WO2003000886A1 - Nouvelle proteine a recepteur couple a une proteine g et son adn - Google Patents

Nouvelle proteine a recepteur couple a une proteine g et son adn Download PDF

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Publication number
WO2003000886A1
WO2003000886A1 PCT/JP2002/006356 JP0206356W WO03000886A1 WO 2003000886 A1 WO2003000886 A1 WO 2003000886A1 JP 0206356 W JP0206356 W JP 0206356W WO 03000886 A1 WO03000886 A1 WO 03000886A1
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Prior art keywords
protein
receptor protein
salt
coupled receptor
present
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PCT/JP2002/006356
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English (en)
Japanese (ja)
Inventor
Masanori Miwa
Takashi Ito
Yasushi Shintani
Nobuyuki Miyajima
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Takeda Chemical Industries, Ltd.
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Publication of WO2003000886A1 publication Critical patent/WO2003000886A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention relates to a novel G protein-coupled receptor protein derived from a human heart or a salt thereof, a polynucleotide encoding the same, and the like. Furthermore, the present invention relates to a screening method and a screening kit using the novel G protein-coupled receptor protein of the present invention or a salt thereof, a compound obtained by using the screening method or the screening kit, a salt thereof, and the like. Background art
  • G proteins Rereru guanine nucleotide one binding protein of (hereinafter sometimes abbreviated as G proteins) performs signal transduction in cells through activation of, also, Because of their common structure with seven transmembrane domains, they are collectively referred to as G protein-coupled receptor proteins or seven transmembrane receptor proteins (7 TMRs).
  • 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.
  • Physiological functions are regulated under the control of substances or bioactive substances.
  • physiologically active substances exist in various parts of the body, and regulate their physiological functions through their corresponding receptor proteins.
  • receptor proteins There are many unknown hormones, neurotransmitters, and other physiologically active substances in the living body, and the structures of their receptor proteins have not been reported so far. Furthermore, it is often unknown whether subtypes exist in known receptor proteins.
  • Clarifying the relationship between substances that regulate complex functions in living organisms and their specific receptor proteins is a very important tool for drug development.
  • the functions of receptor protein genes expressed in vivo must be elucidated and expressed in an appropriate expression system. Was necessary.
  • the G protein-coupled receptor is useful for searching for a new physiologically active substance (that is, a ligand) and for searching for an agonist or antagonist for the receptor using its signal transduction as an index.
  • a physiological ligand that is, a ligand
  • an agogoest or an antagonist to the receptor it is possible to produce an agogoest or an antagonist to the receptor.
  • These ligands, agonists or antagonists to these receptors can be expected to be used as preventive or therapeutic agents for diseases associated with dysfunction of G protein-coupled receptors.
  • 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.
  • the nucleotide sequence of the receptor is indispensable information for examining the presence or absence of a deletion or mutation in the gene
  • the gene of the receptor is a prophylactic / therapeutic agent for a disease associated with dysfunction of the receptor. And 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, and a polynucleotide containing a polynucleotide (DNA, RNA and a derivative thereof) encoding the G protein-coupled receptor protein or a partial peptide thereof Nucleotides (DNA, RNA and their derivatives), a recombinant vector containing the polynucleotide, a transformant carrying the recombinant vector, the G protein-coupled receptor protein, A method for producing 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, a method for screening a compound (antagonist, agonist) or a salt thereof that alters the binding property between a ligand and the G protein-coup
  • a compound (antagonist, agonist) or a salt thereof that alters the binding between the ligand and the G protein-coupled receptor protein that can be obtained by using the compound, and the binding between the ligand and the G protein-coupled receptor protein It is intended to provide a medicament comprising a compound (antagonist, agonist) that alters or a compound that alters the expression level of the G protein-coupled receptor protein or a salt thereof.
  • the present inventors have isolated cDNA encoding a novel G protein-coupled receptor protein derived from human adipocytes and succeeded in analyzing the entire nucleotide sequence thereof.
  • this nucleotide sequence was translated into an amino acid sequence, the first to seventh transmembrane regions were confirmed on the hydrophobicity plot, and the protein encoded by these cDNAs was a seven-transmembrane G protein. It was confirmed to be a conjugated receptor protein.
  • the present inventors have conducted further studies based on these findings, and have completed the present invention. That is, the present invention
  • 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: 5;
  • a method for producing a G protein-coupled receptor protein or a salt thereof A method for producing a G protein-coupled receptor protein or a salt thereof,
  • the antibody according to (11) which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to (1);
  • the G protein-coupled receptor protein according to (1) or a salt thereof which can be obtained by using the G protein-coupled receptor protein according to (11) or the partial peptide according to (4) or a salt thereof.
  • a ligand comprising the G protein-coupled receptor protein according to the above (1) or the partial peptide or a salt thereof according to the above (4), and a G protein-coupled receptor protein according to the above (1) Or a screening kit for a compound or a salt thereof that changes the binding property to a salt thereof,
  • a method for screening a compound or a salt thereof that changes the amount of the G protein-coupled receptor protein according to (1) in a cell membrane which comprises using the quantification method according to (25);
  • the medicament according to the above (21), 21, 31 or 32 which is a therapeutic agent for central disease, inflammatory disease, cardiovascular disease, cancer, metabolic disease, immune system disease or digestive system disease.
  • a ligand obtainable by using the screening method described in (18) or the screening kit described in (19) above, and a G protein-coupled receptor protein described in (1) above or a mammal thereof.
  • Prevention of central disease, inflammatory disease, circulatory disease, cancer, metabolic disease, immune system disease or digestive system disease characterized by administering an effective amount of a compound or a salt thereof that alters the binding to a salt.
  • a compound or a salt thereof that alters the binding between the ligand obtainable by the screening method or the screening kit described in (19) and the G protein-coupled receptor protein or salt thereof described in (1) above;
  • the protein is: (1) an amino acid sequence represented by SEQ ID NO: 5, one or more in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30, more preferably 1 to 30) An amino acid sequence in which about 9 amino acids have been deleted, more preferably several (1 to 5) amino acids; and 2 or more amino acids (preferably 1 to 30 amino acids) in the amino acid sequence represented by SEQ ID NO: 5 Number, more preferably about 1 to 10, and still more preferably several (1 to 5) amino acid sequences to which amino acid is added.
  • the ligand is, for example, angiotensin, bombesin, cannabinoid, cholecystokinin, gnoletamine, serothene, melatonin, neuuropeptide Y, opioid, purine, pasoplethsin, human xytocin, PACAP (e.g., PAC AP 27, P ACAP 38), secretin, glucagon, calcitonin, kyodonomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (passoactive intestinanole polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin , CGRP (force ⁇ / cytotonine relayed peptide), leukotriene, pancreastatin, prostaglandin, tropoxane, adenosine, adrenaline, chemokine superfamily (eg, IL-18, G
  • Labeling when contacted with the G protein-coupled receptor protein described in (1) or a salt thereof or the partial peptide described in (4) or a salt thereof is measured and compared with the ligand characterized in that 1) A method for screening a compound or a salt thereof which alters the binding property to the G protein-coupled receptor protein or a salt thereof according to the above,
  • a compound that activates the G protein-coupled receptor protein or a salt thereof described in (1) above and a test compound are contacted with cells containing the G protein-coupled receptor protein described in (1) above.
  • the cell stimulating activity mediated by 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 changed.
  • the compound that activates the G protein-coupled receptor protein described in (1) above is angiotensin, bombesin, cannabinoid, cholecystokun, guzoletamine, serotonin, melatonin, neuropeptide Y, opioid, pre- , Vasoprescin, oxytocin, PACAP (e.g., PACAP 27, PAC ⁇ 38), secretin, glucagon, canolecithonin, admrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (vasoactive intestinal poly) Peptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene, ha.
  • PACAP e.g., PACAP 27, PAC ⁇ 38
  • secretin secretin
  • glucagon canolecithonin
  • a compound or a salt thereof that alters the binding property between the ligand obtainable by the screening method according to any of (43) to (50) and the G protein-coupled receptor protein or its salt according to (1).
  • FIG. 1 is a hydrophobicity plot of TGR39.
  • FIG. 2 is a hydrophobicity plot of hTGR39A.
  • FIG. 3 is a distribution diagram of TGR39 expression. BEST MODE FOR CARRYING OUT THE INVENTION
  • the G protein-coupled receptor protein of the present invention (hereinafter sometimes abbreviated as receptor protein) has the same amino acid sequence as SEQ ID NO: 5. Or a receptor protein containing substantially the same amino acid sequence.
  • the receptor protein of the present invention can be used, for example, in any cells (eg, spleen cells, nerve cells, neurons, etc.) of mammals (eg, humans, guinea pigs, rats, mice, rabbits, pigs, sheep, whales, monkeys, etc.).
  • brain various parts of the brain (e.g., olfactory bulb, squamous nucleus, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus nucleus, cerebral cortex, medulla, cerebellum, occip
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 5 includes, for example, about 50% or more, preferably about 60% or more, of the amino acid sequence represented by SEQ ID NO: 5,
  • the amino acid sequence preferably has about 70% or more, particularly preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
  • Examples of a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 5 of the present invention include, for example, an amino acid substantially identical to the amino acid sequence represented by SEQ ID NO: 5
  • a protein containing a sequence and having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 5 is preferred.
  • substantially the same activity examples include a ligand binding activity and a signal transduction activity. Substantially the same means that their activities are the same in nature. Therefore, the activities such as the ligand binding activity and the signal transduction activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably (Approximately 0.5 to 2 times), but the quantitative factors such as the degree of activity and the molecular weight of the protein may be different.
  • the measurement of the activity such as the ligand binding activity and the signal transduction action can be performed according to a known method.
  • the activity can be measured according to the ligand determination method described below, which is described in the SCREENING method.
  • protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 5 include a protein containing the amino acid sequence represented by SEQ ID NO: 1, and the like. .
  • the receptor protein of the present invention includes: 1) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 5 (preferably, about 1 to 30, more preferably 1 to: L 0 Amino acid sequence in which several (1 to 5) amino acids have been deleted, and more preferably 1 or 2 or more amino acids (preferably 1 to 5 amino acids) in the amino acid sequence represented by SEQ ID NO: 5.
  • amino acid sequence having about 30 amino acids, more preferably about 1 to 10 amino acids, and still more preferably several (1 to 5) amino acids; 3 one or more amino acids in the amino acid sequence represented by SEQ ID NO: 5 Amino in which two or more (preferably about 1 to 30, preferably 1 to 10 and more preferably several (1 to 5)) amino acids are substituted with another amino acid Proteins containing acid sequences or amino acid sequences combining them It may also be used.
