WO2002059304A1 - Novel g protein-coupled receptor protein and dna thereof - Google Patents

Abstract

It is intended to provide a novel protein which is useful in screening an agonist/an antagonist, etc. More specifically, a human-origin protein or its salt, a DNA encoding this protein, a method of determining a ligand to the protein, a method/a kit for screening a compound altering the binding properties of the ligand to the protein, a compound obtained by the screening or its salt, etc. are provided. The human-origin protein as described above and the DNA encoding the same are usable in, for example,: (1) determining a ligand to the protein; (2) preventives and/or remedies for diseases in association with the hypofunction of the protein; and (3) screening a compound (an agonist, an antagonist, etc.) altering the binding properties of the protein to the ligand.

Description

 Specification

 New G protein-coupled receptor protein and its DNA technology

 The present invention relates to a novel G protein-coupled receptor protein derived from human brain or a salt thereof, and a DNA encoding the same. Background art

Many physiologically active substances, such as hormone ゃ neurotransmitters, regulate the functions of living organisms through specific receptor proteins present on cell membranes. By increasing f or conjugate of these receptions tartan Nono ⁰ click proteins by Rereru guanine nucleotide- binding protein (hereinafter, abbreviated as G proteins) no rows intracellular signaling through activation I spoon of, also, seven It has a common structure with the same transmembrane domain, and is collectively called G protein-coupled receptor protein or seven-transmembrane receptor protein (7 TMR).

 G protein-coupled receptor proteins are present on the surface of various functional cells of living cells and organs, and are used as physiological targets as molecules that regulate the functions of those cells and organs, such as hormones, neurotransmitters and bioactive substances. Plays an important role. The receptor transmits a signal into a cell through binding to a physiologically active substance, and this signal causes various reactions such as suppression of activation and activation of the cell.

 To clarify the relationship between substances that regulate complex functions in cells and organs of various living organisms and their specific receptor proteins, especially G protein-coupled receptor proteins, It will elucidate the functions of cells and provide a very important means for drug development closely related to those functions.

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

 Elucidation of the relationship between substances that regulate complex functions in living organisms and their specific receptor proteins is a very important means for the development of drugs containing agonists and antagonists for receptor proteins. However, conventionally, in order to efficiently screen for agonists and antagonists against receptor proteins and to develop pharmaceuticals, the functions of receptor protein genes expressed in vivo must be elucidated, and the appropriate expression system must be used. Expression was required.

 In recent years, as a means of analyzing genes expressed in vivo, studies on the random analysis of cDNA sequences have been actively conducted. Registered in the database as a Sequence Tag (EST) and published. However, most ESTs contain only sequence information, and it is difficult to estimate their functions. Conventionally, substances that inhibit the binding of a G protein-coupled receptor to its ligand, a biologically active substance, and substances that bind to cause signal transduction similar to that of a biologically active substance, As an agonist, it has been used as a medicine to regulate biological functions. Therefore, finding new G protein-coupled receptor proteins that are not only important in physiological expression in vivo but also become targets for drug development, and cloning their genes (for example, cDNA), This is a very important tool for finding specific ligands, agonists and antagonists for novel G protein-coupled receptor proteins.

However, not all G protein-coupled receptors have been found. At present, unknown G protein-coupled receptors and their ligands have not been identified. There is a great deal of demand for searching for new G protein-coupled receptors and elucidating their functions. G protein-coupled receptors are useful for searching for new ligands (physiologically active substances), and for searching for agonists or antagonists to the receptor, using the signal transduction action as an index. On the other hand, even if a physiological ligand is not found, an agonist or an antagonist for the receptor is produced 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 these receptors can be expected to be used as preventive or therapeutic agents for diseases associated with impaired function of G protein-coupled receptors. Furthermore, a decrease or increase in the function of a G protein-coupled receptor in a living body due to a genetic mutation often causes some disease. In this case, not only administration of an antagonist or agonist to the receptor, but also gene therapy by introducing the receptor gene into a living body (or a specific organ) or by introducing an antisense nucleic acid against the receptor gene. It can also be applied to In this case, the nucleotide sequence of the receptor is indispensable information for examining the presence or absence of a deletion or mutation on the gene. It can also be applied to diagnostics.

The present invention provides a novel G protein-coupled receptor protein useful as described above. That is, a novel G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, a polynucleotide containing a polynucleotide (DNA, RNA and a derivative thereof) encoding the G protein-coupled receptor protein or a partial peptide thereof (DNA, RNA and derivatives thereof), a recombinant vector containing the polynucleotide, a transformant carrying the recombinant vector, a method for producing the G protein-coupled receptor protein or a salt thereof, An antibody against the G protein-coupled receptor protein or a partial peptide thereof or a salt thereof; a compound that changes the expression level of the G protein-coupled receptor protein; a method for determining a ligand for the G protein-coupled receptor; And the G protein Compounds that alter the binding property between the mold receptor protein (antagonist Agonisuto) or disk of a salt thereof Compounds (antagonists, agonists) that alter the binding between the ligand obtainable by using the screening method, the screening kit, the screening method or the screening kit, and the G protein-coupled receptor protein, or a compound thereof A salt, a compound (antagonist, agonist) that changes the binding property between the ligand and the G protein-coupled receptor protein, a compound that changes the expression level of the G protein-coupled receptor protein, or a salt thereof. Provide medicine and the like. Disclosure of the invention

 As a result of intensive studies, the present inventors have succeeded in isolating cDNA encoding a novel G protein-coupled receptor protein derived from human brain and analyzing the entire nucleotide sequence thereof. Then, when this base sequence was translated into an amino acid sequence, the first to seventh transmembrane regions were confirmed on the hydrophobicity plot shown in FIG. 1, and the protein encoded by these cDNAs was transmembrane seven times. It was confirmed that it was a G-protein-coupled receptor protein. The present inventors have conducted further studies based on these findings, and as a result, have completed the present invention.

 That is, the present invention

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

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

 (3) the G protein-coupled receptor protein according to (1), wherein the substantially identical amino acid sequence is the amino acid sequence represented by SEQ ID NO: 2;

 (4) partial peptides or salts thereof of the G protein-coupled receptor protein described in (1) above,

 (5) a polynucleotide comprising a polynucleotide encoding the G protein-coupled receptor protein according to (1),

( ⁶ ) the polynucleotide according to (5), which is a DNA, (7) the polynucleotide according to the above (5), which has a base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4,

 (8) a recombinant vector containing the polynucleotide according to (5),

 (9) The transformant transformed with the recombinant vector according to the above (8),

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

 (11) a pharmaceutical comprising the G protein-coupled receptor protein according to (1) or the partial peptide according to (4) or a salt thereof;

 (12) a medicament comprising the polynucleotide according to the above (5),

 (13) an antibody against the G protein-coupled receptor protein according to the above (1) or the partial peptide or a salt thereof according to the above (4),

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

 (15) a diagnostic agent comprising the antibody according to (13),

 (16) a medicament comprising the antibody according to (13),

 (17) The G protein-coupled receptor according to (1), which can be obtained by using the G protein-coupled receptor protein according to (1) or the partial peptide according to (4) or a salt thereof. Ligands for proteins or their salts,

 (18) The medicament comprising the ligand according to (17),

 (19) The G protein-coupled receptor protein described in (1) or a partial peptide thereof described in (4) or a salt thereof, wherein the G protein-coupled receptor protein described in (1) or a partial peptide thereof is used. How to determine the ligand for the salt,

(20) A ligand characterized by using the G protein-coupled receptor protein described in (1) or the partial peptide described in (4) or a salt thereof, and a G protein-coupled receptor protein described in (1). Or a method of screening for a compound or a salt thereof that alters the binding with a salt thereof, (21) A ligand comprising the G protein-coupled receptor protein described in (1) or the partial peptide described in (4) or a salt thereof, and the G protein-coupled receptor described in (1) A kit for screening a compound or a salt thereof that changes the binding property to a protein or a salt thereof,

 (22) Binding ability of the ligand obtainable by using the screening method described in (20) or the screening kit described in (21) to the G protein-coupled receptor protein or a salt thereof described in (1). Or a compound that alters

* · * Ιτπ.

 (23) Binding ability of the ligand obtainable by using the screening method according to (20) or the screening kit according to (21) to the G protein-coupled receptor protein or the salt thereof according to (1). A pharmaceutical comprising a compound or a salt thereof that changes

 (24) a polynucleotide comprising the polynucleotide of (5) or a base sequence complementary to the polynucleotide of (5) or a part thereof under high stringency conditions with the polynucleotide of (5);

 (26) The method for quantifying mRNR of a G protein-coupled receptor protein according to (1), wherein the polynucleotide according to (5) or a part thereof is used.

 (27) The method for quantifying a G protein-coupled receptor protein according to (1), wherein the antibody according to (13) is used.

 (28) The method for diagnosing a disease associated with the function of the G protein-coupled receptor according to (1), wherein the quantification method according to (26) or (27) is used,

(29) A method for screening a compound or a salt thereof that alters the expression level of a G protein-coupled receptor protein according to (1), which comprises using the quantification method according to (26);

(30) A method for screening a compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein according to the above (1) in a cell membrane, characterized by using the quantification method according to the above (27), (31) a compound or a salt thereof that alters the expression level of the G protein-coupled receptor protein according to (1), which can be obtained by using the screening method according to (29);

 (32) a compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein according to (1) in a cell membrane obtainable by using the screening method according to (30);

 (33) A medicament comprising a compound capable of changing the expression level of the G protein-coupled receptor protein according to (1) or a salt thereof, which can be obtained by using the screening method according to (29)!

 (34) a medicament comprising a compound or a salt thereof that 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 (30) above,

 (35) The above-mentioned (23) which is an agent for preventing or treating central diseases, endocrine diseases, metabolic diseases, cancer, inflammatory diseases, cardiovascular diseases, respiratory diseases, digestive diseases, immune diseases or infectious diseases. The medicament according to (33) or (34),

 (36) Central disease, endocrine disease, metabolic disease, characterized by administering to a mammal an effective amount of the compound according to (22), (31) or (32) or a salt thereof. , Cancer, inflammatory diseases, cardiovascular diseases, respiratory diseases, digestive diseases, immune system diseases or infectious diseases,

 (37) To manufacture a preventive or therapeutic agent for central, endocrine, metabolic, cancer, inflammatory, circulatory, respiratory, digestive, immune, or infectious diseases The present invention relates to the use of the compound according to (22), (31) or (32) or a salt thereof.

 The invention further provides:

 (38) The protein is: (1) an amino acid sequence represented by SEQ ID NO: 1;

1 or 2 or more (preferably about 1 to 30, more preferably 1 to about 10, and more preferably several (1 to 5)) amino acids in the amino acid sequence represented by 1 are deleted The amino acid sequence represented by SEQ ID NO: 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10, more preferably Preferably, the amino acid sequence is obtained by adding several (1 to 5) amino acids. ③ One or more (preferably about 1 to 30, more preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 1. Is a protein containing an amino acid sequence in which 1 to 10 amino acids are substituted with another amino acid, and more preferably about 10 amino acids, more preferably several (1 to 5) amino acids. The G protein-coupled receptor protein or a salt thereof according to (1) above,

 (39) The method according to the above (19), wherein the G protein-coupled receptor protein or the salt thereof according to the above (1) or the partial peptide or the salt thereof according to the above (4) is brought into contact with a test compound. How to determine the ligand of

 (40) When the ligand is, for example, angiotensin, bombesin, cannabinoid, cholecystokinin, gnoletamine, serotonin, melatonin, neuropeptide Y, opioid, purine, pasoprescin, oxotocin, PACAP (e.g., PAC ΑΡ 27, P ACAP 3 8), secretin, glucagon, calcitonin, adore nomedulin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (vasoactive intestinal polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcinine) Rated peptide), leukotriene, pancreatastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfamily (eg, IL-18, GRO a GRO, GRO γ NAP — 2, EN A—78, GC P—2, PF 4, IP—10, Mig, CXC chemokine subfamily such as PBSF / SDF-1, MCAF / MCP-1, MC P—2, MC P_ 3, MC P—4, eotaxin, RANTE S, MIP—la, MI P — 1] 3, HCC — 1, MIP-3a / LARC, MIP-3β / ELC, 1—309, TARC, MIPF—1 , MIPF-2 / eotaxin-2, MDC, DC-CK1 / PARC, SLC and other CC chemokines subfamily;

C chemokine subfamily such as lymphotactin; CX 3 C chemokine subfamily such as fractalkine), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (L PA) or sphingosine monophosphate, the method for determining the ligand according to the above (39), (41) (i) when the ligand is brought into contact with the G protein-coupled receptor protein or the salt thereof described in (1) above or the partial peptide or the salt thereof described in (4) above, and (ii) ) To compare the G protein-coupled receptor protein described in the above (1) or a salt thereof or the partial peptide described in the above (4) or a salt thereof with a ligand and a test compound. The screening method according to the above (20), which is characterized in that:

