine

T thymine

G Guanin

C Sitshin

RNA ribonucleic acid

mRNA messenger ribonucleic acid

d ATP Deoxyadenosine triphosphate

dTTP Deoxythymidine triphosphate

dGTP Deoxyguanosine triphosphate

dCTP Deoxycytidine triphosphate

ATP 'triphosphate

EDTA: tetraacetic acid

SD S Sodium dodecyl sulfate

G 1 y Daricin

A 1 a Alanin

V a 1 Valine

Le u

I 1 e isoleucine

S e r serine

Th r threonine

C y s Sistine

Me t Methionin G 1 u: Glutamic acid

As p: Aspartic acid

Ly s: Lysine

A r g: Arginine

H i s: Histidine

P he: feniralanin

Ty r: Tyrosine

T r p: Tribute fan

Pro: Proline

As n: Asparagine

G in: glutamine

pG 1 u: pyroglutamic acid

*: Corresponding to the stop codon

Me: methyl group

E t: ethyl group

B u: butyl group

P h: phenyl group

TC: thiazolidine—4 (R) —carboxamide group

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

T 0 s p-toluenesulfonyl

CHO Holmill

B z 1 benzyl

Cl ₂ Bzl 2, 6—dichlorobenzyl

Bom: benzyloxymethyl

Z: benzyloxycarbonyl

C1-Z: 2-cyclobenzyloxycarbonyl

B r -Z: 2-bromobenzyloxycarbonyl

Bo c: t-butoxycarbonyl DNP: dinitrophenol

Trt: Trityl

Bum: t-butoxymethyl

Fmo c: N_ 9 _fluorenylmethoxycarbonyl

HOB t: 1-hydroxybenztriazole

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

1,2,3-benzotriazine

HONB: Trihydroxy-5-norpolene-2,3-dicarpoxyimide

DCC: N, N'-dicyclohexylcarbodiimide The sequence numbers in the sequence listing in this specification indicate the following sequences.

SEQ ID NO: 1

1 shows the amino acid sequence of the novel human-derived G protein-coupled receptor protein TGR 20-1 of the present invention.

SEQ ID NO: 2

1 shows the nucleotide sequence of cDNA encoding the novel human G protein-coupled receptor protein TGR20-11 of the present invention.

SEQ ID NO: 3

The base sequence of primer 1 used in the PCR reaction in Examples 1, 2 and 3 below is shown.

SEQ ID NO: 4

1 shows the nucleotide sequence of primer 2 used in the PCR reaction in Example 1 below. SEQ ID NO: 5

1 shows the amino acid sequence of a novel human-derived G protein-coupled receptor protein TGR20-2 of the present invention.

SEQ ID NO: 6

1 shows the nucleotide sequence of cDNA encoding the novel human-derived G protein-coupled receptor protein TGR 20-2 of the present invention.

SEQ ID NO: 7 The base sequence of the primer 13 used in the PCR reaction in Examples 2 and 3 below is shown.

SEQ ID NO: 8

FIG. 3 shows the nucleotide sequence of a positive primer TGR20TQF used in Example 3 below. SEQ ID NO: 9

The base sequence of the reverse primer TGR20TQR used in Example 3 below is shown. SEQ ID NO: 10

7 shows the nucleotide sequence of probe TGR20TQP used in Example 3 below. The transformant obtained in Example 1 below, Escherichia coli

T0P10 / pCR2.1-TGR20-1 is an independent administrative corporation of Chuo No. 6 (Zip code 305-8566), 1-chome, Tsukuba-Higashi, Ibaraki, Japan since April 19, 2001 At the National Institute of Advanced Industrial Science and Technology, Patent Depositary Depositary No. FE RM BP-7552, 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka, Osaka from April 11, 2001 (postal code 532) -8686) has been deposited with the Fermentation Research Institute (IFO) under the deposit number IF〇16612.