  • the receptor protein has an N-terminus (amino terminus) at the left end and a C-terminus (carboxyl terminus) at the right end in accordance with the convention of peptide labeling.
  • the receptor proteins of the present invention including the receptor protein containing the amino acid sequence represented by SEQ ID NO: 5, have a carboxyl group at the C-terminus (-COOH), a carboxylate (one COO—), an amide (one CO It may be either NH 2 ) or an ester (_COOR).
  • R in the ester e.g., methyl, Echiru, n- propyl, Isopuropiru or n-C M alkyl group such as heptyl, for example, consequent opening pentyl, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, (X- naphthyl C 6 _ 12 Ariru group such as, for example, benzyl, a one Nafuchiru CM alkyl such as phenylene Lou C J.2 alkyl or ⁇ - naphthylmethyl such phenethyl C 7 such groups - such as 14 Ararukiru Bibaroi Ruokishimechiru groups commonly used as ho force oral ester group is used.
  • the receptor protein of the present invention has a carboxyl group (or hydroxyl group) at a position other than the C-terminus
  • a protein in which the carboxyl group is amidated or esterified is also included in the receptor protein of the present invention.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the receptor protein of the present invention is the protein mentioned above, protected with Amino group protecting group of Mechionin residues of N-terminal (e.g., formyl group, etc. Ashiru groups such as any C 2 _ 6 Arukanoiru group of Asechiru) Glutamyl group generated by cleavage of the N-terminus in vivo, and glutamine oxidized in the mouth, substituents on the side chains of amino acids in the molecule (for example, OH, _SH, amino group, imida tetrazole group, indole group, those such Guanijino group) is protected with a suitable protecting group (e.g., formyl group, etc.
  • Cw Ashiru group such as C 2 _ 6 Arukanoiru group such Asechiru
  • a sugar chain bound Complex proteins such as so-called glycoproteins are also included.
  • receptor protein of the present invention include, for example, a receptor protein having the amino acid sequence represented by SEQ ID NO: 5, a receptor protein having the amino acid sequence represented by SEQ ID NO: 1, and the like.
  • the partial peptide of the receptor protein of the present invention may be any of the above-mentioned partial peptides of the receptor protein of the present invention.
  • the receptor protein molecules of the present invention those which are exposed outside the cell membrane and have receptor binding activity are used.
  • a portion analyzed as an extracellular region (hydrophilic site) in a hydrophobicity plot analysis was used. Including peptides. Further, a peptide partially containing a hydrophobic site can also be used. A peptide containing individual domains may be used, but a peptide containing a plurality of domains at the same time may be used.
  • the number of amino acids of the partial peptides of the present invention is determined by the above-described receptor protein of the present invention. Peptides having at least 20 or more, preferably 50 or more, more preferably 100 or more amino acid sequences among the amino acid sequences constituting the protein are preferred.
  • Substantially identical amino acid sequences refer to those amino acid sequences of about 50% or more, preferably about 60% or more, more preferably about 70% or more, and still more preferably about 80% or more. However, the amino acid sequence preferably has a homology of about 90% or more, and most preferably about 95% or more.
  • one or more (preferably about 1 to 10 and more preferably several (1 to 5)) amino acids in the above amino acid sequence are deleted. Or 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.
  • One or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the amino acid sequence of the rice cake It may be substituted.
  • the C-terminus carboxyl group of the present invention (one CO OH), Cal Bokishireto (_ COO-), amide (one CO NH 2) or an ester - may be either (COOR).
  • the partial peptide of the present invention has a N-terminal methionine residue in which the amino group of the methionine residue is protected with a protecting group, and the N-terminal side is cleaved in vivo to form a peptide.
  • G 1 n obtained by pyroglutamic acid substitution, those in which the substituent on the side chain of the amino acid in the molecule is protected by an appropriate protecting group, and those in which a sugar chain is bonded to a complex peptide such as a so-called glycopeptide. included.
  • Examples of the salt of the receptor protein or its partial peptide of the present invention include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable.
  • Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) You.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, oxalic acid,
  • the receptor protein of the present invention or a salt thereof can be produced from the above-mentioned mammalian cell or tissue by a known method for purifying a receptor protein, or a DNA encoding the receptor protein of the present invention described later. Can also be produced by culturing a transformant containing Also, the protein can be produced by the protein synthesis method described later or according to the method.
  • a commercially available resin for protein synthesis can be usually used.
  • a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydrogen resin.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target protein according to various known condensation methods.
  • the protein is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is carried out in a highly diluted solution to obtain the target protein or its amide.
  • various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable.
  • carpimides examples include DCC, N, N'-diisopropylcarboimide, and N-ethyl-1-N, 1- (3-dimethylaminoprolyl) carpimide.
  • active dans a protected amino acid is directly added to the resin together with a racemic dan suppression additive (for example, HO Bt, HO OB t), or a symmetric acid anhydride or HO is added.
  • the protected amino acid can be added as a Bt ester or HOOBt ester to the resin after activation of the protected amino acid in advance.
  • 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.
  • acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylvirolidone, halogenated hydrocarbons such as methylene chloride and chloroform, trifluorofluoride Alcohols such as ethanol, sulfoxides such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof.
  • the reaction temperature is appropriately selected from the range known to be usable for the protein bond formation reaction, and is usually selected from the range of about 120 ° C to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the starting amino group include Z, Boc, tertiary pentoxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, C 1 _Z, Br—Z, ⁇ Damantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, dipheninolephosphinothioinole, Fmoc and the like are used.
  • Carboxyl groups can be, for example, alkyl esterified (e.g., methyl, ethyl, propynole, petitnole, tertiary petitnole, cyclopentinole, cyclohexyl / cyclohexyl, cycloheptyl, cyclootatyl, 2-adamantyl, etc.) , Branched or cyclic alkyl esterification), aralkyl esterification (for example, benzinolestenol, 4-12 trobenzinolestenol, 4-methoxybenzylislenol, 4 benzyl) Ester, benzhydryl esterification), phenacyl esterification, benziloxycanoleboninolehydrazide, tertiary butoxy force / report hydrazide And trityl hydrazide.
  • alkyl esterified e.g., methyl, e
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for this esterification for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group, and the like are used.
  • Examples of a group suitable for ethereal are a benzyl group, a tetrahydrobiranyl group, a t-butyl group, and the like.
  • Examples of protecting groups for Fuwenoru hydroxyl group of tyrosine for example, B z and C 1 2 -
  • the protecting groups for histidine imidazo include, for example, Tos, 4-methoxy-
  • activated carboxyl groups in the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol) , Cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOB t)].
  • active esters eg, pentachlorophenol, 2,4,5-trichloromouth phenol, 2,4-dinitrophenol
  • Cyanomethyl alcohol eg, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOB t
  • the activated amino group of the raw material for example, a corresponding phosphoric amide is used.
  • Methods for removing (eliminating) protecting groups include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd—black or Pd—carbon; Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and sodium in liquid ammonia Reduction by acetic acid is also used.
  • the elimination reaction by the above-mentioned acid treatment is generally carried out at a temperature of about -20 ° C to 40 ° C.
  • a force-thione scavenger such as dimethinoresanolide, 1,4-butanedithiol or 1,2-ethanedithiol. It is also used as an imidazole protecting group for histidine.
  • the 2,4-dinitrophenyl group used is removed by thiophenol treatment, and the formyl group used as an indole protecting group for tryptophan is treated by acid treatment in the presence of 1,2-ethanedithiol and 1,4-butanedithiol. In addition to deprotection, it is also removed by alkaline treatment with dilute sodium hydroxide solution, dilute ammonia and the like.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • a peptide (protein) chain is added to the amino group side to a desired chain length.
  • a protein in which only the protecting group for the amino group at the ⁇ -terminus of the peptide chain was removed and a protein in which only the protecting group for the carboxy group at the C-terminus was removed were prepared. Condensate in a mixed solvent. Details of the condensation reaction are the same as described above. After purifying the protected protein obtained by the condensation, all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein. The crude protein is purified using various known purification means, and the main fraction is lyophilized to obtain an amide of the desired protein.
  • an esterol form of the desired protein is obtained in the same manner as the protein amide. Can be obtained.
  • the partial peptide of the protein of the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptide.
  • a method for synthesizing a peptide for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, a partial peptide or amino acid capable of constituting the protein of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the desired peptide.
  • Known condensation methods and elimination of protecting groups include, for example, those described in the following 1 to 5 Law.
  • the partial peptide of the present invention can be purified and isolated by a combination of ordinary purification methods, for example, solvent extraction, distillation, column chromatography, 'liquid chromatography', and recrystallization.
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained as a salt, it is converted to a free form by a known method. be able to.
  • any polynucleotide may be used 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 DNA, 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).
  • the mRNA of the receptor protein of the present invention can be quantified.
  • Examples of the DNA encoding the receptor protein of the present invention include genomic DNA, a genomic DNA library, the above-described cell / tissue-derived cDNA, the above-described cell / tissue-derived cDNA library, and synthetic DNA. Either may be used.
  • Libra The vector used in the plasmid may be any of pateriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT_PCR method) using a preparation of a total RNA or mRNA fraction from the above-mentioned cells and tissues.
  • RT_PCR method reverse transcriptase polymerase chain reaction
  • the DNA encoding the receptor protein of the present invention includes, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 6 or a DNA containing the nucleotide sequence represented by SEQ ID NO: 6 Having a DNA that hybridizes under high stringent conditions to DNA, and having substantially the same activity as a receptor protein containing an amino acid sequence represented by SEQ ID NO: 5 (eg, ligand binding activity, Any DNA may be used as long as it encodes a receptor protein having a signal information transduction action.
  • Examples of the DNA that hybridizes with the DNA containing the nucleotide sequence represented by SEQ ID NO: 6 under high stringent conditions include, for example, about 70% or more, preferably A DNA containing a nucleotide sequence having a homology of about 80% or more, more preferably about 90% or more, and most preferably about 95% or more is used. Specific examples include SEQ ID NO: DNA containing the base sequence represented by 2 and the like.
  • Hybridization is performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). be able to. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual. More preferably, the reaction can be performed under high stringent conditions.
  • the high stringency conditions include, for example, a sodium concentration of about 19 to 40 ⁇ , preferably about 19 to 20 mM, and a temperature of about 50 to 70 ° C, preferably about 60 to The conditions at 65 ° C are shown. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C is most preferable.