 (42) (i) the case where the labeled ligand is brought into contact with the G protein-coupled receptor one protein or the salt thereof described in (1) above or the partial peptide or the salt thereof described in (4) above; When the labeled ligand and test compound are 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 (4), the above (1) ) The amount of binding to the G protein-coupled receptor protein described above or a salt thereof or the partial peptide described in (4) or a salt thereof is measured and compared with the ligand described in (1) above. A protein-coupled receptor, a method for screening a compound or a salt thereof that changes the binding property to a protein or a salt thereof,

 (43) (i) When the labeled ligand is brought into contact with cells containing the G protein-coupled receptor protein described in (1) above, and (ii) When the labeled ligand and the test compound are The ligand and the G protein according to (1), wherein the amount of binding of the labeled ligand to the cell when the cell is contacted with the cell containing the G protein-coupled receptor protein is measured and compared. A method for screening a compound or a salt thereof that changes the binding property to a conjugated receptor protein or a salt thereof,

(44) (i) contacting the labeled ligand with the membrane fraction of the cell containing the G protein-coupled receptor protein described in (1); (1) measuring and comparing the amount of labeled ligand bound to the membrane fraction of the cell when it is brought into contact with the membrane fraction of the cell containing the G protein-coupled receptor protein described in (1). Between the ligand described above and the G protein-coupled receptor protein described in (1) or a salt thereof A method for screening a compound or a salt thereof that changes the sex,

 (45) (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 (9) above; ii) When the labeled ligand and test compound are brought into contact with a G protein-coupled receptor protein expressed on the cell membrane of the transformant by culturing the transformant according to (9) above, The amount of binding of the labeled ligand to the G protein-coupled receptor protein is measured and compared, and the binding between the ligand and the G protein-coupled receptor protein or the salt thereof described in (1) above is determined. A method for screening the compound to be changed or a salt thereof,

 (46) (i) When 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 described in (1) above or a salt thereof and a test compound containing the G protein-coupled receptor protein described in (1) above. A ligand characterized by measuring and comparing cell stimulating activity via a G protein-coupled receptor protein when brought into contact with cells, and a G protein-coupled receptor protein or a salt thereof according to (1) above. A method for screening a compound or a salt thereof that changes the binding property of

(47) A compound that activates the G protein-coupled receptor protein or a salt thereof described in (1) above, and cultivates the transformant described in (9) above to express the G protein coupled to the cell membrane of the transformant. The G protein-coupled receptor protein described in (1) above or a compound that activates the salt thereof and a test compound are cultured in the transformant described in (9) above. Thus, a ligand characterized by measuring and comparing cell stimulating activity via a 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 a compound that alters the binding property to the G protein-coupled receptor protein or a salt thereof according to (1) above, or Is a method for screening its salts, (48) The compound that activates the G protein-coupled receptor protein according to the above (1) is angiotensin, bombesin, cannabinoid, cholecystokinin, gnoretamine, serotonin, melatonin, neuropeptide Y, opioid, or primin. , Vasoprescin, Oxytocin, PACAP (e.g., PACAP27, PCAPAP38), Secretin, Gourgon, Force Λ · ^ Cytonin, Adrenomedullin, Somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokai Superfamily _{(e.g., IL-8, GROa s GROj3} , GR0 NAP- 2, ENA- 78, GCP- 2, PF4, IP- 10, M ig, CXC chemokine subfamily such as PBSF / SDF- 1; MCAF / MCP -1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, MI P-1α, MI P-1, HCC-1, MIP-3 / LARC, MIP-3β / ELC I-309, TARC, MIPF-1, MIPF-2 / eotaxin-2, MDC, DC-CK1 / PARC, CC chemokine subfamily such as SLC; C chemokine subfamily such as lymphotactin; CX3C chemokine subfamily such as fractalkine) (46) or (47), which is endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA) or sphingosine 1-phosphate. ,

 (49) A compound or a salt thereof, which alters the binding between the ligand obtainable by the screening method according to any of (41) to (48) and the G protein-coupled receptor protein or a salt thereof according to (1).

 (50) A compound or a salt thereof that alters the binding property between the ligand obtainable by the screening method according to any of (41) to (48) and the G protein-coupled receptor protein or its salt according to (1). A medicament characterized by containing

(51) The screening kit according to (21), which comprises a cell containing the G protein-coupled receptor protein according to (1). (52) The screening kit according to (21), which comprises a membrane fraction of a cell containing the G protein-coupled receptor protein according to (1).

 (53) The screening method according to (21), which comprises a G protein-coupled receptor protein expressed in the cell membrane of the transformant by culturing the transformant according to (9). Kit,

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

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

(56) The antibody according to (1), wherein the antibody according to (13) is contacted with the G protein-coupled receptor protein according to (1) or the partial peptide according to (4) or a salt thereof. )) Or the method for quantifying a partial peptide or a salt thereof according to (4) above,

 (57) The antibody of (13) is competitively competent with the test solution and the labeled G protein-coupled receptor protein of (1) or the partial peptide of (4) or a salt thereof. Reacting, and measuring the ratio of the labeled G protein-coupled receptor protein according to (1) or the partial peptide or salt thereof according to (4), which is bound to the antibody. And the method for quantifying the G protein-coupled receptor protein described in the above (1) or the partial peptide or the salt thereof described in the above (4).

(58) The test solution and the antibody according to (13) insoluble on the carrier and the labeled antibody according to (13) are reacted simultaneously or continuously, and then labeled on the insolubilized carrier. A method for measuring the activity of an agent, the method for quantifying the G protein-coupled receptor protein described in (1) above, the partial peptide described in (4) above, or a salt thereof in a test solution. . BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a hydrophobicity plot of TGR34L.

 FIG. 2 is a hydrophobicity plot of TGR34V. BEST MODE FOR CARRYING OUT THE INVENTION

The G protein-coupled receptor protein of the present invention (hereinafter sometimes abbreviated as receptor protein) is a receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. is there. The receptor protein of the present invention includes, for example, all cells (eg, spleen cells, neurons, glial) of human non-human mammals (eg, guinea pigs, rats, mice, rabbits, pigs, sheep, birds, monkeys, etc.) Cells, germ cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, fat cells, immune cells (eg, macrophages, Τ cells, Β cells) , Natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, breast cells, hepatocytes Or stromal cells, or their precursors, stem cells, or cancer cells), blood cells, or any group in which these cells are present Tissue, for example, brain, each part of the brain (e.g., olfactory bulb, acrosomal nucleus, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus nucleus, cerebral cortex, medulla, cerebellum, occipital lobe, frontal lobe, temporal lobe, Putamen, caudate nucleus, brain stain, substantia nigra, spinal cord, pituitary, stomach, kidney, kidney, liver, gonad, thyroid, gall bladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, colon) , Small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, peripheral blood cells, prostate, testicle, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. Also, it may be a synthetic protein. Examples of the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, about 50% or more, preferably about 60% or more, more preferably about 70% Above, more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more amino acid sequence having homology. 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 A protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1 is preferred.

 As an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1, more specifically, for example, an amino acid sequence represented by SEQ ID NO: 2, etc. Examples thereof include ligand binding activity and signal transduction. 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 2 times). However, 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.

 In addition, the receptor protein of the present invention includes (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30, more preferably about 1 to 10, and more preferably about 1 to 10; (1-5) amino acid sequences, and (2) one or more (preferably about 1 to 30, more preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 1. An amino acid sequence to which about 1 to 10, more preferably several (1 to 5) amino acids have been added; and ③ one or more (preferably 1 or more) amino acids in the amino acid sequence represented by SEQ ID NO: 1 About 30 amino acids, more preferably about 1 to about 10 amino acids, and even more preferably several (1 to 5) amino acids substituted with another amino acid, or a combination thereof. Proteins containing amino acid sequences Used by all.

In the present specification, the amino acid sequence of the receptor protein is N-terminal (amino terminal) on the left end and C-terminal (carboxyl terminal) on the right end according to the convention of peptide notation. Contains the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 The receptor protein of the present invention including the receptor protein is as follows. The terminal may be any of a carboxyl group (-C00H), phenolic oleboxylate (—C00—), an amide (one C0NH ₂ ) or an ester (—C00R).

Here, as R in the ester, e.g., methyl, Echiru, n- propyl, alkyl groups such as isopropyl, n- butyl, Shikuropen chill, C ₃ _ ₈ cycloalkyl group such as cyclohexyl, for example, phenyl, alpha-Na C ₆ _ ₁₂ Ariru group such Fuchiru, for example, benzyl, phenyl, such as phenethyl - a C ₇ _ ₁₄ Ararukiru groups such as C w alpha-Nafuchiru C w alkyl Le group such as an alkyl group or an alpha-naphthylmethyl A pivaloyloxymethyl group commonly used as an ester for oral use and oral use is used.

 When the receptor protein of the present invention has a lipoxyl group (or carboxylate) other than the C-terminus, the receptor protein of the present invention includes a lipoxyl group amide or esterified. . As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

Furthermore, the receptor protein of the present invention is the protein mentioned above, Amino group protecting groups Mechionin residues of N-terminal (e.g., formyl group, etc. C _M Ashiru group such as any C ₂ _ ₆ Arukanoiru group of Asechiru) , The daltamyl group formed by cleavage of the N-terminus in vivo and pyroglutamine oxidation, the substituent on the side chain of the amino acid in the molecule (for example, — 0H, one SH, amino group, imidazole group, indole group, etc. Guanijino group) appropriate protecting groups (e.g., formyl group, C ₂ such Asechiru - ₆ such Ashiru groups such Arukanoiru groups) are protected by shall or sugar chain bound Also included are complex proteins such as so-called glycoproteins.

 As a specific example of the receptor protein of the present invention, for example, a receptor protein containing an amino acid sequence represented by the sequence No. 1 or SEQ ID NO. 2 is used.

The partial peptide of the receptor protein of the present invention (hereinafter sometimes abbreviated as a partial peptide) may be any of the above partial peptides of the receptor protein of the present invention. The receptor protein of the present invention Among the molecular molecules, those that are exposed outside the cell membrane and have substantially the same activity are used.

 Here, “substantially the same activity” indicates, for example, ligand binding activity. The measurement of the ligand binding activity can be performed in the same manner as described above.

 Specifically, as a partial peptide of the receptor protein having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, the extracellular region (hydrophilicity) in the hydrophobicity plot analysis shown in FIG. 1 or FIG. (Hydrophilic) site). In addition, a peptide partially containing a hydrophobic (Hydrophobic) site can also be used. A peptide containing individual domains individually may be used, but a peptide containing a plurality of domains simultaneously 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 and the like are preferred.

 A substantially identical amino acid sequence is defined as about 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, and particularly preferably about 80% or more, of these amino acid sequences. An amino acid sequence having 90% or more, most preferably about 95% or more homology is shown.

 In addition, the partial peptide of the present invention is characterized in that: (1) one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence are deleted; (2) One or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids are added to the amino acid sequence, Or ③ One or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the above amino acid sequence may be substituted with another amino acid .

In the partial peptide of the present invention, the C-terminus may be any of a carboxyl group (one C00H), a carboxylate (one C00—), an amide (one C0NH ₂ ) or an ester (one C00R) (where R is Show the same significance). When the partial peptide of the present invention has a carboxyl group (or carboxylate) other than at the C-terminus, the partial peptide of the present invention includes a carboxyl group amidated or esterified. You. As the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.

 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. Also included are those in which Gin is pyroglutamic acid, 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.

 Examples of the salt of the receptor protein of the present invention or its partial peptide include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable. 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, fumanoleic 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 human or non-human mammal cell or silk tissue by a known method for purifying a receptor protein, or the present invention described below. It can also be produced by culturing a transformant containing DNA encoding the receptor protein. Also, the protein can be produced by the protein synthesis method described later or according to the method. When producing from tissues or cells of a human non-human mammal, the tissues or cells of the human non-human mammal are homogenized, extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, Purification and isolation can be achieved by combining chromatography such as ion exchange chromatography.

For the 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 usually used. Examples of such resins include chloromethinole resin, hydroxymethinole resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, and PAM resin. , 4-hydroxymethylmethylphenylacetamide methyl resin, polyacrylamide tree Luminous, 4- (2 ', 4'-dimethoxyphenylhydroxymethyl) phenoxy tree Luminous, 4- (2', 4, dimethoxyphenyl-mocaminoethyl) phenoxy resin it can. Using such a resin, an amino acid having a suitably protected amino group and side chain functional group is condensed on the resin according to various known condensation methods in accordance with the amino acid sequence of the target protein or peptide. At the end of the reaction, the protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed. To get.

 Regarding the condensation of the protected amino acids described above, various activation reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carpimides, DCC, N, N'-diisopropylcarpimide, N-ethyl_Ν '-(3-dimethylaminoprolyl) carpimide, and the like are used. Activation by these involves the ability to add the protected amino acid directly to the resin together with a racemization inhibitor additive (eg, HOBt, HOOBt), or as a symmetric anhydride or HOBt ester or HOOBt ester. After the protected amino acid is activated in advance, it can be added to the resin.

The solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, methylform, and trifluoroethanol Alcohols such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; and esters such as methyl acetate and ethyl acetate. And the like. The reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about 120 ° C to 50 ° C. The activated amino acid derivative is usually used in a 1.5 to 4-fold excess. As a result of the test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be carried out by repeating the condensation reaction without removing the protecting group. Even if the reaction is repeated, sufficient condensation If not obtained, unreacted amino acid can be acetylated using acetic anhydride or acetylimidazole.