The transformant obtained in Example 2 below, Escherichia coli

TOP10 / pCR2.TGR20-2 is an independent administrative corporation of Tsukuba-Higashi 1-chome 1 Chuo No. 6 (Zip code 305-8566) from April 19, 2001 (Heisei 13) Deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the deposit number FERM BP-7553, from April 11, 2001, 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (Postal code 532-8686) ) And deposited with the Fermentation Research Institute (IFO) under the deposit number IF〇16613. Example

Hereinafter, the present invention will be described in more detail with reference to Examples, but these do not limit the scope of the present invention. In addition, the gene using Escherichia coli followed the method described in Molecular'cloning (Molecular cloning). Example 1 Cloning of cDNA encoding G protein-coupled receptor Yuichi protein derived from human testis and determination of nucleotide sequence

Using human testis cDNA (CL0NTECH) as type III, PCR reaction was performed using two primers, primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4). The composition of the reaction solution for this reaction was as follows, using the above cDNA as type 3 / 、, 11 volumes of Advantage-GC2 Polymerase Mix (CL0NTECH), primer 1 (SEQ ID NO: 3) and primer 1 (SEQ ID NO: 2). : 4) was added to each of 0.5 zM, dNTPs to 200 zM, buffer attached to the enzyme to 101, and GCMdt to 51 to make a liquid volume of 50 l. After 1 minute at 95, 5 cycles of 95 minutes, 30 seconds, 68 minutes of 2 minutes, 95 cycles of 30 seconds, 66 cycles of 30 seconds, 68 cycles of 2 minutes, 5 times A cycle of 95 for 30 seconds, 64 for 30 seconds, and 68 for 2 minutes was repeated 30 times, and a final extension reaction of 68 for 7 minutes was performed. The PCR reaction product was subcloned into a plasmid vector pCR2.1 (Invitrogen) according to the prescription of a T0P0-TA cloning kit (Invitrogen). This was introduced into E. coli TOP10, and clones having cDNA were selected on LB agar medium containing ampicillin. As a result of analyzing the sequence of each clone, a cDNA sequence (SEQ ID NO: 2) encoding a novel G protein-coupled receptor Yuichi protein was obtained. A novel G protein-coupled receptor protein containing these amino acid sequences was named TGR20-1. The transformant was named Escherichia coli T0P10 / pCR2.1-TGR20-1.

The hydrophobicity plot of TGR 20-1 is shown in FIG. Example 2 Cloning of cDNA encoding human testis-derived G protein-coupled receptor Yuichi protein and determination of nucleotide sequence

Using human testis cDNA (CL0NTECH) as type I, a PCR reaction was performed using two primers, primer 1 (SEQ ID NO: 3) and primer 3 (SEQ ID NO: 7). The composition of the reaction solution used in the reaction was as follows, using the above cDNA as a 3 l 铸 form, the amount of Advantage-GC2 Polymerase Mix (CL0NTECH), primer 1 (SEQ ID NO: 3) and primer 1 (SEQ ID NO: 7). 0.5 ^ M each, dNTPs 200 μl, buffer attached to the enzyme 101 and GCMelt 51 were added to make a volume of 501. After 95 minutes, the PCR reaction was repeated 5 times at 95 · 30 seconds, 68 682 minutes, 95 9530 seconds, 66 で 30 seconds, 68 · The 2-minute cycle was repeated five times, 95:30 seconds, 64-30 seconds, and 68. The 2-minute cycle was repeated 30 times, and an extension reaction was performed at 68 for 7 minutes. The PCR reaction product was subcloned into a plasmid vector pCR2.1 (Invitrogen) according to the prescription of a T0P0-TA cloning kit (Invitrogen). This was introduced into E. coli T0P10, and clones having cDNA were selected on LB agar medium containing ampicillin. As a result of analyzing the sequence of each clone, a cDNA sequence (SEQ ID NO: 6) encoding a novel G protein-coupled receptor Yuichi protein was obtained. The novel G protein-coupled receptor protein containing these amino acid sequences was named TGR20-2. The transformant was named Escherichia coli (fod? 7'd? / 'A coli) T0P10 / pCR2. TGR20-2.

The hydrophobicity plot of TGR 20-2 is shown in FIG.