  • a receptor containing the amino acid sequence represented by SEQ ID NO: 5 examples include a DNA having the base sequence represented by SEQ ID NO: 6, and the like. Examples of the DNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 1 include the sequence: For example, DNA having the base sequence represented by No. 2 is used.
  • a part of the nucleotide sequence of the DNA encoding the receptor protein of the present invention or a polynucleotide containing a part of the nucleotide sequence complementary to the DNA is a polynucleotide encoding the following partial peptide of the present invention. It is used to mean not only DNA but also RNA.
  • an antisense polynucleotide 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 base sequence information of the encoding DNA.
  • a polynucleotide can hybridize to RNA of the G protein-coupled receptor protein gene and has the ability to inhibit the synthesis or function of the RNA, or the G protein-coupled receptor protein-related RNA. It can regulate and control the expression of G protein-coupled receptor protein gene through interaction with.
  • G-protein coupled receptor protein-related polynucleotides that are complementary to a selected sequence of RNA and polynucleotides that can specifically hybridize to G-protein coupled receptor protein-related RNA ⁇ 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.
  • corresponding means having homology or being complementary to a particular sequence of nucleotides, base sequences or nucleic acids, including genes.
  • “Corresponding” between a nucleotide, base sequence or nucleic acid and a peptide (protein) usually refers to the amino acid of the peptide (protein) in the instructions derived from the nucleotide (nucleic acid) sequence or its complement. ing. 5, end hairpin loop, 5, end 6—base ba 'repeat, 5' end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation termination codon, 3, end untranslated region, 3, end palindrome
  • the region, and the three-terminal hairpin loop can be selected as preferred regions of interest, but any region within the G protein-coupled receptor protein gene can be selected as the region of interest.
  • the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region is as follows.
  • Antisense polynucleotides are polynucleotides that contain 2-dexoxy D-ribose, polynucleotides that contain D-ribose, or other types of polynucleotides that are 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 found in DNA or RNA). Base pairing (including nucleotides having a configuration that allows base attachment)).
  • RNA hybrids can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can also be unmodified polynucleotides (or unmodified oligonucleotides), In addition, those with known modifications, such as those with a label known in the art, capped, methylated, one or more natural nucleotides substituted with analogs, Modified with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphorothioate) , Phosphorodithioate, etc., such as proteins (nucleases, nucleases' inhibitors, Those with side groups such as toxins, antibodies, signal peptides, poly-L-lysine, etc.
  • nucleoside include not only purine and pyrimidine bases but also modified Other compounds having a heterocyclic base may be included. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles.
  • Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with halogens, aliphatic groups, etc., or functionalities such as ethers, amines, etc. It may be converted to a group.
  • the antisense 'polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • 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.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes or microspheres, may be applied by gene therapy, It could be given in additional form.
  • 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 fats to be added include cholesterol and its derivatives (eg, cholesterol rectal formate, cholic acid, etc.).
  • nucleic acids can be attached to the 3, 5 'or 5' end of nucleic acids and attached via bases, sugars, or intramolecular nucleoside bonds. Can be worn.
  • Other groups include cap groups that are specifically located at the 3 'end or the 5' end of a nucleic acid and that prevent degradation by nucleases such as exonuclease and RNase. Examples of such a capping group include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycol and tetraethylene dalicol.
  • the inhibitory activity of an antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of a G protein-coupled receptor protein. it can.
  • the nucleic acid can be applied to cells by various known methods.
  • the DNA encoding the partial peptide of the present invention may be any DNA as long as it contains the above-described nucleotide sequence encoding the partial peptide of the present invention.
  • any of the genomic DNA, the genomic DNA library, the above-described cDNA derived from the cells and the DNA, the above-described cDNA library derived from the cells and tissues, and the synthetic DNA may be used.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can be directly amplified by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using an mRNA fraction prepared from the cells and tissues described above.
  • RT-PCR method Reverse Transcriptase Polymerase Chain Reaction
  • the DNA encoding the partial peptide of the present invention for example,
  • a DNA having a partial nucleotide sequence of DNA encoding a receptor protein having Examples of the DNA that hybridizes with the DNA containing the nucleotide sequence represented by SEQ ID NO: 6 under high stringent conditions include, for example, about 70% or more and the nucleotide sequence represented by SEQ ID NO: 6, preferably Is about 80% or more, more preferably about 90% or less.
  • a DNA containing a nucleotide sequence having about 95% or more homology is used, and specific examples include a DNA containing the nucleotide sequence represented by SEQ ID NO: 6
  • Cloning of DNA that completely encodes the receptor protein of the present invention or a partial peptide thereof includes DNA encoding the receptor protein of the present invention.
  • DNA that codes for a part or all of the receptor protein of the present invention is a DNA that is amplified by a PCR method using a synthetic DNA primer having a partial nucleotide sequence of Selection can be performed by hybridization with fragments or those labeled with synthetic DNA.
  • Hybridization methods include, for example, molecular cloning.
  • Conversion of the DNA sequence of DNA is performed by PCR or a known kit, for example, Mutan TM -super Express Km (Takara Shuzo), Mutan TM -K (Takara Shuzo), etc., using the 0DA-LA PCR method, the Gapped duplex method, and the Kunkel method. It can be carried out according to a known method such as a method or a method analogous thereto.
  • the DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or can be used by digesting with a restriction enzyme or adding a linker if desired.
  • the DNA may have ATG as a translation initiation codon on its 5, terminal side, and may have TAA, TGA or TAG on its 3, terminal side as a translation termination codon. These translation initiation codon and translation termination codon can also be added using a suitable 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 DNA encoding the receptor protein of the present invention; It can be produced by ligating downstream of the promoter.
  • vectors include Escherichia coli-derived plasmids (eg, pCR4, pCR2.1, pBR322, pBR325 pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pCl94), yeast-derived plasmid (Eg, pSH19, pSH15), bacteriophage such as ⁇ phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc., ⁇ A1-11, pXT1, Rc / CMV, pRc / RSV, pcDNAI / Neo, etc. are used.
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SRa promoter when animal cells are used as host, SRa promoter, SV40 promoter, LTR motor, CMV promoter, HSV-TK promoter and the like can be mentioned. Of these, it is preferable to use the CMV promoter, SRa promoter and the like.
  • the host is Escherichia, trp promoter, 1 ac promoter, recA promoter; LP, promoter, lpp promoter, etc.
  • SPOL promoter When the host is Bacillus, SPOL promoter, SP02 promoter , such as p en p promoter, if the host is a yeast, PH05-flops opening motor, PGK promoter, GAP promoter, etc. ADH promoter are preferred.
  • SPOL promoter When the host is Bacillus, SPOL promoter, SP02 promoter , such as p en p promoter, if the host is a yeast, PH05-flops opening motor, PGK promoter, GAP promoter, etc. ADH promoter are preferred.
  • SPOL promoter When the host is Bacillus, SPOL promoter, SP02 promoter , such as p en p promoter, if the host is a yeast, PH05-flops opening motor, PGK promoter, GAP promoter, etc. ADH promoter are preferred.
  • the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
  • the expression vector may further include an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter, sometimes abbreviated as SV40 ori), and the like, if desired.
  • SV40 ori an SV40 replication origin
  • selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dh fr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter sometimes abbreviated as Am), neomycin resistance gene (hereinafter sometimes abbreviated as Ne o r, G418 resistance).
  • the target gene when used as a selection marker using CHO (dh fr-) cells, the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host may be replaced with the receptor protein of the present invention. Add to the N terminal side of the park.
  • the host is a bacterium belonging to the genus Escherichia, a Ph A A signal sequence, an Omp A signal sequence, or the like is used. If the host is yeast, use the MFc signal sequence, SUC2 signal sequence, etc.If the host is an animal cell, use the insulin signal sequence, interferon signal sequence, antibody molecule, signal sequence, etc. it can.
  • a transformant can be produced.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells, and the like are used.
  • Escherichia bacteria include, but are not limited to, Escherichia coli Kl 2 ⁇ DH1 [Prosings, Ob, The National, Academy, Ob, * Sciences, Ob, The * U.S.A. Acad. Sci. USA), Vol. 60, 160 (1968)], JM103 [Nucleic Acids Research, Vol. 9, 309 (1 981)], JA221 (Journal 7) ⁇ Ob * molecular ⁇ / Journal of Molecular Biology, 120, 5 17 (1 978)], HB 101 [Journal of Molecular Biology, 41, 459 ( 1969)], C600 [Genetics, 39, 440 (1954)], DH5a
  • Bacillus genus examples include Bacillus subtilis (Bacillus
  • subtilis MI 1 14 [Gene, 24 vol., 255 (1 983)], 207- 21 [Journal of Biochemistry, 95 Vol., 87 (1 984)], etc. Is used.
  • yeast examples include Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, 20 B-12, Schizosaccharomyces pombe NC YC 1913, NCYC 2036, Pichia pastoris (Pichia pastoris) and the like are used.
  • insect cells for example, when the virus is Ac NPV, Spodoptera frugiperda cell (Sf cell), MG1 cell derived from the midgut of Trichoplusia ni, High Five derived from egg of Trichoplusia ni when the virus is Ac NPV TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • Sf cell a silkworm-derived cell line (Bombyx mori N; Bm N cell) is used.
  • Sf cell include Sf9 cell (ATCC CRL1711) and Sf21 cell (Vaughn, J.L. et al., In.
  • insects for example, silkworm larvae are used [Maeda et al.
  • animal cells examples include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CH ⁇ cell), dh fr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dfr—) cell).
  • CH ⁇ cell Chinese hamster cell CHO
  • CHO (dfr—) cell dh fr gene-deficient Chinese hamster cell CHO
  • Mouse L cells mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, and the like.
  • Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, 168, 11 (179).
  • Insect cells or insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
  • a liquid medium is suitable as a medium used for the cultivation, and a carbon source necessary for the growth of the transformant is contained therein.
  • Nonrogen sources include, for example, ammonium salts, nitrates, corn chip'liquor, peptone, potato zein, meat extract, soybean meal, potato extract
  • the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride and the like.
  • yeast extract, vitamins, growth promoting factors, etc. may be added.
  • the pH of the medium is preferably about 5-8.
  • a medium for cultivating a bacterium belonging to the genus Escherichia for example, an M9 medium containing glucose and casamino acid [Miller, Journal “Ob” Experiment ”,“ Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972].