 Examples of the protecting group for the starting amino group include Z, B oc, tertiary pentoxy carbonyl, isobornyl oxycarbonyl, 4-methoxybenzinol xycanoleponinole, C1-Z, Br_Z, Adamantinoreoxycanoleboninole, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioi, and the like are used.

 The power boxyl group may be, for example, a linear chain such as an alkyl ester group (eg, methyl, ethyl, propyl, butynole, tertiary butynole, cyclopentinole, cyclohexyl, cyclohepty-cyclooctyl, 2-adamantyl, etc.). , Branched or cyclic alkyl esterification), aralkyl esterification (for example, benzyl ester, 412-ester benzyl ester, 4-methoxybenzyl ester, 4-methyl benzyl ester, benzhi) Drill esterification), phenacinoleestenolation, benzyloxycarbonyl hydrazide, tertiary 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 this esterification, for example, a lower alkanol group such as an acetyl group, an arylo group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group or an ethoxycarbonyl group, and the like are used. In addition, groups suitable for etheric groups include, for example, a benzyl group, a tetrahydrobiranyl group, a t-butyl group, and the like.

As, for example, B z 1, C 1 ₂ protecting group of the phenolic hydroxyl group of tyrosine - B zl, 2-two Torobenjiru, as the B r _ Z, protecting group of the imidazole of histidine such as tertiary butyl is used, For example, Tos, 4-methoxy-1,2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.

Examples of the raw material in which the carboxyl group is activated include, for example, a corresponding acid anhydride, azide, active ester [alcohol (for example, pentachlorophenol, 2 , 4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt). For example, the corresponding phosphoric amide is used as the raw material of the active amino group.

To eliminate (split off) the protecting groups, there are used catalytic reduction under hydrogen gas flow in the presence of (1 over black § Rui 1 ⁵ 3 _-carbon which catalyst and anhydrous hydrogen fluoride Acid treatment with methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, or base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc. The elimination reaction by the above acid treatment is generally performed at a temperature of about 20 ° C to 40 ° C. In the acid treatment, for example, anisol, phenol, thioanisole, etc. Clicks such as, meth-cresolone / re, saccharesoneole, dimethinoresolenolide, 1,4-butanedithiol, 1,2-ethanedithiol, etc. The addition of an on-capture agent is effective, and 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 tryptophan is as described above. In addition to deprotection by acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc.

 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, after amidating and protecting a monocarboxyl group of a carboxy terminal amino acid, a peptide (protein) chain is extended to a desired chain length on the amino group side. Then, a protein from which only the protecting group for the N-terminal a-amino group of the peptide chain was removed and a protein from which only the protecting group for the C-terminal carboxyl group was removed were prepared. Condensation in Details of the condensation reaction are the same as described above. By condensation After purifying the obtained protected protein, all the protecting groups are removed by the above method to obtain a desired crude protein. This crude protein is purified by various known purification means, and the main fraction is lyophilized 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. You can get your body.

 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 peptide. As a peptide synthesis method, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the desired 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 removing the protective group when the product has a protective group. it can. Known condensation methods and elimination of protecting groups include, for example, the methods described in the following ① to ⑤.

 ® M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)

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

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

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

 治 Supervised by Haruaki Yajima, Development of Pharmaceuticals 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. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and when it is obtained as a salt, it can be converted to a free form by a known method. be able to. 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. The polynucleotide is RNA such as DNA or mRNA encoding the receptor protein of the present invention, 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, a method described in the publicly known experimental medicine "New PCR and its application" 15 (7), 1997 or a method analogous thereto, for example, a method such as TaqMan PCR As a result, 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, genomic DNA library, cDNA derived from the above-mentioned cells and tissues, cDNA library derived from the above-described cells and itinerary, and synthetic DNA. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a preparation of total RNA or mRNA fraction from the above-mentioned cell'tissue.

 Specifically, the DNA encoding the receptor protein of the present invention includes, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4, or SEQ ID NO: 3 or SEQ ID NO: 4. It has a DNA that hybridizes under high stringent conditions with DNA having the represented base sequence, and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal signaling activity, etc.) Any DNA can be used as long as it encodes a receptor protein.

Examples of DNA that hybridizes under high stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 include, for example, Contains a nucleotide sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more homology with the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. DNA or the like is used.

 Hybridization is performed by 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). When a commercially available library is used, it can be carried out according to the method described in the attached instruction manual, and more preferably, can be carried out under high stringent conditions.

 The high stringent conditions include, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. Show. In particular, a sodium concentration of about 19 mM and a temperature of about 65 ° C are most preferred.

 More specifically, as a DNA encoding a receptor protein containing an amino acid sequence represented by SEQ ID NO: 1, a DNA containing a base sequence represented by SEQ ID NO: 3; As a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 4, a DNA having the base sequence represented by SEQ ID NO: 4 or the like is used.

 A polynucleotide comprising a part of the base sequence of DNA encoding the receptor protein of the present invention or a part of a base sequence complementary to the DNA is a partial peptide of the present invention described below. It is used to mean not only DNA to be loaded but also RNA.

According to the present invention, an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of a G protein-coupled receptor protein gene is cloned, or a G protein-coupled receptor protein determined. It can be designed and synthesized based on the nucleotide sequence information of the DNA to encode. Such a polynucleotide (nucleic acid) can hybridize with RNA of a G protein-coupled receptor protein gene and has the ability to inhibit ^^ formation or function of the RNA or RNA associated with G protein-coupled receptor protein. With G protein-coupled receptor protein gene expression can be regulated and controlled through interaction. Polynucleotides that are complementary to the selected sequence of G protein-coupled receptor protein-related RNA and polynucleotides that can specifically hybridize with G protein-coupled receptor protein-related RNA are in vivo. It is useful for regulating and controlling the expression of G protein-coupled receptor protein gene in vitro, and is also useful for treating or diagnosing diseases and the like. In addition, the G protein-coupled receptor protein gene has 5 terminal hairpin loops, 5 terminal 6-base pair 'repeat, 5' terminal untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, The 3'-end untranslated region, the 3'-end palindrome region, and the 3'-end hairpin loop can be selected as preferred regions of interest, but any region within the G protein-coupled receptor protein gene can be selected.

The relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region, that is, the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target can be said to be “antisense”. Antisense Polynucleotides are 2-deoxy-D-report-containing polydeoxynucleotides, D-report-containing polynucleotides, and other types of N-glycosides of purine or pyrimidine bases Or other polymers having a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that the polymer is DNA, (Including nucleotides having a configuration that allows base pairing and base attachment as found in RNA). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified Oligonucleotides), and those with known modifications, for example, those with a label known in the art, capped, methylated, and one or more natural nucleotides. Substituted with an intramolecular nucleotide, such as those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.) Having a bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as a protein (nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or a sugar ( For example, those having a side chain group such as monosaccharides, etc., those having an interfering compound (eg, acridine, psoralen, etc.), chelating compounds (eg, metal, radioactive metal, boron) , An oxidizing metal, etc.), an alkylating agent, or a compound having a modified bond (for example, α-anomeric nucleic acid). Here, "nucleoside J,""nucleotide" and "nucleic acid" may not only contain purine and pyrimidine bases, but may also include those based on 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 have modified sugar moieties, e.g., one or more hydroxyl groups have been replaced with halogens, aliphatic groups, or functional groups 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 the modified nucleic acid include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. 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 increase the affinity for the target sense strand, and to antisense if toxic. Make nucleic acids less toxic.

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

The antisense nucleic acids of the present invention may have altered or modified sugars, bases, And may be provided in special forms, such as ribosomes, microspheres, applied by gene therapy, or provided in a cured 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 the uptake of nucleic acids ( 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.). These can be attached to the 3 'end or the 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond. Other groups include capping groups specifically arranged at the 3 'end or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNase. Examples of such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycolone and tetraethyleneglycol.

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

 The DNA encoding the partial peptide of the present invention may be any DNA as long as it contains a base sequence encoding the above-described partial peptide of the present invention. In addition, any of a genomic DNA, a genomic DNA 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 pacteriophage, plasmid, cosmid, and phagemid. Alternatively, it can be directly amplified by the 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) a portion of DNA having a base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. (2) having DNA that hybridizes under high stringent conditions with DNA represented by SEQ ID NO: 3 or SEQ ID NO: 4; For example, DNA having a partial base sequence of DNA encoding a protein having the same activity (eg, ligand binding activity, signal transduction action, etc.) is used.

 Examples of the DNA that hybridizes with the DNA represented by SEQ ID NO: 3 or SEQ ID NO: 4 under high stringency conditions include, for example, about 70% or more, preferably, about 70% or more of the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. DNA having a nucleotide sequence having homology of about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more is used.

 As a means for cloning DNA that completely encodes the receptor protein of the present invention or its partial peptide (hereinafter sometimes abbreviated as the receptor protein of the present invention), the peptide of the present invention may be coded. Amplify by PCR using a synthetic DNA primer having a partial base sequence of the base sequence of DNA, or encode a part or the whole region of the receptor protein of the present invention with DNA incorporated in an appropriate vector. And a DNA fragment labeled with a synthetic DNA fragment or a synthetic DNA. Hybridization can be performed according to, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

The nucleotide sequence of the DNA can be replaced by OD A-LA PCR using PCR or a known kit, for example, Mutan ™ —super Express Km (Takara Shuzo), Mutan ^™ —K (Takara Shuzo), or the like. The method can be carried out according to a known method such as the gapped duplex method, the Kunkel method and the like, or a method analogous thereto.

The DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or can be digested with a restriction enzyme if desired, or can be used after adding a linker. The DNA has AT G at the 5 ′ end as a translation initiation codon, and TAA, TGA or TA at the 3 ′ end as a translation stop codon. It may have G. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

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

 Escherichia coli-derived plasmids (eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, PSH19, pSH15), phage such as λ phage, animal viruses such as retrovirus, vaccinia virus, and baculovirus, pAl-ll, pXTl, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc. 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 hosts, SRa promoter, SV40 promoter, LTR open motor, CMV promoter, HSV-TK promoter and the like can be mentioned.

Of these, it is preferable to use the CMV promoter, SRα promoter and the like. When the host is Eshierihia genus bacterium, trp promoter, 1 ac flop port motor, r _ec A promoter,; LP _L promoter, etc. lpp promoter when the host is Bacillus, SPO 1 promoter, SPO2 When the host is yeast, such as a promoter or Pen P promoter, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, etc. are preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, expression vectors which contain an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV4 Oori), and the like, if desired, may be used. Can be used. Examples of selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene (methotrexate (MTX) resistance), ampicillin resistance Sex gene (hereinafter sometimes abbreviated as Am p ^r), neomycin resistant gene (hereinafter sometimes abbreviated as N eo ^r, G 4 1 8 resistance) and the like. In particular, when the dhfr gene is used as a selection marker using CHO (dhfr-1) cells, the target gene can be selected by using a thymidine-free medium or 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. If the host is Escherichia, the PhoA signal sequence, OmpA signal sequence, etc., if the host is Bacillus, the amylase signal sequence, subtilisin signal sequence, etc., and the host is yeast In some cases, the MFa 'signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, the insulin signal sequence, a-interferon

• Signal sequence, antibody molecule, signal sequence, etc. can be used.

 Using the vector containing the DNA encoding the receptor protein of the present invention thus constructed, a transformant can be produced.

 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 [Procedures of the National Academy of Sciences, _Ob . Acad. Sci. USA), 60 volumes, 160 (1968)], JM103 [Nucleic Acids Research], 9 volumes, 309 (1981)], JA221 [journal 'ob' molecular 'bio Journal of Molecular Biology], Volume 120, 517 (1978)], HB101 [Journal 'Op' Molecular 'Biology, Volume 41, 459 (1969) 3, C600 [Genetics, 39 , 440 (1954)], DH5a [Inoue, H., Nojima, Η. And Okayama, H., Gene, 96, 23-28 (1990)], DH10B [Proceedings. Academy 'Ob S.C., _Ob . The' U.S.A. ( _proc . Natl. Ac ad. Sci. USA), Vol. 87, 4645-4649 (1990)].

Examples of Bacillus bacteria include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Ob'Bioke] For example, yeast such as Saccharomyces cerevisiae AH22, AH22R, NA87-11A, DKD-5D, 20B-12, Zizaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris (Pichia pastoris), etc.

 As insect cells, for example, when the virus is Ac NPV, the cell line derived from the larvae of night moth (Spodoptera frugiperda cell; Sf cell), MG1 cells derived from the midgut of Trichoplusia ni, and eggs derived from eggs of Trichoplusia ni High Five ™ cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmN PV, a cell line derived from silkworm (Bombyx mori N; BmN cell) is used. As the Sf cells, for example, Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217 (1977)) and the like are used. Can be

 As insects, for example, silkworm larvae are used [Maeda et al., Nature, 31δ, 5 (1985)].

 Examples of animal cells include monkey cells COS_7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dhfr-) cell), mouse L Cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc. are used.