Example 3 Analysis of expression tissue distribution of TGR20-1 and TGR20-2 (hereinafter, these may be simply abbreviated as TGR20) using TaqManPCR

First, primers and probes were designed using Primer Express ver.1.0 (PE Biosystems Japan), forward primer TGR20TQF (5'-AAGTC AGAGG AGAGG AGGAC ACA-3 '(SEQ ID NO: 8)), reverse primer TGR20TQR (5'-TGAAT CATTT TGCAG GCCTG-3 '(SEQ ID NO: 9)) and probe TGR20TQP (5, -TGTGT TGGCT GGCAC TCTGT GGAGA A-3' (SEQ ID NO: 10)) were prepared. The reporter dye of the probe added FAM (6-carboxyfluorescein).

For the standard cDNA, a PCR fragment obtained by amplifying pCR2. was purified by], it was used to prepare the 10 ^fl one ¹⁰⁶ copies I 5 ^ 1.

Various cDNA libraries prepared using the SMART RACE Library System of CL0NTECH were used as the cDNA source for each tissue. At the same time as the TGR20 expression level, the TGR20 tissue distribution was compared by measuring the / 3-actin expression level using TaaMan / 3-actin control reagents Mix (PE Biosystems Japan) and normalizing it.

TadMan PCR uses TatiMan Universal PCR Master Mix (PE Biosystems Japan). The reaction was performed using ABI PRISM 7700 Sequence Detection System (PE Biosystems Japan) according to the attached instructions.

The results are shown in FIG. TGR20 was highly expressed in testis and lung. table

Tissue expression

(One copy / β-actin χ ΙΟΟΟΟΟΟ)

m, 32

Fetal brain 0

0

Liver 0

Heart Moon 顾 79

40

228

Lung 88

Thymus 2

Placenta 18

White blood cells 0

mm 0

Pituitary 4

Possibility of industrial use

The G protein-coupled receptor protein of the present invention or its partial peptide or a salt thereof, and the polynucleotide encoding the receptor protein or its partial peptide (for example, DNA, RNA and their derivatives) are: (2) Acquisition of antibodies and antisera, (3) Construction of a recombinant receptor protein overnight expression system, (4) Development of a receptor-integrated atsushi system using the same expression system と Screening of drug candidate compounds, ⑤ Performing drug design based on comparison with structurally similar ligands レ Receptors ⑥ Probes in gene diagnosis ゃ Reagents for creating PCR primers ⑦ Transgeneic It can be used for animal production or as a drug such as a gene therapy agent.

Claims

The scope of the claims

1. A G protein-coupled receptor protein or a salt thereof, which comprises the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or 5.

2. A G protein-coupled receptor protein or a salt thereof, which comprises the amino acid sequence represented by SEQ ID NO: 1 or 5.

3. A partial peptide of the G protein-conjugated receptor according to claim 1, or a salt thereof.

4. A polynucleotide comprising a polynucleotide having a base sequence 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 or 6.

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 G protein-coupled receptor protein according to claim 1, wherein the transformant according to claim 8 is cultured to produce the G protein-coupled receptor protein according to claim 1. Or a method for producing the salt thereof.

10. A medicament 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 4. 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 of claim 1 or the partial peptide of claim 3 or a salt thereof. A ligand for the G protein-coupled receptor of the protein or its salt.

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 or the salt thereof according to claim 3, wherein the G protein-coupled receptor protein or the salt thereof is used. How to determine the ligand for the salt.

18. A ligand characterized by using the G protein-coupled receptor protein according to claim 1 or the partial peptide or a salt thereof according to claim 3, and a G protein-coupled receptor protein according to claim 1. Or a method for screening a compound or a salt thereof that alters the binding property to a salt thereof.

19. A G protein-coupled receptor protein according to claim 1 or a ligand comprising the partial peptide or a salt thereof according to claim 3, and a G protein-coupled receptor protein according to claim 1 or A kit for screening a compound or a salt thereof that changes the binding property to the salt.

20. A ligand obtainable by using the screening method according to claim 18 or the screening kit according to claim 19, and a G protein-coupled receptor protein or a salt thereof according to claim 1. Or a salt thereof, which alters the binding property to the compound.

21. A ligand obtainable by using the screening method according to claim 18 or the screening kit according to claim 19, and a G protein-coupled receptor protein or a salt thereof according to claim 1. A pharmaceutical comprising a compound that alters the binding of a compound or a salt thereof.