  • a drug such as 33-indolylacrylic acid can be added to make the promoter work efficiently.
  • cultivation is usually carried out at about 15-43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be applied.
  • the cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring may be added.
  • a medium for example, Burkholder's minimum medium [Bostian, KL et al., "Procedures of the National Academy of Cultures” Proc. Natl. Acad. Sci. USA, 77, 4505 (1980) "and an SD medium containing 0.5% casamino acid [Bitter, GA et al. Singing's Op. The National Academy Op. Sciences Ob. The U.S.A. (Proc. Natl. Acad. Sci. USA), 81, 5330 (1 984).
  • 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, and if necessary, aeration and stirring are added.
  • the culture medium When culturing an insect cell or a transformant whose host is an insect, the culture medium was immobilized in Grace's Insect Medium (Grace, TCC, Nature, 1995, 788 (1962)). A solution to which an additive such as 10% serum is appropriately added is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. Cultivation is usually carried out at about 27 ° C for about 3 to 5 days, with aeration and stirring as needed.
  • a MEM medium containing about 5 to 20% fetal bovine serum Science, 122, 501 (1952)]
  • DMEM medium [Virology, Vol. 8, 396 (1 959)
  • RPMI 1640 medium Journal of the American Medical Association
  • the pH is preferably about 6-8.
  • the cultivation is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • the G protein-coupled receptor protein of the present invention can be produced in the transformant, inside the cell membrane, or outside the cell.
  • the receptor protein of the present invention 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 ruptured 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 TM.
  • the receptor protein contained in the thus obtained culture supernatant or extract can be purified by appropriately combining known separation and purification methods.
  • known separation and purification methods include methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • a method using the difference in hydrophobicity, a method using the difference in isoelectric point such as isoelectric focusing, and the like are used.
  • the receptor protein thus obtained When the receptor protein thus obtained is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto. 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 the action of an appropriate protein-modifying enzyme before or after purification.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, anoreginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the activity of the receptor protein of the present invention or a salt thereof thus produced is determined by labeling It can be measured by a binding experiment with the ligand thus obtained and an enzymimnoassay using a specific antibody.
  • 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. You may.
  • An antibody against the receptor protein of the present invention or a partial peptide thereof or a salt thereof may be a known antibody or antiserum using the receptor protein of the present invention as an antigen. It can be manufactured according to the manufacturing method.
  • 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.
  • complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability.
  • the administration is usually performed once every 2 to 6 weeks, for a total of 2 to 10 times. Examples of mammals to be used include monkeys, egrets, dogs, guinea pigs, mice, rats, sheep, and goats, and mice and rats are preferably used.
  • monoclonal antibody-producing cells When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with the antigen, for example, an individual with an antibody titer from a mouse, and collect the spleen or lymph node 2 to 5 days after the final immunization By fusing antibody-producing cells contained therein with myeloma cells, monoclonal antibody-producing hybridomas can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting 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.
  • Fusion promoters include, for example, Power such as Renglycol / PEG (PEG) and Sendai virus.
  • PEG is used.
  • myeloma cells examples include NS_1, P3U1, SP2Z0 and the like, and P3U1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and the concentration of PEG (preferably PEG1000 to PEG6000) is about 10 to 80%.
  • PEG preferably PEG1000 to PEG6000
  • hybridomas can be cultured on a solid phase (eg, microplate) onto which an antigen such as a receptor protein has been directly or adsorbed together with a carrier. Then, add an anti-immunoglobulin antibody labeled with a radioactive substance, an enzyme, etc. (if the cells used for cell fusion are mice, use an anti-mouse immunoglobulin antibody).
  • a solid phase eg, microplate
  • an antigen such as a receptor protein has been directly or adsorbed together with a carrier.
  • an anti-immunoglobulin antibody labeled with a radioactive substance, an enzyme, etc. if the cells used for cell fusion are mice, use an anti-mouse immunoglobulin antibody.
  • a method for detecting a monoclonal antibody bound to a solid phase adding a hybridoma culture supernatant to a solid phase to which anti-immune glopurin antibody or protein A is adsorbed, and adding a receptor protein or the like labeled with a radioactive substance or an enzyme, Examples include a method for detecting a monoclonal antibody bound to a solid phase.
  • 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.
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium that can grow a hybridoma can be used.
  • R PMI 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 A serum-free medium for hybridoma culture (SFM_101, Nissui Pharmaceutical Co., Ltd.) or the like can be used.
  • the culture temperature is usually 20 to 40 ° C, preferably about 37 ° C.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • Nourishment can usually be performed under 5% carbon dioxide gas.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies, such as immunoglobulin separation and purification (e.g., salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers). (E.g., DE AE) adsorption / desorption method, ultracentrifugation method, gel filtration method, antigen-bound solid phase or active adsorbent such as protein A or protein G, and only antibody is collected. Specific Purification Method Obtained].
  • immunoglobulin separation and purification e.g., salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchangers.
  • DE AE adsorption / desorption method
  • ultracentrifugation method ultracentrifugation method
  • gel filtration method antigen-bound solid phase or active adsorbent such as protein A or protein G
  • the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (an antigen such as the receptor protein of the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody.
  • the antibody can be produced by collecting the antibody-containing substance against the antibody and the like and separating and purifying the antibody.
  • the type of carrier protein and the mixing ratio of the carrier and the hapten are different from those of the hapten immunized by cross-linking the carrier. If antibodies can be efficiently produced, any kind of antibody can be cross-linked at any ratio.For example, serum albumin, thyroglobulin, keyhorne 'limpet' hemocyanin, etc. A method of pulling the hapten at a ratio of about 0.1 to 20 and preferably about 1 to 5 with respect to hapten 1 is used.
  • Various condensing agents can be used for force coupling between the hapten and the carrier.
  • glutaraldehyde-carboimide, 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 capable of producing antibodies.
  • 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.
  • Polyclonal antibodies can be obtained from the blood, ascites, etc. of a mammal immunized by the above method. Preferably, it can be collected from blood.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. Separation and purification of the polyclonal antibody can be performed according to the same method for separation and purification of immunoglobulins as in the above-described separation and purification of monoclonal antibodies.
  • the receptor protein of the present invention or a salt thereof, a partial peptide or a salt thereof, and a DNA encoding the receptor protein or a partial peptide thereof are:
  • determining a ligand (agonist) for the G protein-coupled receptor protein of the present invention (2) preventing and / or treating a disease associated with dysfunction of the G protein-coupled receptor protein of the present invention; (3) a gene diagnostic agent, (4) a method for screening a compound that changes the expression level of the receptor protein or its partial peptide of the present invention, (5) a compound that changes the expression level of the receptor protein or its partial peptide of the present invention
  • a prophylactic and / or therapeutic agent for various diseases comprising: (6) a method for quantifying a ligand for the G protein-coupled receptor protein of the present invention; (7) a method for determining the relationship between the ligand and the G protein-coupled receptor protein of the present invention; Screening methods for compounds that alter binding (eg, agonists, antagonists, etc.); ) A preventive and / or therapeutic agent for various diseases containing a compound (argonist, antagonist) that alters the binding between a G protein-coupled receptor protein and a lig
  • a compound that changes the binding property of a ligand to a G protein-coupled receptor specific to a mammal by using a receptor-binding Atsuy system using the expression system of the recombinant G protein-coupled receptor protein of the present invention can be used as an agent for preventing or treating various diseases.
  • the receptor protein or partial peptide of the present invention or a salt thereof 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 may be abbreviated as DNA
  • the use of the antibody against the receptor protein or the like of the present invention hereinafter sometimes abbreviated as the antibody of the present invention
  • the receptor protein of the present invention or a salt thereof or the partial peptide or a salt thereof of the present invention is useful as a reagent for searching for or determining a ligand for the receptor protein of the present invention or a salt thereof.
  • 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 (e.g., angiotensin, bombesin, canapinoid, cholecystokinin, gnoletamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxotocin, ⁇ ACAP (e.g., PACAP 27, PACAP 38), secretin, gnore ligone, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (Pasoactive Intestinal and Rerated Polypeptide), somatostatin, dopamine, motilin, amylin , Bradycun, CGRP (calcitonin gene relayed peptide), leukotriene, no, ° creatastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine super Family (eg, IL-1 8, G
  • Fami Lee Fami Lee; MCAF / MCP—1, MCP-2, MCP—3, MCP—4, eotaxin, R ANTES, MI P—la, MIP—1, HCC-1, MI P-3 ⁇ / LARC, MI P— 3 ⁇ / ELC, 1-309, TARC, MI PF-l, MI PF-2 / eotaxi n-2, MDC, DC—CK chemokine subfamily such as CKl / PARC, SLC; 1 C chemo such as ymp hotactin Cain subfamily one; CX 3 C chemokine subfamily such as fractalkine, etc.), endothelin, enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingocin 1_
  • tissue extracts of mammals eg, human, mouse, rat, pig, magpie, hid
  • the ligand determination method of the present invention uses the receptor protein of the present invention or a partial peptide thereof or a salt thereof, or constructs an expression system for a recombinant receptor protein, and uses the expression system.
  • a receptor-binding assay system it binds to the receptor protein of the present invention and has a cell stimulating activity (for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, Inhibition of intracellular c AMP production, intracellular c GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity to suppress or reduce H)
  • a cell stimulating activity for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, Inhibition of intracellular c AMP production, intracellular c GMP production, inositol phosphate production, cell membrane
  • the test compound 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.
  • the present invention provides
  • the labeled test compound is transferred to the receptor protein of the present invention or its salt or Is a method for measuring the amount of binding of a labeled test compound to the protein or a salt thereof, or to the partial peptide or a salt thereof when the sample is brought into contact with the partial peptide or a salt thereof of the present invention.
  • a method for determining a ligand for a receptor protein or a salt thereof
  • a method for determining a ligand for a receptor protein of the present invention which comprises measuring a binding amount of a compound to the receptor protein or a salt thereof;
  • a cell stimulating activity via the receptor protein eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular Activities that promote cAMP generation, suppression of intracellular cAMP production, intracellular cGMP generation, inositol phosphate production, fluctuation of cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease of pH, etc.
  • a cell stimulating activity via the receptor protein eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular Activities that promote cAMP generation, suppression of intracellular cAMP production, intracellular cGMP generation, inositol phosphate production, fluctuation of cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease of pH, etc.