 For transformation of Escherichia sp., For example, Proc. Natl. Acad. Sci. USA, Proc. Natl. Acad. Sci. USA, 69 vol. , 2110 (1972) and Gene (17), 107 (1982).

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

To transform yeast, for example, Mesos in Enzymology (Meth ods in Enzymology), vol. 194, 182—187 (1991), Proc. Natl. Acad. Sci. Proc. Natl. Acad. Sci. USA), Vol. 75, 1929 (1978).

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

 Transformation of animal cells can be performed, for example, by the methods described in Cell Engineering Annex 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973). It can be performed according to the method.

 Thus, a transformant transformed with the expression vector containing DNA encoding the G protein-coupled receptor protein is obtained.

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

 As a medium for culturing Escherichia sp., For example, M9 medium containing glucose and casamino acid (Miller, Journal 'Ob' Experimen'in ',' Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3j3-indolyl atalilic acid can be added in order to make the promoter work efficiently.

When the host is a bacterium belonging to the genus Escherichia, the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be added. When the host is a bacterium belonging to the genus Bacillus, the cultivation is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.

When culturing a transformant in which the host is yeast, for example, Burkholder's minimal medium [Bostian, KL et al., "Procedures. Natl. Acad. Sci. USA, 77, 4505 (1980) "and an SD medium containing 0.5% casamino acid [Bitter, GA et al., Proc. _Nat'l Acad. Sci. USA, 81, 5330 (1984) J. The pH is preferably adjusted to about 5 to 8. Culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours.

 When culturing a transformant in which the host is an insect cell or an insect, the culture medium is Grace's Insect Medium (Grace, TCCC, Nature, 195, 788 (1962)).

Suitable additives such as immobilized 10% serum are used. The pH of the culture medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C 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, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], DMEM Medium [Virology, 8 volumes, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association (1992), 519 (1967) 3, 199 medium [Proceding of the Society for the Biological Medicine], 73, 1 (1950)], etc. The culture is preferably about 6 to 8. Culture is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and agitation are used as necessary.

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

To separate and purify the receptor protein of the present invention from the above culture, for example, It can be performed by the following method.

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

 Purification of the receptor protein contained in the culture supernatant or extract thus obtained can be carried out by appropriately combining known separation and purification methods. These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method using difference in molecular weight, Method using charge difference such as ion exchange chromatography, Method using specific affinity such as affinity mouth chromatography, Reverse phase high-performance liquid mouth chromatography, etc. A method utilizing the difference in the hydrophobicity of the polymer, a method utilizing the difference in the 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 into a salt by a known method or a method analogous thereto. It can be converted into a free form or another salt by an analogous method.

 The receptor protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein-modifying enzyme before or after purification. As the protein-modifying enzyme, for example, trypsin, chymotrypsin, arginyl endopeptidase, proteinase, glycosidase and the like are used.

The activity of the receptor protein of the present invention or a salt thereof thus produced can be determined by a binding experiment with a labeled ligand and an enzyme antibody using a specific antibody. It can be measured by means such as a sail.

 The antibody against the receptor protein of the present invention or its partial peptide or its salt may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the receptor protein or its partial peptide or its salt of the present invention. I ’m sorry.

 The receptor protein of the present invention or its partial peptide or its salt (hereinafter referred to as

The antibody against (there may be abbreviated as the receptor protein or the like of the present invention) can be produced according to a known antibody or antiserum production method using the receptor protein or the like of the present invention as an antigen.

 [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 about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mouse and rat are preferably used.

When producing monoclonal antibody-producing cells, a warm-blooded animal immunized with the antigen, for example, a mouse with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization. By fusing 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 a labeled receptor protein or the like described below with the antiserum, 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 Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used. Myeloma cells include, for example, NS-1, P3U1, SP2 / 0, etc. i P 3 U 1 is preferably used. 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 PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%. The cell fusion can be carried out efficiently by incubating at about 20 to 40 ° C, preferably about 30 to 37 ° C for about 1 to 10 minutes.

 Various methods can be used for screening monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is collected on a solid phase (eg, a microplate) to which antigens such as receptor proteins are directly or adsorbed together with a carrier. Then, an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice) or protein A labeled with a radioactive substance or an enzyme, and then added to the solid phase Monoclonal antibody detection method, hybridoma culture supernatant was added to a solid phase to which anti-immune globulin antibody or protein A was adsorbed, and a receptor protein etc. labeled with radioactive substances, enzymes, etc. were added and bound to the solid phase. A method for detecting a monoclonal antibody is exemplified.

 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 1-20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or hybridoma culture A serum-free medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used. The culturing temperature is usually 20 to 40 ° (preferably about 37 ° C.) The culturing time is usually 5 days to 3 weeks, preferably 1 to 2 weeks. The antibody titer of the culture supernatant of the hybridoma can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.

 (b) Purification of monoclonal antibody

Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies. , DEAE) adsorption / desorption method, ultracentrifugation method, Filtration, an antigen-binding solid phase or a specific purification method in which an antibody alone is collected using an active adsorbent such as protein A or protein G, and the bond is dissociated to obtain the antibody.

 (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 (antigen such as the protein of the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting an antibody-containing substance against a protein or the like and separating and purifying the antibody.

 Regarding the complex of the carrier protein and the immunizing antigen used to immunize mammals, the mixing ratio of the carrier protein and the hapten is different from that of the hapten immunized by cross-linking the carrier. Any antibody may be cross-linked at any ratio as long as it can be efficiently used.For example, the weight ratio of serum albumin, thyroglobulin, key hornore limpet, hemocyanin, etc. A method of coupling about 0.1 to 20, preferably about 1 to 5 with respect to 1 hapten is used.

 In addition, various condensing agents can be used for force coupling between the hapten and the carrier. For example, glutaraldehyde ゃ carpoimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like 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 polyclonal antibodies can be performed according to the same immunoglobulin separation and purification method as described above for monoclonal antibodies. You.

 The receptor protein or its salt, its partial peptide or its salt, and the DNA encoding the receptor protein or its partial peptide of the present invention are as follows: (1) The ligand (for the G protein-coupled receptor protein of the present invention) (2) prophylactic and / or therapeutic agents for diseases associated with dysfunction of the G protein-coupled receptor protein of the present invention, (3) genetic diagnostic agents,

(4) a screening method for a compound that changes the expression level of the receptor protein or its partial peptide of the present invention; (5) prevention of various diseases containing a compound that changes the expression level of the receptor protein or its partial peptide of the present invention. And Z or a therapeutic agent, (6) a method for quantifying a ligand for the G protein-coupled receptor protein of the present invention, (7) a compound that changes the binding property between the ligand and the G protein-coupled receptor protein of the present invention ( (8) Various methods containing compounds (agonist, antagonist) that change the binding property between the G protein-coupled receptor protein and the ligand of the present invention. A preventive and / or therapeutic agent for a disease, (9) the receptor protein of the present invention or a portion thereof; (10) a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, (11) the amount of the receptor protein of the present invention or its partial peptide in the cell membrane A prophylactic and / or therapeutic agent for various diseases containing a compound that alters the following: (12) neutralization by an antibody against the receptor protein of the present invention or its partial peptide or a salt thereof; (13) the G protein-conjugated type of the present invention It can be used for producing non-human transgenic animals having DNA encoding the receptor protein.

In particular, by using the receptor-binding assay system using the recombinant G protein-coupled receptor protein expression system of the present invention, ligands for G protein-coupled receptors specific to human and non-human mammals can be obtained. Compounds that alter the binding properties (eg, agonists, antagonists, etc.) can be screened, and the agonists or antagonists can be used as agents for preventing or treating various diseases. The receptor protein or partial peptide of the present invention or a salt thereof (hereinafter, may be abbreviated as the receptor protein of the present invention, etc.), the DNA encoding the receptor protein of the present invention or the partial peptide thereof (hereinafter, referred to as the present invention). The use of an antibody against the receptor protein or the like of the present invention (hereinafter abbreviated as the antibody of the present invention) may be specifically described below.

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

 The receptor protein of the present invention or its salt or the partial peptide or its salt of the present invention is useful as a reagent for searching for or determining a ligand (agonist) for the receptor protein of the present invention or its salt. It is.

That is, the present invention provides a method for determining a ligand to the receptor protein of the present invention, which comprises contacting the receptor protein of the present invention or a salt thereof or a partial peptide of the present invention or a salt thereof with a test compound. . Test compounds include known ligands (eg, angiotensin, bombesin, cannabinoid, cholecystokinin, gnoletamine, serotonin, melatonin, neuropeptide Y, opioid, purine, pasoprescin, oxytocin, ΡACAP (eg, PACAP 27 , PACAP 38), secretin, glucagon, calciton, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (Pasoactive Intestinal and Rerated Polypeptide), somatostatin, dopamine, motilin, amiline, Bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfiber Millie (eg, CXC chemokine subfamily such as IL_8, GROa, GRO / 3, GROy, NAP-2, ENA-78, GCP-2, PF4, IP-10, Mig, PBSF / SDF-1; MCAF / MCP_1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, ΜΙ Ρ-1α, ΜΙ Ρ-1 | 8, HCC-1, 1, ΜΙΡ-3α / LARC, ΜΙΡ-3β / ELC , I- 309, TARC, MIPF-1, MIPF-2 / eotaxin-2, MDC, DC-CK1 / PARC, SLC, etc. CC chemokine subfamily; C chemokine subfamily such as lymphotactin; CX 3 chemokine such as fractalkine Subfamily, etc.), endoselin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosine monomonophosphate, etc.) Tissue extracts of human or non-human mammals (eg, mouse, rat, pig, porcine, sheep, monkey, etc.), cell culture supernatant, and the like are used. For example, the tissue extract, cell culture supernatant and the like are added to the receptor protein of the present invention, and fractionation is performed while measuring cell stimulating activity and the like, so that a single ligand can be finally obtained.

Specifically, the method for determining a ligand of the present invention comprises constructing an expression system for a mosquito or recombinant receptor protein using the receptor protein of the present invention or a partial peptide thereof or a salt thereof, and using the expression system. By using the receptor-binding Atsei system, which binds to the receptor protein of the present invention, it stimulates cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular c A compound having an activity of promoting or suppressing GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. (for example, peptides, Protein, non-peptidic 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 a partial peptide thereof, for example, the amount of the test compound bound to the receptor protein or the partial peptide, It is characterized by measuring irritation activity.

 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 a salt thereof or the partial peptide of the present invention or a salt thereof, the labeled test compound or the salt thereof, or the salt thereof. The receptor protein of the present invention, which is characterized by measuring the amount of binding to a partial peptide or a salt thereof. Is the method of determining the ligand for the salt,

 (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 receptor protein or a salt thereof of the present invention,

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

(4) When a test compound is brought into contact with a cell containing the receptor protein of the present invention, cell stimulating activity via the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular c AMP production, intracellular c GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. A method for determining a ligand for the receptor protein or a salt thereof of the present invention, which comprises measuring

細胞 Receptor protein-mediated cell stimulating activity when a test compound is brought into contact with a receptor protein expressed on a cell membrane by culturing a transformant containing DNA encoding the receptor protein of the present invention. (For example, 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, c — A method for determining a ligand for the receptor protein or a salt thereof of the present invention, which comprises measuring the activity of promoting or suppressing fos activation, reduction of H, and the like.

In particular, it is preferable to carry out the above-mentioned tests 1 to 3 after conducting the tests 1 to 3 above and confirming that the test conjugate binds to the receptor protein of the present invention. First, the receptor protein used in the method for determining a ligand may be any containing the above-described receptor protein of the present invention or the partial peptide of the present invention. Any receptor protein may be used, but a receptor protein expressed in large amounts using animal cells is suitable.

 The receptor protein of the present invention is preferably produced by expressing DNA encoding the 1S receptor protein, which is used in the above expression method, in mammalian cells or insect cells. As the DNA fragment encoding the protein portion of interest, cDNA is usually used, but is not necessarily limited thereto. For example, 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 a host animal cell and express them efficiently, the nuclear polyhedrosis virus belonging to baculovirus using the DNA fragment as an insect host is required. (Nuclear polyhedrosis virus; NPV) polyhedrin promoter, SV40-derived promoter, retrovirus / less promoter, metamouth thionine promoter, human heat shock promoter, cytomegaloy / less promoter, SRα promoter, etc. It is preferably incorporated downstream. 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. Chera.), 267, 19555-19559, 1992]. it can.

 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 fixed with daltaraldehyde, formalin, or the like. The fixing method can be performed according to a known method.

 The cell containing the receptor protein of the present invention refers to a host cell expressing the receptor protein of the present invention. As the host cell, Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like are used.

Many cell membrane fractions are obtained by crushing cells and then using known methods. Refers to the fraction included. The cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Warlinda blender-Polytron (Kinema tica), crushing by ultrasonic waves, pressing the cells while pressing with a French press, etc. There is a broken frame by ejecting from a thin nozzle. For the cell membrane fractionation, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further spun at a higher speed (15000 to 30000 rm) for 30 minutes to 2 hours. After centrifugation, the obtained precipitate is used as a membrane fraction. The membrane fraction is rich in the expressed receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.