22. A polynucleotide that hybridizes with the polynucleotide of claim 4 under high stringency conditions.

23. A polynucleotide comprising a nucleotide sequence complementary to the polynucleotide of claim 4 or a part thereof.

2 4. The method for quantifying mRNA of the 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 protein according to claim 1, wherein the antibody according to claim 12 is used. A method for quantifying protein coupled receptor Yuichi 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. A 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. A method for screening a 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.

29. The 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, which alters the amount of a G protein-coupled receptor protein in a cell membrane obtainable by using the screening method according to claim 28.

31. A medicament comprising a compound that changes the expression level of the G protein-coupled receptor protein of claim 1 or a salt thereof, which can be obtained by using the screening method of claim 27.

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 test compound is contacted with a G protein-coupled receptor protein of claim 1 or a salt thereof or a partial peptide of claim 3 or a salt thereof and a test compound. A method for determining a ligand as described above.

3 4. (i) Contacting the ligand with the G protein-coupled receptor protein of claim 1 or its salt or the partial peptide or its salt according to claim 3, and (ii) claim A comparison is made between the case where the G protein-coupled receptor protein or the salt thereof according to the item 1 or the partial peptide or the salt thereof according to the item 3 is contacted with a ligand and a test compound. 18. The screening method according to 18.

35. (i) contacting the labeled ligand with the G protein-coupled receptor protein or its salt according to claim 1 or the partial peptide or its salt according to claim 3, and (ii) the labeled ligand The G protein according to claim 1, wherein the ligand and the test compound are contacted with the G protein-coupled receptor protein according to claim 1 or a salt thereof or the partial peptide according to claim 3 or a salt thereof. A ligand characterized by measuring and comparing the amount of binding to a conjugated receptor protein or a salt thereof or the partial peptide or a salt thereof according to claim 3, and a G protein-coupled receptor according to claim 1. A method for screening a compound or a salt thereof that changes the binding property to a protein or a salt thereof.

36. (i) contacting the labeled ligand with a cell containing the G protein-coupled receptor protein according to claim 1; and (ii) labeling the labeled ligand and the test compound with the G protein according to claim 1. The G protein-coupled receptor according to claim 1, wherein the amount of the labeled ligand bound to the cell when the cell is brought into contact with a cell containing the protein-coupled receptor protein is measured and compared. A method for screening a compound or a salt thereof that changes the binding property to one protein or a salt thereof.

37. (i) When the labeled ligand is brought into contact with the membrane fraction of the cell containing the G protein-coupled receptor protein according to claim 1, and (ii) the labeled ligand and the test compound are described in claim 1. A ligand which is characterized in that the amount of a labeled ligand bound to a membrane fraction of the cell when the G protein-coupled receptor protein is brought into contact with the membrane fraction is measured and compared. Item 6. A method for screening a compound or a salt thereof that alters the binding property to the G protein-coupled receptor protein or a salt thereof according to Item 1.

38. (i) When the labeled ligand is brought into contact with the G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to claim 8, ii) G protein expressed on the cell membrane of the transformant by culturing the transformant according to claim 8 with a labeled ligand and a test compound. The ligand and the G protein according to claim 1, wherein the amount of the labeled ligand bound to the G protein-coupled receptor protein is measured and compared when the ligand is brought into contact with the protein coupled receptor protein. Conjugated Receptor A method for screening a compound or a salt thereof that changes the binding property to a protein or a salt thereof.

39. (i) A compound that activates the G protein-coupled receptor protein or a salt thereof according to claim 1 was contacted with a cell containing the G protein-coupled receptor protein according to claim 1. And (ii) a compound and a test compound that activate the G protein-coupled receptor protein of claim 1 or a salt thereof and a test compound are added to cells containing the G protein-coupled receptor protein of claim 1. The ligand and the G protein-coupled receptor protein according to claim 1, wherein the cell stimulating activity via the G protein-coupled receptor protein and the ligand are measured and compared with each other. A method for screening a compound or a salt thereof that changes the binding property to the salt.