  • a method for determining a ligand for the receptor protein or a salt thereof of the present invention e.g, arachidonic acid release, ace
  • Cell stimulating activity via a receptor protein when a test compound is brought into contact with a receptor protein expressed on a cell membrane by culturing a transformant containing a DNA encoding the receptor protein of the present invention for example, Arachidonic acid release, Acetylcholine release, Intracellular Ca 2+ release, Intracellular cAMP production, Intracellular cAMP production suppression, Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein Phosphorylation, activation of c-fos, activity of suppressing or reducing pH, etc.
  • a transformant containing a DNA encoding the receptor protein of the present invention for example, Arachidonic acid release, Acetylcholine release, Intracellular Ca 2+ release, Intracellular cAMP production, Intracellular cAMP production suppression, Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein Phosphorylation, activation of
  • any receptor protein used in the method for determining a ligand may be used as long as it contains the above-described receptor protein of the present invention or the partial peptide of the present invention. Suitable receptor proteins are suitable.
  • the receptor protein of the present invention is preferably produced by expressing DNA encoding the receptor protein in mammalian cells or insect cells using the above expression method.
  • a complementary DNA is usually used as the DNA fragment encoding the protein portion of interest, but is not necessarily limited to this.
  • a gene fragment or a synthetic DNA may be used.
  • the nuclear polyhedrosis virus belonging to baculovirus using the DNA fragment as an insect host is required.
  • NPV nuclear polyhedrosis virus
  • SV40-derived promoter SV40-derived promoter
  • retrovirus promoter metamouth thionine promoter
  • human heat shock promoter cytomegalovirus promoter
  • SR ⁇ promoter SR ⁇ promoter
  • Inspection of the amount and quality of the expressed receptor can be performed by a known method. For example, the method is carried out according to the method described in the literature [Nambi, P. et al., The Journal of Biological 'Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992]. be able to.
  • the receptor protein of the present invention or a partial peptide thereof or a salt thereof may be a receptor protein or a partial peptide thereof or a salt thereof purified according to a known method. Or a cell containing the receptor protein or a cell membrane fraction thereof may be used.
  • the cell When a cell containing the receptor protein of the present invention is used in the method for determining a ligand of the present invention, the cell may be immobilized with datalaldehyde, formalin, or the like.
  • the immobilization 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.
  • Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells and the like are used as the host cell.
  • the cell membrane fraction refers to a cell membrane-rich fraction obtained by disrupting cells and then obtained by a known method.
  • Cell crushing methods include a method of crushing cells with a Potter-Elvehjem type homogenizer, a pelleting blender and a polytron.
  • centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • cell lithotripsy is centrifuged at low speed (500 rpm to 300 rpm) for a short period of time (usually about 1 to 10 minutes), and the supernatant is further sped up (150 rpm to 1000 rpm).
  • the mixture is centrifuged at 300 rpm for 30 minutes to 2 hours, and the resulting precipitate is used as a membrane fraction.
  • the membrane fraction is rich in the expressed receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the receptor protein of the cells or during the membrane fraction containing the receptor protein is preferably from 1 0 3 to 1 0 8 molecules per cell, which is the one 0 5-1 0 7 minutes terminal Is preferred.
  • receptor protein fraction a natural receptor protein fraction or a recombinant receptor fraction having an activity equivalent thereto is desirable.
  • equivalent activity refers to equivalent ligand binding activity, signal transduction activity and the like.
  • the method for determining a ligand for the receptor protein or a salt thereof first, cells or a membrane fraction of the cell containing the receptor protein of the present invention are suspended in a buffer suitable for the method for determination.
  • the buffer may be any buffer such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris monohydrochloride buffer which does not inhibit the binding between the ligand and the receptor protein.
  • various proteins such as detergents such as CHAPS, Tween-80 TM (Kao-Ichi Atlas), digitonin, and deoxycholate, and serum albumin and gelatin are buffered.
  • PMSF peroxisome proliferative protein
  • leptin peroxisome proliferative protein
  • PMSF peroxisome proliferative protein
  • Protease inhibitors such as E-64 (manufactured by Peptide Research Laboratories) and pepstatin can also be added.
  • 0. 0 1 ml to 1 0 to ml of said receptor solution a certain amount (5 0 0 0 cpm ⁇ 5 0 0 0 0 0 cpm) of [3 H :), [125 1], [14 C] [ 35 S] coexist test compound labeled with a.
  • the reaction is carried out at about 0 ° C. to 50 ° C., preferably about 4 ° C. to 37 ° C., for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
  • filter with a glass fiber filter or the like wash with an appropriate amount of the same buffer, and measure the radioactivity remaining on the glass fiber filter with a liquid scintillation counter or y -counter.
  • a test compound in which the count (B-NSB) obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) exceeds 0 cpm is defined as a ligand (agonist) for the receptor protein of the present invention or a salt thereof. You can choose.
  • a cell stimulating activity via the receptor protein for example, arachidonic acid release, acetylcholine release, intracellular Ca 2 + Release, intracellular cAMP production, intracellular cAMP production inhibition, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, cell-protein phosphorylation, c-fOS activation, pH Activity that promotes or suppresses the decrease of the protein
  • a cell stimulating activity via the receptor protein for example, arachidonic acid release, acetylcholine release, intracellular Ca 2 + Release, intracellular cAMP production, intracellular cAMP production inhibition, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuations, cell-protein phosphorylation, c-fOS activation, pH Activity that promotes or suppresses the decrease of the protein
  • a known method or a commercially available measurement kit for example, arachidonic acid release, acetylcholine release, intracellular Ca 2 + Release, intracellular c
  • the ligand Before determining the ligand, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells, add the test conjugate, incubate for a certain period of time, and then extract the cells or collect the supernatant. Then, the produced product is quantified according to each method. If the production of a substance (for example, arachidonic acid) as an indicator of the cell stimulating activity is difficult to be assayed by a degrading enzyme contained in a cell, an inhibitor for the degrading enzyme may be added to perform the assay. In addition, activities such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production has been increased by forskolin or the like.
  • a substance for example, arachidonic acid
  • Kit for determining ligand binding to receptor protein of the present invention or salt thereof Contains a receptor protein of the present invention or a salt thereof, a partial peptide or a salt thereof of the present invention, a cell containing the receptor protein of the present invention, or a membrane fraction of a cell containing the receptor protein of the present invention. Is what you do.
  • kits for determining a ligand of the present invention include the following.
  • CHO cells expressing the receptor protein of the present invention 1 2-well plates and passaged 5 X 1 0 5 or Z holes, 2 days of culture at 3 7 ° C, 5% C 0 2, 9 5% air What did.
  • Test conjugates that are poorly soluble in water are dissolved in dimethylformamide, DMSO, methanol and the like.
  • the same as the labeling compound is prepared at a concentration of 100 to L000 times higher.
  • 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 brain, pituitary, heart, knee, adipose tissue, mammary gland, testis, and the like. Specifically, angiotensin, bombesin, canapinoid, cholecystokine, glutamine, serotonin, melatonin, nucleated peptide ⁇ , opioid, pudding, vasopressin, xyxitocin, PAC ⁇ (eg, PACAP 27, PACAP 38 ), Secretin, glucagon, calcitonin, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (passoactive intestinale and related polypeptide), somatostatin, dopamine, motilin, amylin, RP (Calcitonin Gene Rated peptide), leukotriene, pancreastatin, prostaglan
  • a preventive and / or therapeutic agent for a disease associated with dysfunction of the G protein-coupled receptor protein of the present invention if the ligand for the receptor protein of the present invention is identified, then, depending on the action of the ligand, (1) the receptor protein of the present invention or (2) DNA encoding the receptor protein may be: 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.
  • the receptor protein of the present invention 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), (1) administering the receptor protein of the present invention to the patient, By supplementing the amount of receptor protein, or (2) administering the DNA encoding the receptor protein of the present invention to the patient and expressing it, or (mouth) transferring the receptor of the present invention to target cells.
  • the amount of the receptor protein in the patient's body can be increased by, for example, transplanting the cells into the patient, so that the effect of the ligand can be fully exerted. it can. That is, DNA encoding the receptor protein of the present invention is useful as a safe and low-toxic agent for preventing and / or treating diseases associated with dysfunction of the receptor protein of the present invention.
  • the receptor protein of the present invention is a G protein-coupled receptor protein, hOT7T09 (Japanese Patent Application Laid-Open No. 2000-279183), which has an amino acid sequence level of 35.7. It is a novel seven-transmembrane receptor protein with approximately% homology.
  • the receptor protein of the present invention or DNA encoding the receptor protein may be used for central diseases (eg, Alzheimer's disease, dementia, eating disorders, etc.), inflammatory diseases (eg, allergy, asthma, rheumatism, etc.), cardiovascular diseases (eg, Hypertension, cardiac hypertrophy, angina, arteriosclerosis, 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.) , Metabolic diseases (eg, diabetes, diabetic complications, obesity, arteriosclerosis, gout, cataract, etc.), immune system diseases (eg, autoimmune disease, etc.), digestive system diseases (eg, gastric ulcer, duodenal ulcer, It is useful for prevention and treatment of gastritis, reflux esophagitis, etc.).
  • central diseases eg, Alzheimer's disease, dementia, eating disorders, etc.
  • inflammatory diseases eg, allergy, asthma, rheumatism, etc.
  • the receptor protein of the present invention When used as the above-mentioned prophylactic or therapeutic agent, It can be formulated according to conventional methods.
  • the DNA of the present invention when used as the above-mentioned prophylactic / therapeutic agent, the DNA of the present invention may be used alone or in a retroviral vector. After insertion into a suitable vector, such as an adenovirus vector, an adenovirus associated virus vector, etc., 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 uptake, using a gene gun or a catheter such as a hydrogel catheter.
  • the receptor protein of the present invention or (2) DNA encoding the receptor protein may be orally administered as sugar-coated tablets, capsules, elixirs, microcapsules, or the like, or water or other substances. It can be used parenterally in the form of an injection, such as a sterile solution with a pharmaceutically acceptable liquid or a suspension.
  • 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.
  • the formulations can be prepared by mixing them 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. And sucrose, lactose or saccharine, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
  • 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 are formulated according to standard pharmaceutical practice, such as by 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.
  • aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, Thorium, etc.), and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbate 80 TM) It may be used in combination with HCO-50).
  • solubilizers 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 It may be combined with serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • buffers eg, phosphate buffer, sodium acetate buffer
  • soothing agents eg, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers eg, human It may be combined with serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg.g, antioxidants, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and have low toxicity, so they can be administered to mammals (e
  • the dosage of the receptor protein of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration for example, in a cancer patient (as 60 kg), it is generally required to be administered per day. It is about 0.1 mg to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg.