The amount of the receptor protein in the cells containing the receptor protein or in the membrane fraction thereof is preferably 10 ³ to 10 ⁸ molecules, and more preferably 10 ⁵ to 10 ⁷ molecules per cell. 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. .

 In order to carry out the above 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.

The labeled test compound, ^[3 H], ^[125 I], ^[14 C], ^[35 S] labeled angiotensin etc., bombesin, cannabinoid, cholecystokinin, grayed Noretamin, serotonin, melatonin, neuropeptide Peptide Y, opioids, purines, vasopressin, oxotocin, PACAP (e.g., PACAP 27, PACA P38), secretin, glucagon, kardinin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (vasoak) Active Intestinal and Retained Polypeptides), Somatosta Chin, dopamine, motilin, amylin, bradykinin, CGRP

Peptide, leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine one-perfamily (eg, IL-8, GROa, GRO] 3, GROy _s NAP—2, ENA— 7 8, CXC chemokine subfamily such as GC P-2, PF 4, IP-10, Mig, PBSF / SDF-1; MCAF / MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, ΜΙ Ρ_1α, MIP—lj3, HCC—1, MIP-3a / LARC, MIP-3β / ELC, 1—309, TARC, MIPF—1, MIPF-2 / eot axin-2, MDC, DC-CK1 / PARC, CCLC chemokines such as SLC, etc .; 1 Cmpokine subfamily such as ymphotactin; CX3 chemo such as fractalkine. , Histamine, neurotensin, TRH, pancreatic po Bae Putai de, galanin, Li Zohosufachijin acid (LPA), including Sufuingoshin 1 monophosphate is preferred. Specifically, to carry out the method for determining a ligand for the receptor protein of the present invention or a salt thereof, first, a cell or a membrane fraction of the cell containing the receptor protein of the present invention is suspended in a buffer suitable for the determination method. Prepare a sample of the receptor by turbidity. The buffer may be any buffer such as a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-HCl buffer which does not inhibit the binding between the ligand and the receptor protein. To reduce non-specific binding, various proteins such as surfactants such as CHAPS, Tween-80 ™ (Kao Ichi Atlas), digitonin, and deoxycholate, serum albumin, and gelatin are used as buffers. Can be added. Furthermore, a protease inhibitor such as PMSF, leptin, E-64 (manufactured by Peptide Research Institute), or pepstatin can be added for the purpose of suppressing the degradation of the receptor and ligand by the protease. 0.01: to the 10 m 1 of the receptor solution, a certain amount (5,000 to 500,000 c pm) of ^[3 H], ^[125 I], ^[14 C], coexist test compounds labeled with a ^[35 S] Let it. Prepare a reaction tube containing a large excess of unlabeled test compound to determine the amount of non-specific binding (NSB). The reaction is carried out at about O ° C to 50 ° C, preferably at about 4 ° (: up to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours. After filtering with a filter paper and washing with an appropriate amount of the same buffer, the radioactivity remaining on the glass fiber filter paper is measured with a liquid scintillation counter or /-counter. A test compound having a count (B-NSB) of less than 0 cpm obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) is used as a ligand (agonist) for the receptor protein of the present invention or a salt thereof. ) Can be selected as

In order to carry out the above-mentioned methods (1) to (4) for determining a ligand for the receptor protein or a salt thereof of the present invention, a cell stimulating activity via the receptor protein (for example, arachidonic acid release, acetylcholine release, intracellular Ca ^{2 +} Release, promotes intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease in pH, etc. Activity or inhibitory activity) can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured in a multiwell plate or the like. Before determining the ligand, replace the medium with a fresh buffer, which is not toxic to cells, and add a test compound, etc., incubate for a certain time, and extract the cells. Alternatively, collect the supernatant, and quantify the generated product according to each method. If 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 the 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 basic production has been increased by forskolin or the like.

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

 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) with 0.05% Min (made by Sigma).

 Sterilize by filtration through a filter with a pore size of 0.45 μπι, and store at 4 ° C, or prepare it before use.

 ② G protein-coupled receptor protein sample

CHO cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at ⁵ × 10 ⁵ holes and cultured for 2 days at 37 ° C., 5% CO ₂ and 95% air.

 ③ Labeled test compound

Commercially available ^[3 H], ^[125 I], ^[14 C], ^[35 S] those labeled with a compound labeled or in a suitable way, such as

 Store the solution in an aqueous solution at 4 ° C or 120 ° C, and dilute to 1 μΜ with the measurement buffer before use. For test compounds that are poorly soluble in water, dissolve in dimethylformamide, DMSO, methanol, etc.

 ④Unlabeled test compound

 Prepare the same labeled compound as 100-1000 times concentrated.

 2. Measurement method

 (1) Wash CHO cells expressing the receptor protein of the present invention cultured in a 12-well tissue culture plate twice with 1 ml of measurement buffer, and add 490 μl of measurement buffer to each well.

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

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

 す る Measure radioactivity using a liquid scintillation counter (Beckman Coulter).

The ligand capable of binding to the receptor protein of the present invention or a salt thereof includes, for example, substances specifically present in the hypothalamus, cerebral cortex, colon cancer, lung cancer, heart, placenta, lung, and the like. Typically, angiotensin, bombesin, cannabinoid, cholecystokinin, glutamine, serotonin, melatonin , Neuropeptide Y, opioid, purine, pasoplethsin, oxotocin, PACAP (e.g., PACAP 27, PACAP 38), secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH , VIP (Vasoactive Intestinal and Retained Polypeptide), Somatostatin, Dopamine, Motilin, Amylin, Bradykinin, CGRP (Calcitonin Gene Reited Peptide), Leuco Trien, Pancreastatin, Prostaglandin, Thromboxane, Adenosine, Adrenaline, Chemokine Superfamily (eg, IL-8, GROa, GRO] 3, GROγ, NAP-2, ΕΝΑ-78, GCP-2, PF4, IP-10, Mig, PBSF / SDF -1, such as CXC chemokine subfamily "; MCAF / MCP-1, MC P-2, MC P-3, MC P_4, eotaxin ヽ RANTE S, MIP-1 CK, MI P-1 j3 _s HCC-1, MIP-3a / LARC MIP-3β / ELC I-309, TARC, MIPF-1 , MIPF-2 / eotaxin-2, MDC, DC-CK1 / PARC, SLC, etc .; CC chemokine subfamily; lymphotactin, etc., C chemokine subfamily; fractalkine, etc., CX3 chemokine subfamily, etc.), endotherin, e Non-gastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), and sphingosine 1-phosphate are used.

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

 In the above method (1), if the ligand for the receptor protein of the present invention becomes clear, depending on the action of the ligand, (1) the receptor protein of the present invention or (2) DNA encoding the receptor protein may be replaced with the present invention. It can be used as a medicament such as a preventive and / or therapeutic agent for diseases 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 due to a decrease in the receptor protein of the present invention in the living body (deficiency of the receptor protein): To supplement the amount of the receptor protein, and (2) (a) encoding the receptor protein of the present invention. The cells are transplanted into the patient by administering the DNA to the patient and expressing it, or (mouth) after introducing and expressing the DNA encoding the receptor protein of the present invention in the target cells. As a result, the amount of receptor protein in the body of the patient can be increased, and the effect of the ligand can be sufficiently exerted. That is, DNA encoding the receptor protein of the present invention is useful as a safe and low-toxic agent for the prevention and / or treatment of diseases associated with dysfunction of the receptor protein of the present invention.

 The receptor protein of the present invention is a novel seven-transmembrane receptor protein in which the orphan receptor RE2 (GenBank accession number AF091890) has about 32% homology at the amino acid sequence level.

 The receptor protein of the present invention or the DNA encoding the receptor protein 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 cell types, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic diseases (eg, diabetes, dyslipidemia, 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 (e.g., allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.), cardiovascular diseases (e.g., hypertension, heart Hypertrophy, angina, myocardial infarction, arteriosclerosis, 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.) , Diabetic respiratory disease (eg, sclera syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis / pulmonary embolism, etc.), digestive system disease (eg, gastric ulcer, duodenal ulcer, gastritis, reflux esophagitis, knee) Inflammation), immune system diseases (for example, autoimmune diseases), infectious diseases (for example, immune dysfunction, pneumonia, citmegavirus, influenza virus, herpes virus, etc., rickettsial infections) , Bacterial infections, etc.). Among them, they are particularly useful for the prevention and / or treatment of Alzheimer's disease.

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

 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-mentioned prophylactic or therapeutic agent, the DNA of the present invention may be used alone or in a retrovirus vector. After insertion into an appropriate vector such as an adenovirus vector or an adenovirus-associated virus vector, it can be carried out according to a conventional method. The DNA of the present invention can be administered as it is or together with an adjuvant for promoting ingestion, using a gene gun or a catheter such as Hyde-mouth gel force table.

 For example, (1) the receptor protein of the present invention or (2) DNA encoding the receptor protein may be orally administered as tablets, capsules, elixirs, microcapsenoles, or the like, or coated with water, if necessary. 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 recognized together with known physiologically recognized carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like. 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. Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing pudose and other adjuvants (eg, D-sorbitol, D-mannitol, Thorium, etc.), and suitable solubilizers, for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 ™, HCO — 50) may be used in combination. 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 therapeutic agents include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), anti-acidic agents and the like. The prepared injection solution is usually filled in a suitable ampoule. The preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.

 The dosage of the receptor protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like. However, in the case of oral administration, for example, in a cancer patient (as 60 kg), the dose is usually 1 day. About 0.1 to: 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. For example, in the case of an injection, it is usually used, for example, in a cancer patient (as 60 kg). It is convenient to administer about 0.01 to 30 mg / day, preferably about 0.1 to 20 mg / day, more preferably about 0.1 to 10 mg / day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

The dose of the DNA of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like. About 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the subject to be administered, target organ, symptoms, administration method, and the like. Is about 0.01 to 3 per day It is convenient to administer about 0 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, a dose equivalent to 60 kg can be administered. (3) Gene diagnostic agent

 The DNA of the present invention can be used as a probe to produce the receptor of the present invention in human or non-human mammals (eg, rat, mouse, rabbit, sheep, pig, pig, cat, dog, monkey, etc.). Abnormality (genetic abnormality) of DNA or mRNA encoding a protein or a partial peptide thereof can be detected. For example, damage, mutation or reduced expression of the DNA or mRNA, or decrease of the DNA or It is also useful as a gene diagnostic agent for increasing or overexpressing mRNA.

 The above-described genetic diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Prosessing Ops. The National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America), Vol. 86, pp. 2766-2770 (1989)) be able to. (4) A method for screening a compound that changes the expression level of the receptor protein or a partial peptide thereof of the present invention

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

 That is, the present invention provides, for example, (i) non-human mammal blood, and specific organs.

(3) Tissue or cells isolated from an organ, or (ii) Receptor protein of the present invention contained in a transformant or the like. Receptor protein of the present invention or a portion thereof by measuring the mRNA amount of peptide. A method for screening a compound that changes the expression level of a peptide is provided. The measurement of the mRNA amount 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 (eg, mice, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerotic rabbits) 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 tissue or cells isolated from a specific organ (eg, brain, lung, colon, etc.) or organ power is obtained.

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

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

 Screening of a compound that changes the expression level of the receptor protein or a partial peptide thereof according to the present invention includes:

 (i) A predetermined time before giving a drug or physical stress to a normal or disease model non-human mammal (30 minutes to 24 hours before, preferably 30 minutes to 12 hours before, more preferably 1 hour to 12 hours before Hours 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 simultaneously with drug or physical stress The test compound is administered, 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 receptor of the present invention contained in cells Quantification and analysis of the amount of mRNA: of the protein or its partial peptide,

(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 to 7 days, preferably 1 to 3 days, more preferably Two to three days later), it can be performed 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 is a compound having an action of changing the expression level of the receptor protein or a partial peptide thereof of the present invention. By increasing the expression level of the receptor protein or its partial peptide, the cell stimulating activity via the G protein-coupled receptor (eg, arachidonic acid release, acetylcholine release, intracellular ^{Ca2 +} release, intracellular c) AMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity to promote or suppress pH reduction, etc.) By reducing the expression level of the receptor protein of the present invention or its partial peptide, Is a compound that attenuates stimulating activity.

 Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like, and 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 attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein of the present invention.

 When a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, 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-described pharmaceuticals containing the receptor protein of the present invention.

 The preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.

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 cancer patients (as 60 kg), about 0 .; to 100 mg per day, preferably about 1.0 to 50 mg per day. More preferably about 1.0-20 mg. In the case of parenteral administration, the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc. 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 alters the expression level of the receptor protein or its partial peptide of the present invention.

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

 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, brown) Cell types, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic disorders (eg, diabetes, dyslipidemia, diabetic complications, obesity, Gout, cataract, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), inflammatory disease ( For example, allergies, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc., cardiovascular diseases (eg, hypertension, cardiac hypertrophy, stenosis) Diseases, myocardial infarction, arteriosclerosis, etc.), respiratory diseases (eg, cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis, pulmonary embolism, etc.), digestive system diseases (eg, gastric ulcer, duodenal ulcer, gastritis) , Reflux esophagitis, knee inflammation, etc.), immune system disease (eg, autoimmune disease, etc.), or infectious disease (eg, immune dysfunction, pneumonia, cytomegal virus, influenza virus)

> Viral infections, rickettsial infections, bacterial infections, etc.) And the like. Among these, it is particularly useful for prevention or treatment of Alzheimer's disease.