40. A G protein-coupled receptor expressed by the compound of claim 1 or a compound that activates a salt thereof, which is expressed in the cell membrane of the transformant by culturing the transformant of claim 8. A compound that activates the G protein-coupled receptor protein or a salt thereof according to claim 1 and a test compound, by culturing the transformant according to claim 8. G protein-coupled receptor expressed on the cell membrane of the transformant. Estimated cell stimulating activity mediated by G protein-coupled receptor when exposed to protein. A compound that changes the binding property between a ligand and the G protein-coupled receptor protein of claim 1 or a salt thereof, or a compound thereof. Screening method of.

41. The compound that activates the G protein-coupled receptor protein according to claim 1, wherein the angiotensin, bombesin, canapinoid, cholecystokinin, glumin, serotonin, melatonin, neuropeptide Y, obioid, Purine, vasopleucine, oxytocin, PACAP, secretin, glucagon, calcitonin, adrenomedullin, somatosintin, GHRH, CRF, ACTH, GRP, PTH, VIP, Somatos, Chitin, Dopamine, Motilin, Amylin, Bradykinin, CGRP, Leukotriene, Pancreastatin, Pross, Darandin, Trompoxane, Adenosine, Adrenaline, Chemokines Superfamily, Endothelin, Enterogastrin The screening method according to claim 39 or 40, wherein the screening method is histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA) or sphingosine 1-phosphate.

42. A compound that alters the binding between the ligand obtainable by the screening method according to any one of claims 34 to 41 and the G protein-coupled receptor protein or a salt thereof according to claim 1. Or its salt.

43. A compound that alters the binding between the ligand obtainable by the screening method according to any one of claims 34 to 41 and the G protein-coupled receptor protein or a salt thereof according to claim 1. Or a medicament characterized by containing a salt thereof.

44. The screening kit according to claim 19, comprising a cell containing the G protein-coupled receptor protein according to claim 1.

45. A screening kit according to claim 19, comprising a membrane fraction of a cell containing the G protein-coupled receptor protein according to claim 1.

46. The screening method according to claim 19, further comprising a G protein-coupled receptor protein expressed in the cell membrane of the transformant by culturing the transformant according to claim 8. kit.

47. A ligand and the G protein-coupled receptor according to claim 1, which can be obtained by using the screening kit according to any one of claims 44 to 46, and a protein or a salt thereof. Or a salt thereof, which changes the binding property of

48. A ligand, a G protein-coupled receptor according to claim 1, and a protein or a salt thereof, which can be obtained by using the screening kit according to any one of claims 44 to 46. A pharmaceutical comprising a compound that alters the binding of a compound or a salt thereof.

49. The antibody according to claim 12 and the G protein-coupled receptor according to claim 1. The method for quantifying a G protein-coupled receptor protein according to claim 1, or a partial peptide according to claim 3, or a salt thereof, which is brought into contact with a protein or a partial peptide according to claim 3 or a salt thereof.

50. Competition between the antibody according to claim 12 and the test solution and the labeled G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof. Wherein the ratio of the labeled G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 3 or a salt thereof, which is bound to the antibody, is measured. A method for quantifying the G protein-conjugated receptor according to claim 1 or the partial peptide or the salt thereof according to claim 3 in a liquid.

51. The test solution is insolubilized on the carrier and the antibody according to claim 12 and the labeled antibody according to claim 12 are reacted simultaneously or continuously, and then the labeling agent on the insolubilized carrier is used. 4. The method for quantifying the G protein-coupled receptor protein of claim 1 or the partial peptide of claim 3 or a salt thereof in a test solution, wherein the activity of the protein is measured.

5 2. Claims to prevent and / or treat central diseases, endocrine diseases, metabolic diseases, cancer, inflammatory diseases, cardiovascular diseases, respiratory diseases, digestive diseases, immune diseases or infectious diseases. The medicament according to claim 21, claim 31, or claim 32.

53. Central disease, endocrine disease, metabolic disease characterized by administering an effective amount of the compound according to claim 20, claim 29 or claim 30 or a salt thereof to a mammal. A method for preventing and / or treating cancer, inflammatory diseases, cardiovascular diseases, respiratory diseases, digestive diseases, immune system diseases or infectious diseases.

5 4. Manufacturing preventive and / or therapeutic agents for central, endocrine, metabolic, cancer, inflammatory, circulatory, respiratory, digestive, immune, or infectious diseases Use of the compound according to claim 20, claim 29 or claim 30 or a salt thereof.