  • 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 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg by intravenous injection. .
  • the amount converted per 60 kg can be administered.
  • the dosage of the DNA of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like, in the case of oral administration, for example, in a cancer patient (as 6 O kg), for example, From about 0.1 mg per day: L 00 mg, preferably from about 1.0 to 5 Omg, more preferably from about 1.0 to 20 mg.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • an injection usually, for example, in a cancer patient (as 60 kg), about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 0.1 mg / day. It is convenient to administer ⁇ 1 Omg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the DNA of the present invention can be used as a probe to produce the receptor protein of the present invention or a portion thereof in mammals (eg, human, rat, mouse, egret, sheep, pig, pig, cat, dog, monkey, etc.).
  • mammals eg, human, rat, mouse, egret, sheep, pig, pig, cat, dog, monkey, etc.
  • Abnormalities (gene abnormalities) in the DNA or mRNA encoding the peptide can be detected. For example, damage, mutation, or reduced expression of the DNA or mRNA, or increased or excessive expression of the DNA or mRNA. It is useful as a diagnostic agent for genes.
  • the above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the well-known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989)). Proceedings of the National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America), 8 o ;, pp. 2766-2770 (1 989 )).
  • the DNA of the present invention 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.
  • the present invention provides, for example, (i) non-human mammal blood, specific organs,
  • the receptor protein of the present invention or a portion thereof obtained by measuring the amount of mRNA of the receptor protein of the present invention or a portion thereof contained in a tissue or cell isolated from an organ, or (ii) a transformant or the like.
  • a method for screening a compound that changes the expression level of a peptide is specifically performed as follows.
  • non-human mammals eg, mice, rats, rabbits, sheep, sheep, pigs, rabbits, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, arteriosclerotic rabbits
  • Drugs eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
  • blood or a specific organ eg, adipose tissue, mammary gland, testis, etc.
  • a tissue or a cell isolated from the organ is obtained.
  • the mRNA of the receptor protein of the present invention or a partial peptide thereof contained in the obtained cells can be quantified by, for example, extracting mRNA from cells or the like by a usual method and using, for example, a technique such as TaqManPCR.
  • the analysis can also be performed by performing a Northern blot using a known means.
  • 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 similarly prepared. Quantification and analysis.
  • Screening for a compound that alters the expression level of the receptor protein or its partial peptide of the present invention comprises:
  • a given time before drug or physical stress is applied to a normal or disease model non-human mammal (30 minutes to 24 hours before, preferably 30 minutes to 12 hours before, Preferably 1 hour to 6 hours before) or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or drug or physical
  • the test compound is administered simultaneously with the target stress, and after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours 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 cells,
  • the test compound is mixed in the medium, After a certain period of culture (after 1 day to 7 days, preferably after 1 day to 3 days, more preferably after 2 days to 30 days), m of the receptor protein of the present invention or its partial peptide contained in the transformant is obtained. It can be performed by quantifying and analyzing the amount of RNA.
  • 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 the partial peptide thereof of the present invention.
  • G protein-coupled receptor e.g., arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular Promotes cAMP production, suppression of intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, reduction of pH, etc.
  • G protein-coupled receptor e.g., arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular Promotes cAMP production, suppression of intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, reduction of pH, etc.
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like. These compounds may be novel compounds or known compounds.
  • the compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein or the like of the present invention.
  • the compound that attenuates the cell stimulating activity is useful as a safe and low-toxic I I-living drug for decreasing the physiological activity of the receptor protein or the like of the present invention.
  • 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 conventional means.
  • 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 have low toxicity, so they can be administered to mammals (eg, humans, rats, mice, puppies, sheep, pigs, puppies, cats, dogs, monkeys, etc.). can do.
  • the dosage of the compound or its salt varies depending on the administration subject, target organ, symptoms, administration method, and the like. In this case, the amount is about 0.1 to 10 Omg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 nig per day.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. It is convenient to administer about 0.01 to 3 Omg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 1 Omg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • a preventive and / or therapeutic agent for various diseases containing a compound that changes the expression level of the receptor protein of the present invention or its partial peptide.
  • 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.
  • the conjugate When used as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention, it can be formulated according to conventional means.
  • 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.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by the following. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • additives that can be mixed with tablets, capsenoles, etc.
  • Zera Binders such as tin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sucrose, Sweetening agents such as lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cheese are used.
  • 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 liquid for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
  • Auxiliaries such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 TM, HCO-50) Good.
  • 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.
  • 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 It may be blended with albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • buffers for example, phosphate buffer and sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers for example, human serum It may be blended with albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, benzyl alcohol, phenol, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and have low toxicity, so they can be administered to mammals (eg
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, condition, administration method, and the like.
  • oral administration for example, in a patient with cancer (as 60 kg), one dose is generally used. It is about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • about 0.01 to 3 Omg per day for example, in cancer patients (as 6 O kg)
  • about 0.01 to 3 Omg per day preferably about 0.1 to 20 mg, more preferably about 0.1 to 20 mg per day. It is convenient to administer about 1 to 1 Omg by intravenous injection.
  • the dose can be administered in terms of 60 kg.
  • 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.
  • the ligand and the receptor protein or the like of the present invention can be obtained.
  • the ligand and the receptor protein or the like of the present invention can be obtained.
  • peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, etc. or salts thereof can be efficiently screened.
  • Such compounds include (i) cell stimulating activity via G protein-coupled receptors (eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production, intracellular cAMP production Suppression, Intracellular c GMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, c-fos A compound having an activity of promoting or suppressing activation, reduction of pH, etc.
  • G protein-coupled receptors eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production, intracellular cAMP production Suppression, Intracellular c GMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, c-fos
  • the compound (a) is preferably screened by the ligand determination method described above).
  • 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 contacted with a ligand; and (ii) a receptor protein of the present invention or its partial peptide or a salt thereof, Ligand and test Screening of a compound or a salt thereof which changes the binding property between the ligand and the receptor protein of the present invention or a partial peptide thereof or a salt thereof, which is compared with the case where the compound is brought into contact with the compound.
  • a receptor protein of the present invention or its partial peptide or a salt thereof Ligand and test Screening of a compound or a salt thereof which changes the binding property between the ligand and the receptor protein of the present invention or a partial peptide thereof or a salt thereof, which is compared with the case where the compound is brought into contact with the compound.
  • the screening method of the present invention is characterized in that, in the cases (i) and (ii), for example, the amount of a ligand bound to the receptor protein or the like, the cell stimulating activity, and the like are measured and compared.
  • the present invention provides
  • Receptor-mediated cell stimulating activity e.g., arachidonic acid release, acetylcholine release, intracellular Ca
  • 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
  • a transformant containing the DNA of the present invention is cultured on a cell membrane by culturing a transformant containing the DNA of the present invention when a compound that activates the receptor protein or the like of the present invention and a test compound are contacted with a receptor protein or the like.
  • Receptor-mediated cell stimulating activity eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production, intracellular cAMP production when brought into contact with the receptor protein or the like of the present invention
  • the comparison ligand of the present invention which is characterized in that the receptacle Provided is a method for screening a compound or a salt thereof that changes the binding property to a putter protein or the like.
  • a candidate compound is obtained (primary screening), and then a test (secondary screening) is performed to confirm whether or not the target compound actually inhibits the binding of a human G protein-coupled receptor protein to a ligand. ) was required. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins will be mixed in, so it has been difficult to actually screen for an agonist or an antagonist for the target receptor protein.
  • the human-derived receptor protein of the present invention 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. Furthermore, whether the screened compound is an agonist or an antagonist can be easily evaluated.
  • 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.
  • human receptor proteins and the like that are expressed in large amounts using recombinants are suitable for screening.
  • the above method is used to produce the receptor protein or the like of the present invention, but it is preferably carried out by expressing the DNA of the present invention in mammalian cells or insect cells.
  • Complementary DNA is used for the DNA fragment encoding the target protein portion.
  • the force is not necessarily limited to this.
  • gene fragments or synthetic DNA may be used.
  • the DNA fragment is Nuclear polyhedrosis virus (nuclear)
  • polyhedrosis virus (NPV) polyhedrin promoter SV40-derived motor, retroporinores promoter, metallotill promoter, human heat shock promoter, cytomegaloinores promoter, SRa promoter and the like.
  • the amount and quality of the expressed receptor can be examined by a known method. For example, it can be carried out according to the method described in the literature [Nambi, P. et al., J. Biol. Biological 'Chemistry, Vol. 267, pp. 19555-19559, 1992]. it can.
  • 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. Alternatively, a membrane fraction of cells containing the receptor protein or the like may be used.
  • 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 or the like of the present invention is preferably a force S that refers to a host cell that has expressed 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 fraction containing a large amount of cell membrane obtained by breaking a cell and then using a known method.
  • Cell crushing methods include a method of crushing cells with a Potter-Elvehjem type homogenizer, a Perling blender and a polytron.
  • fractionation by centrifugal force such as fractionation centrifugation or density gradient centrifugation is mainly used.
  • the cell lysate is centrifuged at low speed (500 rpm to 3000 rpm) for a short time (usually about 1 to 10 minutes), and the supernatant is further centrifuged at high speed (15,000 to 30000 rpm). Usually, centrifuge for 30 minutes to 2 hours, and use the resulting precipitate as the membrane fraction.
  • the membrane fraction is rich in expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.
  • the 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 cells per cell, 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 allows a large number of samples Be able to measure.
  • an appropriate receptor-one protein fraction and a labeled ligand are required.
  • receptor protein fraction a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto is desirable.
  • equivalent activity refers to equivalent ligand binding activity, signal transduction action, and the like.
  • labeled ligand a labeled ligand, a labeled ligand analog compound and the like are used.
  • a cell or a membrane fraction of the cell containing the receptor protein of the present invention is first screened.
  • 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-HCl.
  • a surfactant such as CHAPS, Tween-80 TM (Kaoichi Atlas), digitonin, or dexcholate can be added to the buffer.
  • protease inhibitors such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and pepstatin can be added to suppress the degradation of receptors and ligands by proteases.
  • 0.01Ml ⁇ to the receptor solution
  • L OML added labeled ligand a certain amount (5000 cpm ⁇ 500000 cm), coexist simultaneously 10- 4 M to test compounds of 10- 10 M.
  • NBS non-specific binding
  • a reaction tube to which the labeled ligand has been added. The reaction is carried out at about 0 ° C. to 50 ° C., preferably about 4 ° C.