 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.

 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 can be used together with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in a unit dosage form generally required for the practice of preparations. It can be manufactured by mixing with. 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. And sucrose, lactose or saccharine, 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 solution for injection include physiological saline, isotonic solution containing glucose and other auxiliary agents (eg, D-sorbitol, D-mannitol, sodium salt, etc.) and the like. In combination with solubilizers, such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), non-ionic surfactants (eg, Polysorbate 80 ™, HCO-50) Is also good. 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. Les, o

 Examples of the prophylactic and therapeutic agents include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (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. The preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, horses, 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. However, in the case of oral administration, for example, in a cancer patient (as 60 kg), the daily dose is generally one day. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like.For example, in the form of an injection, it is usually used, for example, in patients with cancer (60 kg). 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 amount converted per 60 kg can be administered.

(6) Ligand for G Protein-Coupled Receptor Protein of the Present Invention Since the receptor protein and the like of the present invention have a binding property to the ligand, the ligand concentration in the living body can be quantified with high sensitivity. .

 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 test sample can be measured by bringing the test sample into contact with the receptor protein of the present invention. Specifically, for example, it can be used in accordance with the method described in (1) or (2) below or a method analogous thereto.

(1) Hiro Irie "Radio No Tsutsui" (Kodansha, published in 1974) 2) Hiroshi Irie, "Radio Imano 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

By constructing an expression system for the receptor protein or the like of the present invention or a recombinant receptor protein or the like, and using a receptor binding system using the expression system, the ligand and the receptor protein or the like of the present invention can be obtained. (Eg, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc.) or salts thereof can be efficiently screened for such compounds. Cell stimulating activity via G protein-coupled receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential Fluctuation, phosphorylation of intracellular proteins, activation of c-fos, reduction of pH, etc. (A so-called agonist against the receptor protein of the present invention), (mouth) a compound not having the cell stimulating activity (a so-called antagonist against the receptor protein of the present invention), (no) Includes compounds that enhance the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (2) compounds that decrease the binding force between the ligand and the G protein-coupled receptor protein of the present invention. The compound (a) is preferably screened by the ligand determination method described above).

That is, the present invention relates to (i) a 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 (ii) a receptor protein of the present invention or its partial peptide or a salt thereof, Ligand and test A method for screening a compound or a salt thereof that alters the binding property between a ligand and a receptor protein or a partial peptide thereof or a salt thereof of the present invention, which is compared with the case of contacting with a compound. I will provide a. The screening method of the present invention is characterized in that in the cases (i) and (i), for example, the amount of a ligand bound to the receptor protein or the like, the cell stimulating activity, and the like are measured and compared.

 More specifically, the present invention provides

 (1) Binding of the labeled ligand to the receptor protein, etc., when the labeled ligand is brought into contact with the receptor protein, etc. of the present invention, and when the labeled ligand and test compound are brought into contact with the receptor protein, etc., of the present invention. A method for screening a compound or a salt thereof that changes the binding property between a ligand and a receptor protein or the like of the present invention, which is characterized by measuring and comparing the amount of the ligand; When the cells are brought into contact with the cells containing them or the membrane fraction of the cells, and when the labeled ligand and the test compound are brought into contact with the cells containing the receptor protein of the present invention or the membrane fraction of the cells. The amount of the labeled ligand bound to the cell or the membrane fraction is measured and compared. Ligand and method of screening a compound or its salt that alters the binding property between the receptor protein or the like of the present invention, characterized in that,

 (3) When the labeled ligand is brought into contact with a receptor protein or the like expressed on the cell membrane by culturing the transformant containing the DNA of the present invention, and when the labeled ligand and the test compound contain the DNA of the present invention. When the transformant to be cultured 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 or the like is measured and compared. A method for screening a compound or a salt thereof that alters the binding between the ligand defined as ^ and the receptor protein of the present invention,

化合物 A compound that activates the receptor protein of the present invention (eg, a ligand for the receptor protein of the present invention) is brought into contact with a cell containing the receptor protein of the present invention. Receptor-mediated cell stimulating activity (e.g., arachidonic acid release, acetylcholine release, intracellular C a ²⁺ release, intracellular ^cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein And the activity of promoting or suppressing c-fos, c-fos reduction, pH reduction, etc. A method for screening a compound or a salt thereof that changes the binding property of

の The present invention, in which 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 expressed on a cell membrane by culturing a transformant containing the DNA of the present invention. A compound that activates the receptor protein or the like of the present invention and a test compound were expressed on the cell membrane by culturing a transformant containing the DNA of the present invention when the compound was brought into contact with the receptor protein or the like, and when the transformant containing the DNA of the present invention was cultured. Receptor-mediated cell stimulating activity (for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular c when brought into contact with the receptor protein or the like of the present invention) GMP production, inositolonoleic acid production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c_fos, decrease in pH The present invention provides a method for screening a compound or a salt thereof, which alters the binding property between a ligand and a receptor protein of the present invention, which is characterized by measuring and comparing the activity of promoting or suppressing the thrombosis.

 Prior to obtaining the receptor protein or the like of the present invention, when screening for G protein-coupled receptor agonists or antagonists, first, cells, such as rats, containing G protein-coupled receptor protein, mitogen, or a cell membrane fraction thereof are used. To obtain candidate compounds (primary screening), and then to test whether the candidate compounds actually inhibit the binding between human G protein-coupled receptor protein and ligand (secondary screening) ) Was required. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins will also be present, so it has been difficult to directly screen an agonist or an antagonist for the target receptor protein in practice.

However, for example, by using the human-derived receptor protein of the present invention, primary screening is not required, and a compound that inhibits binding between a ligand and a G protein-coupled receptor protein can be efficiently screened. In addition, if the compound screened is an agonist or antago It is possible to easily evaluate whether it is a nest.

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

 First, the receptor protein of the present invention used in the screening method of the present invention may be any as long as it contains the above-described receptor protein of the present invention. A cell membrane fraction of a mammalian organ containing However, since human-derived organs are particularly difficult to obtain, it is suitable to use human-derived receptor proteins and the like, which are expressed in large amounts using recombinants, for screening.

 The above method is used for producing the receptor protein and the like of the present invention, but it is preferable to carry out the expression by expressing the DNA of the present invention in mammalian cells and insect cells. CDNA is used as the DNA fragment encoding the protein portion of interest, but is not necessarily limited thereto. For example, a gene fragment or a synthetic DNA may be used. In order to introduce the DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, the DNA fragment must be prepared by transferring the DNA fragment to a nuclear polyhedrosis virus (nuclear polyhedrosis virus belonging to baculovirus using an insect as a host). Hedrosis virus (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus / less promoter, meta-mouth thionine promoter, human heat shock promoter, cytomegaloinores promoter, downstream of SRa promoter, etc. Incorporation is preferred. The amount and quality of the expressed receptor can be examined by a known method. For example, the method can be carried out according to the method described in the literature [Nambi, P. et al., The 'Journal of Ob, Biological' Chem., 267, 19555-19559, 1992]. it can.

Therefore, in the screening method of the present invention, the protein containing the receptor protein or the like of the present invention may be a receptor protein or the like purified according to a known method, or a cell containing the receptor protein or the like. May be used, or a membrane fraction of cells containing the receptor protein or the like may be used. Fixed with aldehyde, formalin, etc. You can do it. The immobilization method can be performed according to a known method.

 The cell containing the receptor protein or the like of the present invention refers to a host cell that expresses the receptor protein or the like, and the host cell is preferably Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, or the like.

 The cell membrane fraction refers to a cell membrane-rich fraction obtained by disrupting cells and then obtained by a known method. Cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Warlinda blender-Polytron (manufactured by Kinema tica), crushing by ultrasonic waves, pressing cells with a French press, etc. And crushing by ejecting from a thin nozzle. For the cell membrane fractionation, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1-10 minutes), and the supernatant is further centrifuged at a high speed (15000-30000 rpm) for 30 minutes to 2 hours. The resulting precipitate is used as the membrane fraction. The membrane fraction is rich in expressed components such as receptor protein and cell-derived phospholipids and membrane proteins.

The amount of the receptor protein in the cell or membrane fraction containing the receptor protein or the like is preferably 10 ³ to 10 ⁸ molecules per cell, and more preferably 10 ⁵ to 10 ⁷ molecules per cell. 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 port. Become

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

 As the receptor protein fraction, a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto is desirable. 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, [], [ ¹²⁵ 1], [ ¹⁴ C], [ ³⁵ S], etc. .

 61 Known ligands are used.

Specifically, to screen for a compound that alters the binding between the ligand and the receptor protein of the present invention, a cell containing the receptor protein of the present invention or a membrane fraction of the cell is first screened. Prepare the receptor protein preparation by suspending it in a suitable buffer. The buffer may be any buffer that does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8) and a buffer of tris-hydrochloride. In addition, a surfactant such as CHAPS, Tween-80 ™ (Kao Ichi Atlas), digitonin, and dexcholate can be added to the buffer for the purpose of reducing non-specific binding. Furthermore, a protease inhibitor such as PMS F, leptin, E-64 (manufactured by Peptide Research Laboratories), or papstatin can be added for the purpose of suppressing the degradation of the receptor or ligand by the protease. 0.01 to:. 10 m to 1 of the receptor solution, a certain amount to添Ka卩the labeled ligand (5,000 to 500,000 cpm), nonspecific coexist 1 (gamma ⁴ to 10-test compound ^1Q M simultaneously Prepare a reaction tube containing a large excess of unlabeled ligand to determine the target binding (NSB) The reaction is performed at about 0 ° C to 50 ° C, preferably at about 4 ° C to 37 ° C. The reaction is performed for 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours After the reaction, the mixture is filtered with a glass fiber filter paper and the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured by a liquid scintillation counter or γ- When the non-specific binding amount (NS.) Minus the non-specific binding amount (NSB) is subtracted from the count when no antagonist is present (Β.), The specific binding amount (B— NSB) Power It can be selected as a powerful candidate substance.

In order to carry out the above-mentioned methods (1) to (4) for screening a compound which changes the binding property between a ligand and the receptor protein of the present invention, for example, cell stimulating activity via the receptor protein (eg, arachidonic acid release, acetylcholine Release, intracellular ^{Ca2 +} release, intracellular cAMP generation, intracellular cGMP generation, inositol monophosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, decrease in pH, etc. Activity to promote or inhibit) Alternatively, the measurement can be performed using a commercially available measurement kit.

 Specifically, first, cells containing the receptor protein or the like of the present invention 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 the cells or collect the supernatant. The products produced are quantified according to the respective method. If the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult due to the presence of a degrading enzyme contained in the cells, an inhibitor for the degrading enzyme is added to the mixture to perform an assay. Well ,. In addition, an activity such as cAMP production suppression can be detected as a production suppression effect on cells whose basal production has been increased with forskolin or the like.

 For screening by measuring the cell stimulating activity, cells expressing an appropriate receptor protein are required. As a cell expressing the receptor protein of the present invention, a cell line having the natural type receptor protein of the present invention, a cell line expressing the above-mentioned recombinant receptor protein or the like is desirable.

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

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

 Examples of the screening kit of the present invention include the following. 1. Screening reagent

 ①Measurement buffer and washing buffer

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

Sterilize through a 0.45 πι filter and store at 4 ° C or use It may be prepared.

 ② G protein-coupled receptor preparation

CHO cells expressing the receptor protein of the present invention were subcultured on a 12-well plate with ⁵ × 10 ⁵ Z-wells, and cultured for 2 days at 37 ° C., 5% CO ₂ and 95% air.

 ③ Labeled ligand

Commercially available ^[3 H], ^[125 I], ^[14 C], ^[35 S] those states of labeled ligand solution and stored at 4 ° C or single 20 ° C, etc., measurement buffer at use Dilute to 1 with the liquid.

 ④Ligand standard solution

 Dissolve the ligand in PBS containing 0.1% ゥ serum albumin (manufactured by Sigma) so that the concentration of the ligand becomes 1 mM, and store at 20 ° C.

 2. Measurement method

 (1) Wash CHO cells expressing the receptor protein of the present invention cultured in a 12-well tissue culture plate twice with 1 ml of the measurement buffer, and add 490 μl of the measurement buffer to each well.

^② 10- ³ ~10- 1β after adding test compound solution 5 mu 1 of Micromax, the labeled ligand 5 mu 1 was added and reacted at room temperature for 1 hour. A supplementary 5 1 ligands 10- ³ Micromax in place of the test compound to determine the amount of non-specific binding.

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

 ④ Measure the radioactivity using a liquid scintillation counter (manufactured by Beckman Coulter), and determine the Percent Maximum Binding (PMB) by the following formula.