  • Non-specific binding amount (NS.) Minus non-specific binding amount (NSB) is subtracted from the count ( ⁇ .-one NSB) when 100% is defined as the specific binding amount (B-NSB) force.
  • a test compound having a concentration of 50% or less can be selected as a candidate substance having competitive inhibitory ability.
  • a cell stimulating activity mediated by a receptor protein eg, arachidonic acid release, acetylcholine
  • Intracellular Ca 2+ release Intracellular cAMP production
  • Intracellular cAMP production inhibition Intracellular cGMP production
  • Inositol phosphate production Cell membrane potential fluctuation
  • Intracellular protein phosphorylation activity to promote or suppress fos activation, pH reduction, etc.
  • cells containing the receptor protein or the like of the present invention are cultured on a multi-well plate or the like.
  • replace the cells with fresh medium or an appropriate buffer that is not toxic to the cells, incubate with a test compound etc. for a certain period of time, extract the cells, or collect the supernatant.
  • the products generated 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 a degrading enzyme contained in a cell, the assay may be performed by adding an inhibitor against the degrading enzyme. Good. In addition, activities such as suppression of cAMP production can be detected as production inhibiting effects on cells whose basic production has been increased with forskolin or the like.
  • a substance for example, arachidonic acid
  • Cells expressing an appropriate receptor protein are required.
  • Cells expressing the receptor protein etc. of the present invention include natural type books.
  • a cell line having the receptor protein or the like of the invention, Cell lines expressing the recombinant receptor protein or the like are desirable.
  • test compounds for example, peptides, proteins, non-peptidic compounds, synthetic conjugates, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds are novel compounds. Or a known compound.
  • a screening kit for a compound or a salt thereof that alters the binding 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.
  • screening kit of the present invention examples include the following. 1. Screening reagent
  • CHO cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at 5 ⁇ 10 5 Z-wells, and cultured at 37 ° C., 5% CO 2 , and 95% air for 2 days.
  • Ligand 0.1 0 /. ⁇ Dissolve to 1 mM in PBS containing serum albumin (manufactured by Sigma) and store at _20 ° C
  • test compound solution After adding 5 mu 1 to a labeled ligand 5 beta 1 was added and reacted at room temperature for 1 hour. To determine the amount of non-specific binding, add 10- 3 ⁇ ⁇ ⁇ ⁇ ⁇ ligand (5 ⁇ 1) instead of the test compound.
  • 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 between the ligand and the receptor protein of the present invention.
  • Cell stimulating activity via G protein-coupled receptors e.g., arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular cAMP production, inhibition of intracellular cAMP production, intracellular cGMP production,
  • G protein-coupled receptors e.g., arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular cAMP production, inhibition of intracellular cAMP production, intracellular cGMP production,
  • receptor protein of the present invention (so-called receptor protein of the present invention) ), (Mouth) a compound not having the cell stimulating activity (so-called 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) decreases the binding force between the ligand and the G protein-coupled 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) decreases the binding force between the ligand and the G protein-coupled receptor protein of the present invention.
  • Such compounds include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation
  • the compound may be a novel compound or a known compound.
  • the agonist against the receptor protein or the like of the present invention has the same activity as the physiological activity of the ligand for the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug according to the ligand activity.
  • 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 reduces the binding force between the ligand and the G protein-coupled receptor protein of the present invention is useful as a safe and low-toxic drug for reducing the physiological activity of the ligand for the receptor protein or the like of the present invention. .
  • the compound or its salt obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out according to a conventional method.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as in the above-mentioned 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 mammals (eg, humans, rats, mice, egrets, sheep, pigs, pests, cats, dogs, monkeys, etc.). Can be administered.
  • mammals eg, humans, rats, mice, egrets, sheep, pigs, pests, cats, dogs, monkeys, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, condition, administration method, and the like. However, in the case of oral administration, for example, in a patient with cancer (as 60 kg), one dose is generally used. It is about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. Is about 0.01 to 3 O mg per day, preferably about 0.1 to 20 mg, more Preferably about 0.1 to: about 0 mg L is conveniently administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • 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.
  • the receptor protein of the present invention is considered to play some important roles in vivo, such as central functions, circulatory functions, and digestive functions. Therefore, 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 can be used to prevent diseases associated with dysfunction of the receptor protein of the present invention. Z or can be used as a therapeutic agent.
  • the compound or ligand When used as a preventive 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.
  • the compound or ligand is orally administered as tablets, capsules, elixirs, microcapsenoles, etc., if necessary, with sugar or water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as sterile solutions or suspensions.
  • the compound may be formulated in a unit dosage form required for generally accepted pharmaceutical practice with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like. It can be manufactured by mixing. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Excipients that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline 'I raw cenorelose, corn starch, gelatin, A swelling agent such as alginic acid, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharine, and a flavoring agent such as peppermint, cocoa oil or cherry are used.
  • a liquid carrier such as an oil or fat may be contained.
  • 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 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 alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 TM, HCO-50) You may use together.
  • 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.
  • the above-mentioned prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human It may be combined with serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • 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, so they can be administered to mammals (eg, humans, rats, mice, puppies, sheep, pigs, puppies, cats, dogs, monkeys, etc.). can do.
  • mammals eg, humans, rats, mice, puppies, sheep, pigs, puppies, cats, dogs, monkeys, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, subject ⁇ , symptom, administration method, and the like. It is about 0.1 to 10 Omg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the subject of administration, target organ, symptoms, administration method, and the like. Is about 0.01 to 3 O mg per day, preferably about 0.1 to 20 mg, more It is convenient to administer preferably about 0.1 to 1 Omg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • 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,
  • a test comprising 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.
  • one antibody is an antibody that recognizes the N-terminal of the receptor protein or the like of the present invention
  • the other antibody is an antibody that reacts with the C-terminal of the receptor protein or the like of the present invention.
  • the receptor protein of the present invention can be measured using a monoclonal antibody against the receptor protein or the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), and detection by tissue staining or the like can be performed. You can also.
  • the antibody molecule itself may be used, or the 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 corresponding to the amount of antigen (eg, the amount of receptor protein) in the test solution.
  • any method that detects the amount of the antigen complex by chemical or physical means and calculates this from a standard curve prepared using a standard solution containing a known amount of the antigen can be used. Is also good. For example, nephrometry, competition method, imnomome Rick method and sandwich method are preferably used, but it is particularly preferable to use the sandwich method described later in terms of sensitivity and specificity.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used.
  • the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • the enzyme a stable enzyme having a large specific activity is preferable.
  • the fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin and the like are used.
  • a biotin-avidin system can be used for binding the antibody or antigen to the labeling agent.
  • insolubilization of the antigen or the 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.
  • the carrier for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction the labeled monoclonal antibody of the present invention
  • 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 can be insoluble in the method described above.
  • 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.
  • the monoclonal antibody of the present invention used in the primary reaction and the secondary reaction is a receptor protein.
  • Antibodies having different binding sites such as proteins are 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 the C-terminal. For example, an antibody that recognizes an N-terminal other than 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.
  • a competition method an antigen in a test solution and a labeled antigen are allowed to react competitively with an antibody, and then the unreacted labeled antigen, (F), and the labeled antigen (B) combined with the antibody are combined.
  • F labeled antigen
  • B labeled antigen
  • This reaction method uses a soluble antibody as the antibody, a liquid phase method using polyethylene glycol for the BZF separation, a second antibody against the above antibody, and the ability to use an immobilized antibody as the first antibody, or An immobilization method using a soluble antibody as the first antibody and using an immobilized antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of the labeled antibody, and then the solid phase and the liquid phase are separated.
  • 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 either phase is measured to determine the amount of antigen in the test solution.
  • 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 the antigen in the test solution is small and the amount of sediment is small and the force is not humiliated, laser nephrometry utilizing laser scattering is preferably used.
  • 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.
  • various diseases associated with dysfunction of the receptor protein of the present invention can be diagnosed by quantifying the receptor protein of the present invention or a salt thereof in vivo using the antibody of the present invention.
  • 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, and measurement of the behavior of the receptor protein of the present invention in test cells. Can be used for analysis and the like.
  • the antibody of the present invention can specifically recognize the receptor protein of the present invention or a 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.
  • Non-human mammal 1 blood, 2 specific organs, 3 tissue isolated from ⁇ The cell membrane fraction after isolating the cell membrane fraction, and quantifying the receptor protein of the present invention or a partial peptide thereof contained in the cell membrane fraction, whereby the receptor protein of the present invention or the portion thereof in the cell membrane is determined.
  • 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.
  • Sections of (1) blood, (2) specific organs, and (3) tissues or cells isolated from organs of non-human mammals, and then using immunostaining to obtain the receptor protein on the cell surface A method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in a cell membrane by confirming the protein on the cell membrane by quantifying the degree of staining of the cell.
  • the quantification of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically performed as follows.
  • non-human mammals eg, mice, rats, rabbits, sheep, sheep, pigs, rabbits, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, arteriosclerotic rabbits
  • Drugs eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
  • blood or a specific organ eg, adipose tissue, mammary gland, testis, etc.
  • a tissue or cell isolated from the organ is obtained. The obtained organs, tissues, cells, etc.
  • a cell membrane fraction is obtained using a technique such as force column fractionation.
  • an appropriate buffer for example, Tris-HCl buffer, phosphate buffer, Buffer, etc.
  • a surfactant for example, Triton X-100 TM, Tween 20 TM, etc.
  • a cell membrane fraction is obtained using a technique such as force column fractionation.
  • the cell membrane fraction refers to a fraction containing a large amount of cell membrane obtained by a known method after cell disruption.
  • Cells can be disrupted by crushing cells with a Potter-Elvehjem homogenizer, Waring Plender ⁇ Polytron.
  • centrifugal fractionation methods such as differential centrifugation and density gradient centrifugation are mainly used.
  • the cell lysate is centrifuged at a low speed (500 rpm to 3000 rm) for a short time (typically about 1 to 10 minutes), and the supernatant is further centrifuged at a higher speed (15 OOO r pn! To 30000 rpm). Centrifuge for 30 minutes to 2 hours, and use the resulting precipitate as the membrane fraction.
  • the membrane fraction is rich in expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.
  • the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, western blot analysis, or the like.
  • Such a sandwich immunoassay can be performed in the same manner as described above, and the ⁇ stan blot can be performed by known means.