 PMB = [(B-NSB) / (B. One NSB)] xlOO

 PMB: Percent Maximum Binding

 B: Value when the sample is added

 NS B: Non-specific Binding

B 0: maximum binding amount The compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding property between a ligand and the receptor protein or the like of the present invention. ) Cell stimulating activity via G protein-coupled receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, A compound having an activity of promoting or suppressing cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fOS, reduction of pH, etc. (a so-called agonist against the receptor protein of the present invention) (Mouth) a compound having no cell stimulating activity (so-called antagoni against the receptor protein of the present invention) (C) a compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (2) reduces the binding force between the ligand and the G protein-coupled receptor protein of the present invention. It is a compound.

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

 Since the agonist against the receptor protein or the like of the present invention has the same action 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 depending on the ligand activity.

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

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

 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 of the present invention.

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

 The preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals (eg, rats, egrets, sheep, pigs, pigs, 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. However, in the case of microbial administration, in general, for example, in a cancer patient (60 kg), It is about 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg. For parenteral administration, the single dose varies depending on the subject, target organ, symptoms, administration method, and the like.For example, in the form of an injection, it is usually used, for example, in cancer patients (60 kg). 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 amount converted per 60 kg can be administered.

(8) A prophylactic and / or therapeutic agent for various diseases containing a compound (agonist, antagonist) that alters the binding property between a G protein-coupled receptor protein and a 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, digestive function, and cardiac function. Accordingly, compounds (agonists, antagonists) that alter the binding property between the receptor protein of the present invention and the ligand, and ligands for the receptor protein of the present invention, are useful for diseases associated with dysfunction of the receptor protein of the present invention. It can be used as a prophylactic or therapeutic agent.

Examples of diseases 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, brown) Cell type, primary Rudosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic diseases (for example, diabetes, dyslipidemia, diabetic complications, obesity, gout, cataract, high fat) ), 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 disease (eg, allergy, asthma) , Rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc., cardiovascular diseases (eg, hypertension, cardiac hypertrophy, angina, myocardial infarction, arteriosclerosis, etc.), respiratory diseases ( For example, 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, , Self-exempt Epidemic diseases) or infectious diseases (eg, immune dysfunction, pneumonia, viral infections such as cytomeganoreinoles, influenza virus, herpes virus, rickettsial infections, bacterial infections, etc.). Among these, it is particularly useful for the prevention and / or treatment of Alzheimer's disease.

 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 can be aseptically mixed with tablets, capsules, elixirs ij, microcapsules and the like, if necessary, which are sugar-coated, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections such as aqueous solutions or 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. Which is 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 preparations for injection are formulated according to standard pharmaceutical practice, such as by dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil, and the like. Can be. Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other auxiliary agents (eg, D-sorbitol, D-mannitol, sodium salt, etc.) and the like. Solubilizers, such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 ™, HCO-50) May be. Examples of the oily liquid include sesame oil and soybean oil.

It may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.

 The prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, 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. Further, the above-mentioned prophylactic / therapeutic agent can be used in combination with an appropriate drug, for example, as a DDS preparation specifically targeting an organ or tissue in which the receptor protein of the present invention is highly expressed.

 The preparations obtained in this way are safe and have low toxicity, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, rabbits, cats, dogs, dogs ) 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.:! To 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. Is about 0 per day It is convenient to administer about 01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0 :! to about 10 mg 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 or the like of the present invention, it may be used for quantification of the receptor protein or the like of the present invention in a test solution, particularly for quantification by a sandwich immunoassay. Can be. 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 or the like 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), it is preferable that one antibody is an antibody that recognizes the N-terminal of the receptor protein or the like of the present invention, and the other antibody is an antibody that reacts with the C-terminal of the receptor protein or the like of the present invention. .

In addition to measuring the receptor protein and the like of the present invention 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), detection by tissue staining and the like is also possible. it can. 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 assay method using an antibody against the receptor protein or the like of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of receptor protein) in the test solution. Any method that can be used to detect the amount of γ by chemical or physical means and calculate this from a standard curve prepared using a standard solution containing a known amount of antigen A measuring method may be used. For example, nephelometry, a competitive method, an immunometric method and a sandwich method are preferably 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 specific activity of 1 "is preferable. For example, monogalactosidase, j3-dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. For example, fluorescamine, fluorescein isothiocyanate, etc. As the luminescent substance, for example, luminol, luminol derivative, luciferin, lucigenin, etc. Further, binding of antibody or antigen to labeling agent Alternatively, a biotin-avidin system can be used.

 For the insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes 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, a 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 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 a receptor protein or the like by the sandwich method of the present invention, As the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction, an antibody having a different binding site such as a receptor protein is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is preferably other than the C-terminal. For example, an antibody that recognizes the N-terminal 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, after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated ( (B / F separation) Measure the amount of any of B, F, and label and quantify the amount of antigen in the test solution. In this reaction method, a soluble antibody was used as the antibody, a liquid phase method using polyethylene glycol for B / F separation, a second antibody against the above antibody, and a solid-phased antibody as the first antibody. Alternatively, a solid-phase 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 fixed amount of the labeled antibody, and then the force for separating the solid phase and the liquid phase, or The antigen in the medium is allowed to react with an excessive amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase. Then, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.

 In nephelometry, the amount of insoluble sediment 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 a small amount of sediment cannot be obtained, laser nephrometry utilizing laser scattering is preferably used.

 In applying these individual immunological measurement methods to the measurement 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 the usual conditions and the operation method in each method to the ordinary technical consideration of those skilled in the art. For details of these general technical means, reference can be made to reviews and written books.

(Kodansha, published in 1974), edited by Hiroshi Irie "Radio Simnoase (Kodansha, published in 1979), edited by Eiji Ishikawa et al. “Enzyme immunoassay” (Medical Publishing, published in 1978), Eiji Ishikawa et al. “Enzyme immunoassay” (second edition) (Medical Publishing, (Published in 1977), edited by Eiji Ishikawa et al. “Enzyme immunoassay” (3rd edition) (Medical Publishing, published in 1987), “Methods in ENZYMOLOGY” Vol. 70 ( Vol. 73 (Immunochemical Techniques (Part B)), ibid.Vol. 74 (Immunochemical Techniques (Part C)), ibid.Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid. Ibid.Vol.92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), Ibid.Vol.121 (Immunochemical Techniques (Part I: Hybridoma TechnoloGy and Monoclonal Antibodies)) ) Etc.].

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

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

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

(10) A method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in a cell membrane

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

That is, the present invention, for example, (i) After rupture of (1) blood, (2) specific organ, or (3) tissue or cells isolated from the organ of the non-human mammal, the cell membrane fraction is isolated, and the cell membrane fraction 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 the cell membrane by quantifying the receptor protein or its partial peptide,

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

 (i ii) Non-human mammal's (1) blood, (2) specific organ, (3) tissue or cells isolated from the organ, sectioned and immunostaining is used to obtain the receptor protein on the cell surface. A method of screening for a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane by quantifying the degree of staining of the cell membrane.

(iv) Quantifying the degree of staining of the receptor protein on the cell surface by using immunostaining after sectioning a transformant or the like that expresses the receptor protein of the present invention or a partial peptide thereof. Thus, 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 the cell membrane by confirming the protein on the cell membrane. 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 (for example, mice, rats, rabbits, higgs, stags, puppies, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, atherosclerotic puppies) Drugs (eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, waterlogging stress, electric shock, light / dark, low temperature, etc.) After a certain period of time, blood, or specific organs (eg, brain, lungs, large intestine, etc.) or organs Tissue or cells isolated from the cells. The obtained organ, tissue or cell is suspended in, for example, an appropriate buffer (for example, Tris-HCl buffer, phosphate buffer, or Hase buffer) to destroy the organ, tissue or cell. Then, a cell membrane fraction is obtained by using a surfactant (eg, Triton X100 ™, Tween-20 ™, etc.), and further using a method such as centrifugation, filtration, or column fractionation.

 The cell membrane fraction refers to a cell membrane-rich fraction obtained by disrupting cells and then obtained by a known method. The cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Perling Blender ゃ Polytron (manufactured by Kinema tica), crushing by ultrasonic waves, pressing with a French press, etc. Crushing by ejecting cells from a thin nozzle may be mentioned. For the cell membrane fractionation, a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used. For example, the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short period of time (usually about 1-10 minutes), and the supernatant is further centrifuged at a high speed (15000-30000 rpm) for 30 minutes to 2 hours. 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 phospholipid / 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, 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) A transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method, and the receptor protein of the present invention or a partial peptide thereof contained in the cell membrane fraction can be quantified. .

 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 giving a drug or physical stress to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour to 6 hours before) or after a certain time (30 minutes to 3 days) After the administration, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or the test compound is administered simultaneously with the drug or physical stress, and after a certain time after the administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane,

 (ii) The test compound is mixed with the medium when culturing the transformant according to a conventional method, 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 3 days) can be carried out by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane.

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

 (iii) Normal or disease model non-human mammals (for example, mice, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, arteriosclerotic rabbits) 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 given period of time, blood or a specific organ (eg, heart, placenta, lung, etc.) or tissue or cells isolated from the organ is obtained. The obtained organ, tissue, cell, or the like is used as a thread and tissue section according to a conventional method, and immunostaining is performed using the antibody of the present invention. By quantitatively determining the degree of staining of the receptor protein on the cell surface, and confirming the protein on the cell membrane, the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be quantitatively or qualitatively determined. 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 a partial peptide thereof.

The compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the amount of the receptor protein of the present invention or 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 Cell-stimulating activity via protein-coupled receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential Fluctuations, phosphorylation of intracellular proteins, activation of c-fos, activity of promoting or suppressing pH reduction, etc.), (mouth) receptor protein of the present invention or its portion in cell membrane It is a compound that reduces the cell stimulating activity by reducing the amount of the peptide.

 Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like, and 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 attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein of the present invention.

 When a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, 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-described pharmaceuticals containing the receptor protein of the present invention.

 The preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, horses, 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 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. Is about 0.01-30 mg per day, preferably about 0.1-20 mg, more preferably about 0.1-10 mg per day. Conveniently, about mg is administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.

(11) A prophylactic 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

 As described above, the receptor protein of the present invention is considered to play some important role in a living body such as, for example, heart or central function. Therefore, a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be used as a prophylactic or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention.

 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, brown) Cell types, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic disorders (eg, diabetes, dyslipidemia, diabetic complications, obesity, Gout, cataract, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), inflammatory disease ( For example, allergies, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc., cardiovascular diseases (eg, hypertension, cardiac hypertrophy, stenosis) Diseases, myocardial infarction, arteriosclerosis, etc.), respiratory diseases (eg, dysfunction syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis-pulmonary embolism, etc.), digestive system diseases (eg, gastric ulcer, duodenal ulcer, Gastritis, reflux esophagitis, inflammation, etc., immune system disease (eg, autoimmune disease, etc.), or infectious disease (eg, immune dysfunction, pneumonia, cytomegal virus, influenza virus, herpes virus, etc.) Viral infections, rickettsial infections, bacterial infections, etc.). Among them, they are particularly useful for preventing and / or treating Alzheimer's disease.

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, etc., orally, or aseptic solution with water or another pharmaceutically acceptable liquid, if necessary. 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 mixed with Tablet IJ, Capsenole, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, Swelling agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry 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 the aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium salt, etc.) and the like are used. Use in combination with dissolution aids such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, Polysorbate 80 ™, HCO-50) You may. 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 / therapeutic agents include buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. You may mix it. The prepared injection solution is usually filled in a suitable ampoule. The preparations obtained in this way are safe and low toxic and can be used, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, horses, 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. However, in the case of oral administration, for example, in a cancer patient (as 60 kg), the daily dose is generally one day. About 0.1 to: 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. For example, in the form of an injection, it is usually used, for example, in cancer patients (60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0 :! to about 10 mg per day by intravenous injection. In the case of other animals, the amount converted per 60 kg can be administered. (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 a partial peptide thereof or a salt thereof against the receptor protein or the like means an activity of inactivating a signal transduction function involving the receptor protein. . Therefore, when the antibody has a neutralizing activity, signal transduction associated with the receptor protein, for example, cell stimulating activity via the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release) Activating or suppressing intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, decrease in pH, etc. Activity, etc.) can be inactivated. Therefore, it can be used for prevention and / or treatment of diseases caused by overexpression of the receptor protein and the like.

Examples of diseases caused by overexpression of the receptor protein include central diseases (eg, Alzheimer's disease, dementia, eating disorders, depression, epilepsy, etc.), Endocrine disorders (eg, Addison's disease, Cushing's syndrome, pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonad dysfunction, thyroid dysfunction, pituitary dysfunction, etc.), metabolic disorders (eg, diabetes) , Dyslipidemia, diabetic complications, obesity, gout, cataract, hyperlipidemia, etc., cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon) Cancer, rectal cancer, etc., inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.), cardiovascular diseases (eg, hypertension, cardiac hypertrophy, angina) Diseases, myocardial infarction, arteriosclerosis, etc.), respiratory diseases (eg, cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis, pulmonary embolism, etc.), digestive system diseases (eg, gastric ulcer, Duodenal ulcer, gastritis, reflux esophagitis, spleenitis, etc., immune system disease (eg, autoimmune disease, etc.), or infectious disease (eg, immune dysfunction, pneumonia, cytomegal virus, influenza) Virus, herpes virus, etc., rickettsial infection, bacterial infection, etc.). Among these, it is particularly useful for prevention or treatment of Alzheimer's disease.

(13) Production of transgenic 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 and the like of the present invention can be produced. Examples of animals include mammals (for example, rats, mice, egrets, sheep, stags, puppies, cats, cats, dogs, monkeys, etc.) (hereinafter sometimes abbreviated as animals). And egrets are preferred.

When introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of expressing in animal cells. For example, when introducing the DNA of the present invention derived from egret, the homology is high, and a gene construct linked downstream of various promoters capable of expressing the DNA of the present invention derived from animals in animal cells is used. For example, a DNA-transferred animal that highly produces the receptor protein or the like of the present invention can be produced by injecting into a rabbit fertilized egg by mouth opening. For example, プ ロ モ ー タ ー A ubiquitous expression promoter such as a virus-derived promoter or meta-mouth thionine may be used, but a promoter of a gene specifically expressed in the heart is 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 the DNA transfer means that all the offspring of the produced animal have the receptor protein or the like of the present invention in all of the 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 germinal and somatic cells.

 After confirming that the DNA-introduced animal of the present invention stably retains the gene by mating, the animal having the DNA 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 The animal into which the DNA of the present invention has been introduced has high expression of the receptor protein or the like of the present invention, and thus is useful as an animal for screening for an agonist or antagonist against the receptor protein or the like of the present invention.

 The DNA-introduced animal of the present invention can also be used as a cell source for tissue culture. For example, the present invention can be carried out by directly analyzing DNA or RNA in the tissue of the DNA-introduced mouse of the present invention, or by analyzing the ligament containing the receptor protein of the present invention expressed by a gene. Can be analyzed. The cells of a tissue having the receptor protein of the present invention are cultured by standard tissue culture techniques, and these are used to study the function of cells from a generally difficult tissue such as brain or peripheral tissue. be able to. In addition, by using the cells, for example, a drug that enhances the function of various tissues can be selected. In addition, if there is a high expression cell line, the receptor protein of the present invention can be isolated and purified therefrom.

In the present specification and drawings, when bases and amino acids are indicated by abbreviations, the indications are based on the abbreviations by the IUPAC-IUB Commission on Biochemical Nomenclature. Or based on conventional abbreviations in the art. An example is shown below. When an amino acid can have an optical isomer, the L-form is indicated unless otherwise specified.

 Gly: Glycine

 Ala: Alanin

 Val: Norin

 Leu: Leucine

 He: Isoleucine

 Ser: Serine

 Thr: Sure Sainin

 Cys: Cystine

 Met: Methionin

 Glu: Gunoletamic acid

 Asp: Aspartic acid

 Lys: Lysine

 Arg: Anoreginin

 His: histidine

 Phe: feniralanin

 Tyr: Tyrosine

 Trp: Trip, Tojuan

 Pro: Proline

 Asn: Asparagine

 Gin: G / Letamine

 pGlu: Pyrognoletamic acid

 Me: methyl group

 Et: ethinole group

 Bu: butyl group

 Ph: phenyl group

TC: thiazolidine-1 (R) -carboxamide group In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

 1 o s p

 CHO Holmill

 B z 1 Benzinole

C 1 ₂ B z 1: 2, 6-dichlorobenzene

 Bom benzyloxymethyl

 Z Benzinoleoxycanoleponinole

 C 1-Z 2 _ black mouth venice

 B r-Z 2-bromobenzyloxycarbonyl

 B o c t—Butoxycanole

 DNP dinitrophenol

 T r t Trityl

 Bum t—butoxymethyl

 Fmoc: N-9-fluorenyl methoxycarbonyl

 HOB t: 1-hydroxybenztriazole

 HOOB t: 3, 4-Jihi draw 3-hydroxy-1 4-oxo-1

 1,2,3-Venzotriazine

 HONB: 1-hydroxyl-5-norbornolenene 2,3-dicarboxyimide

 DCC: N, N'-dihexyl hexyl carboximide SEQ ID NO: in the Sequence Listing of this specification shows the following sequence.

 SEQ ID NO: 1

 1 shows the amino acid sequence of human-derived novel G protein-coupled receptor protein TGR 34 L of the present invention.

 SEQ ID NO: 2

1 shows the amino acid sequence of human-derived novel G protein-coupled receptor protein TGR34V of the present invention. SEQ ID NO: 3

 1 shows the nucleotide sequence of cDNA encoding the novel human-derived G protein-coupled receptor protein TGR34L of the present invention.

 SEQ ID NO: 4

 1 shows the nucleotide sequence of cDNA encoding the novel human-derived G protein-coupled receptor protein TGR34V of the present invention.

 SEQ ID NO: 5

 This shows the base sequence of primer 1 used in the PCR reaction in Example 1 below. SEQ ID NO: 6

 Shows the base sequence of primer 2 used in the PCR reaction in Example 1 below.

The transformant Escherichia coli TOPlO / pC R2.1-hTGR34L obtained in Example 1 below was prepared on March 5, 2001 at Tsukuba East, Ibaraki, Japan. 1-chome 1 At the Central Organ No. 6 (zip code 305-8566), National Institute of Advanced Industrial Science and Technology (AIST) (formerly National Institute of Advanced Industrial Science and Technology (NIBH)) Deposit number F ERM BP-7490, from February 20, 2001 (Heisei 13) 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-8686) (IFO) as deposit number IFO 16577.

The transformant Escherichia coli TOPlO / pC R2.1-hTGR34V obtained in the following Example 1 was obtained from March 5 S, 2001, Tsukuba, Ibaraki, Japan, East 1 Deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (former National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology (NIBH)) at Central No. 6 (Zip code 305-8566) No. F ERM BP-7491, from February 20, 2001 (Heisei 13) 2 _ 17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-8686) (FOI) under the deposit number IFO 16578. 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 manipulation method using Escherichia coli followed the method described in Molecular cloning. Example 1 Cloning and Nucleotide Sequence of cDNA Encoding G Protein-Coupled Receptor Protein of Human Brain

Using human brain cDNA (CL0NTECH) as type I, a PCR reaction was performed using two primers, Primer 1 (SEQ ID NO: 5) and Primer 2 (SEQ ID NO: 6). The composition of the reaction solution used in the reaction was 1/50 volume of the above cDNA, and 1/50 volume of Advantage-GC2 Polymerase Mix (CL0NTECH), primer 1 (SEQ ID NO: 5) and primer 2 (SEQ ID NO: 6) was added to each of 0.5 juM, dNTPs 200 μΜ, 1/5 volume of the buffer attached to the enzyme, and 1/5 volume of GC Melt to obtain a volume of 20 μl. The PCR reaction is performed at 94 ° C for 5 minutes, followed by a cycle of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 2 minutes 35 times, and finally at 68 ° C for 5 minutes. An extension reaction was performed. The PCR reaction product was subcloned into a plasmid vector pCR2.1 (Invitrogen) according to the procedure of a TA cloning kit (Invitrogen). This was introduced into Escherichia 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 nucleotide sequence (SEQ ID NO: 3 and 4) of cDNA encoding a novel G protein-coupled receptor protein was obtained. These two types of sequences differ by one base at residue 370, and the derived amino acid sequences are SEQ ID NOs: 1 and 2 in which the amino acid at residue 124 is leucine or palin. Novel G protein-coupled receptor containing sequence One protein was named TGR34L and TGR34V. The two types of transformants E. coli _{(Escherichia coli) T0P10 / P CR2} l_hTGR34L, and (Escherichi a coli) T0P10 / pCR2.1 - was named hTGR34V.. Industrial applicability The G protein-coupled receptor protein of the present invention or a partial peptide thereof or a salt thereof, a polynucleotide encoding the receptor protein or a partial peptide thereof (eg, DNA, RNA and derivatives thereof) can be obtained by the following methods: (1) ligand (agonist) 2) Obtaining antibodies and antiserum, 3) Constructing a recombinant receptor protein expression system, 4) Developing a receptor binding assay system using the expression system and screening drug candidate compounds, 4) Structure Implementation of drug design based on comparison with chemically similar ligands and receptors ⑥Problems in gene diagnosis 試 薬 Reagents for creating PCR primers ⑦Generation of transgenic animals or ⑧Gene therapy agents And the like.

Claims

The scope of the claims I. A G protein-coupled receptor protein or a salt thereof, comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1.  2. A G protein-coupled receptor protein or a salt thereof, comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. 3. The G protein-coupled receptor protein according to claim 1, wherein the substantially identical amino acid sequence is the amino acid sequence represented by SEQ ID NO: 2. 4. A partial peptide of the G protein-coupled receptor protein according to claim 1, or a salt thereof.  5. A polynucleotide comprising a polynucleotide encoding the G protein-coupled receptor protein according to claim 1.  6. The polynucleotide according to claim 5, which is DNA. 7. The polynucleotide according to claim 5, having the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.  8. A recombinant vector containing the polynucleotide according to claim 5.  9. A transformant transformed with the recombinant vector according to claim 8.  10. The G protein-coupled receptor protein according to claim 1 or a salt thereof, wherein the transformant according to claim 9 is cultured to produce the G protein-coupled receptor protein according to claim 1. Manufacturing method.  A pharmaceutical comprising the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 4 or a salt thereof.  12. A pharmaceutical comprising the polynucleotide according to claim 5.  13. An antibody against the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 4 or a salt thereof.  14. The antibody according to claim 13, which is a neutralizing antibody that inactivates the signal transduction of the G protein-coupled receptor protein according to claim 1. 15. A diagnostic agent comprising the antibody according to claim 13. 16. A pharmaceutical comprising the antibody according to claim 13.  17. A ligand for the G protein-coupled receptor protein of claim 1 or a salt thereof, which can be obtained by using the G protein-coupled receptor protein of claim 1 or the partial peptide of claim 4 or a salt thereof. 18. A medicament comprising the ligand according to claim 17.  19. The G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 4 or a salt thereof, wherein the ligand for the G protein-coupled receptor protein according to claim 1 or a salt thereof is used. How to determine.  20. A ligand characterized by using the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 4 or a salt thereof, and a G protein-coupled receptor protein according to claim 1 or a ligand thereof. A method for screening a compound or a salt thereof that changes the binding property to a salt is a Jung method.  21. A ligand comprising the G protein-coupled receptor protein according to claim 1 or the partial peptide according to claim 4 or a salt thereof, and the G protein-coupled receptor protein according to claim 1 or the same. A kit for screening a compound or a salt thereof that changes the binding property to a salt.  22. Binding property between the ligand obtainable by the screening method according to claim 20 or the screening kit according to claim 21 and the G protein-coupled receptor protein or a salt thereof according to claim 1 Or a salt thereof 23. Determine the binding between the ligand obtainable by the screening method according to claim 20 or the screening kit according to claim 21 and the G protein-coupled receptor protein or a salt thereof according to claim 1. A medicament comprising a compound to be changed or a salt thereof. 24. A polynucleotide comprising a nucleotide sequence complementary to the polynucleotide according to claim 5 or a part thereof under high stringency conditions. A polynucleotide comprising the polynucleotide.  26. The method for quantifying mRNA of the G protein-coupled receptor protein according to claim 1, wherein the polynucleotide according to claim 5 or a part thereof is used. 27. The method for quantifying a G protein-coupled receptor protein according to claim 1, wherein the antibody according to claim 13 is used.  28. 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 26 or 27 is used. 29. A method for screening a compound or a salt thereof that changes the expression level of a G protein-coupled receptor protein according to claim 1, wherein the method for quantification according to claim 26 is used.  30. A method for screening a compound or a salt thereof that alters 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 27.  31. A compound or a salt thereof that 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 29.  32. A compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein according to claim 1 in a cell membrane obtainable by using the screening method according to claim 30.  33. A medicament comprising a compound capable of changing the expression level of the G protein-coupled receptor protein according to claim 1 or a salt thereof, which can be obtained by using the screening method according to claim 29.  34. A medicament comprising a compound that alters the amount of the G protein-coupled receptor protein according to claim 1 or a salt thereof in a cell membrane obtainable by using the screening method according to claim 3'0. 35 5. Claim 23 which is a preventive or therapeutic agent for central diseases, endocrine diseases, metabolic diseases, cancer, inflammatory diseases, circulatory diseases, respiratory diseases, digestive diseases, immune diseases or infectious diseases. The medicament according to claim 33 or claim 34. 36. Central disease, endocrine disease, metabolic disease characterized by administering to a mammal an effective amount of the compound according to claim 22, claim 31 or claim 32 or a salt thereof. Prevention and treatment of cancer, inflammatory diseases, cardiovascular diseases, respiratory diseases, digestive diseases, immune system diseases or infectious diseases.  3 7. For the manufacture of preventive and therapeutic agents for central, endocrine, metabolic, cancer, inflammatory, circulatory, respiratory, digestive, immune, or infectious diseases. Use of the compound according to claim 22, claim 31, or claim 32 or a salt thereof.