  • a transformant expressing the receptor protein of the present invention or its partial peptide is prepared according to the above method, and the receptor protein of the present invention or its partial peptide 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:
  • a given time before giving a drug or physical stress to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour Before to 6 hours before) or after a certain time (after 30 minutes to 3 days, preferably after 1 hour to 2 days, more preferably after 1 hour to 24 hours), or at the same time as drug or physical stress Administration
  • the receptor protein of the present invention or its partial peptide in the cell membrane is By quantifying the amount,
  • test compound When culturing the transformant according to a conventional method, the test compound is mixed in a medium, and after culturing for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days) After a day), it can be carried out by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane.
  • the identification of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically performed as follows.
  • non-human mammals eg, mice, rats, rabbits, sheep, sheep, stags, rabbits, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, atherosclerotic rabbits
  • Drugs eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
  • blood or a specific organ eg, adipose tissue, mammary gland, testis, etc.
  • a tissue or cell isolated from the organ is obtained.
  • the obtained organ, tissue or cell is cut into a tissue section according to a conventional method, and immunostained using the antibody of the present invention.
  • the receptor protein of the present invention or a partial peptide thereof on the cell membrane can be quantitatively or qualitatively determined. You can check and confirm the quantity.
  • the conjugate 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.
  • the cell stimulating activity via G protein-coupled receptor for example, arachidonic acid release, acetylcholine release, intracellular C a 2+ release, intracellular c AMP production, intracellular c AMP production Cell growth, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity to promote pH reduction, etc.
  • G protein-coupled receptor for example, arachidonic acid release, acetylcholine release, intracellular C a 2+ release, intracellular c AMP production, intracellular c AMP production Cell growth, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like. These compounds may be novel compounds or known compounds.
  • the compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein or the like of the present invention.
  • the compound that 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.
  • 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.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned pharmaceuticals containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and have low toxicity, so they can be administered to mammals (eg, humans, rats, mice, puppies, sheep, pigs, puppies, cats, dogs, monkeys, etc.). can do.
  • mammals eg, humans, rats, mice, puppies, sheep, pigs, puppies, cats, dogs, monkeys, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • the daily dose is generally one day.
  • About 0.1 to 10 Omg preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • it is usually used, for example, in cancer patients (as 6 Okg).
  • the dose can be administered in terms of 60 kg.
  • the receptor protein of the present invention is, for example, It is considered to play some important role in vivo such as function. Therefore, a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be used as a preventive and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention.
  • the compound when 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.
  • the compound is orally sterilized with water or other pharmaceutically acceptable liquids, such as tablets, capsules, elixirs, microforced tablets and the like, optionally coated with sugar. It can be used parenterally in the form of injections, such as solutions or suspensions.
  • the compound can be used together with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, P-preservatives, stabilizers, binders, and the like, in a unit dosage form generally required for the practice of preparations. It can be manufactured by mixing. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be mixed with tablets, capsenoles, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, and flavoring agents such as peppermint, cocoa oil or cherry.
  • 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 are formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil or coconut oil.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
  • Agents such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 TM, HCO-50) may be used in combination.
  • 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.
  • 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 It may be blended with albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc.
  • buffers for example, phosphate buffer and sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers for example, human serum It may be blended with albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, benzyl alcohol, phenol, etc.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and have low toxicity, so they can be administered, for example, to
  • the dosage of the compound or its salt may vary depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral administration in general, for example, in a cancer patient (as 60 kg), About 0.1 to 10 Omg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 2 Omg.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • injection it is usually used, for example, in cancer patients (as 6 Okg).
  • the amount converted per 60 kg can be administered.
  • the receptor protein of the present invention or a partial peptide thereof or a salt thereof means an activity of inactivating a signal transduction function involving the receptor protein. Therefore, when the antibody has 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) , Intracellular cAMP production, Intracellular cAMP production suppression, Intracellular cGMP production, Inositol phosphate production, Cell membrane potential fluctuation, Intracellular protein phosphorylation, c-fos activation, PH decrease Activity or inhibitory activity). Therefore, it can be used for prevention or treatment of diseases caused by overexpression of the receptor protein.
  • cell stimulating activity via the receptor protein eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release
  • Intracellular cAMP production Intracellular cAMP production suppression, Intracellular cGMP production, Inositol
  • transgenic animals expressing the receptor protein and the like of the present invention can be prepared.
  • Animals include mammals (eg, rats, mice, egrets, sheep, pigs, pigs, cats, dogs, monkeys, etc.)
  • mice may be abbreviated as an animal
  • a heron and the like are particularly preferable.
  • the DNA of the present invention When introducing the DNA of the present invention into a target animal, it is generally advantageous to use the DNA as a gene construct linked downstream of a promoter capable of being expressed in animal cells.
  • a DNA construct of the present invention derived from an animal having a high homology to this is linked to a gene construct linked downstream of various promoters capable of expressing the DNA in animal cells.
  • a ubiquitous expression promoter such as a virus-derived promoter or meta-mouth thionein can be used, but an NGF gene promoter and a phenolase gene promoter that are specifically expressed in the brain are preferably used.
  • the introduction of the DNA of the present invention at the fertilized egg cell stage can be performed by Reserved to be present in all somatic cells.
  • the presence of the receptor protein or the like of the present invention in the germinal cells of the produced animal after the introduction of the DNA indicates that all of the offspring of the produced animal have the receptor protein or the like of the present invention in all of the germinal and somatic cells. Means that.
  • the progeny of such animals that have inherited the gene have the receptor protein of the present invention in all of their germinal and somatic cells.
  • the DNA-introduced animal of the present invention After confirming that the DNA-introduced animal of the present invention stably retains the gene by crossing, it can be reared and subcultured 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 progeny can obtain the DNA. Breeding can be subcultured 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 agonist or antagonist staring 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.
  • tissue culture For example, by directly analyzing DNA or RNA in the tissue of the DNA-introduced mouse of the present invention, or by analyzing the tissue in which the receptor protein of the present invention expressed by the gene is present, the receptor protein of the present invention can be obtained. Can be analyzed.
  • the cells of the 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 generally difficult-to-cultivate tissues such as brain and peripheral tissues. be able to.
  • a drug that enhances the function of various tissues can be selected.
  • the receptor protein of the present invention can be isolated and purified therefrom.
  • the abbreviations based on IUP AC—IUB Commission on Biochemical Nomenclature are based on common abbreviations in the art. If there is an optical isomer of the amino acid, the L-form is indicated unless otherwise specified. JA quinolinfoic acid
  • HONB 1-hydroxy-5-norporene-2,3-zica
  • DCC N, N'-dicyclohexylcarpoimide
  • FIG. 1 shows the amino acid sequence of the novel human-derived G protein-coupled receptor protein TGR39 of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding the novel human G protein-coupled receptor protein TGR39 of the present invention.
  • SEQ ID NO: 4 shows the base sequence of ply 1 used in the PCR reaction in Example 1 below.
  • FIG. 1 shows the amino acid sequence of the human-derived novel G protein-coupled receptor protein hTGR39A of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding the novel human-derived G protein-coupled receptor protein hTGR39A of the present invention.
  • SEQ ID NO: 8 shows the base sequence of ply 3 used in the PCR reaction in Example 2 below.
  • Example 7 shows the nucleotide sequence of a probe used in the PCR reaction in Example 3 below.
  • the transformant E scherichiacoli TOP 10 / p CR 2.1—hTGR39 obtained in Example 1 described below has been used since September 13, 2001, 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka, Osaka Prefecture, Osaka Prefecture. Co., Ltd. (Postal Code 532-8686) with the Foundation and Fermentation Research Institute (IFO) as accession number IFO 16702 1-1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan from September 25, 2001 30 5-8566) at the National Institute of Advanced Industrial Science and Technology (AIST) under the deposit number FERM BP-7775.
  • IFO Foundation and Fermentation Research Institute
  • Example 2 The transformant Escherichiacoli TOP10 / pCR2.1-I hTGR39A obtained in Example 2 described below has been used since September 18, 2001, 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka, Japan ( Contracted with the Fermentation Research Institute (IFO) with a postal code of 532-8686) as an accession number IFO 16706, 1-1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan from October 1, 2001 Deposit No. FE RM BP-7760 at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary.
  • IFO Fermentation Research Institute
  • PCR reaction was performed using two primers, Primer 1 (SEQ ID NO: 3) and Primer 2 (SEQ ID NO: 4).
  • the composition of the reaction solution in the reaction was prepared using the above cDNA as a 1 / 10-volume ⁇ form, and the AdVantage—GC 2 Polymerase Mix (CLONTECH) 1Z50 volume, Primer 1 (SEQ ID NO: 3) and Primer 1 (SEQ ID NO: 4) 0.5 ⁇ ⁇ each, dNTPs 200 ⁇ , and 1/5 volume of the enzyme-supplied buffer, GC] ⁇ 6 1 1; was set to 15 dynamic volumes and a liquid volume of 20 ⁇ l.
  • the PCR reaction is repeated at 94 ° C for 5 minutes, followed by a cycle of 94 ° C for 30 seconds, 60 ° C for 30 seconds, 68 ° C for 2 minutes 35 times, and finally for 68 ° C for 5 minutes. Reaction was performed.
  • the PCR reaction product was subcloned into a plasmid vector: CR 2.1 (Invitrogen) according to the formulation of a TA cloning kit (Invitrogen). This was introduced into E. coli TOP10, and a clone having cDNA was selected on an LB agar medium containing ampicillin.
  • the nucleotide sequence (SEQ ID NO: 2) of the cDNA encoding the novel G protein-coupled receptor protein was obtained.
  • a novel G protein-coupled receptor protein having this amino acid sequence (SEQ ID NO: 1) was named TGR39.
  • the transformant was named Escherichia coli TOP10 / pCR2.l-hTGR39.
  • a PCR reaction was performed using two primers, Primer 1 (SEQ ID NO: 7) and Primer 2 (SEQ ID NO: 4).
  • the composition of the reaction solution in this reaction was determined using the above cDNA as lZlO type I, Advantage-GC 2 Polymerase Mix (CLONTECH) 1Z50 amount, primer 1 (SEQ ID NO: 7) and primer 2 (SEQ ID NO: 4) was added to each of 0.5 / zM, dNTPs 200 ⁇ M, 1/5 volume of buffer attached to enzyme, 1Z5 volume of GCMelt, and added 20 ⁇ 1 did.
  • the PCR reaction is performed at 94 ° C for 5 minutes, followed by 94 ° C for 30 seconds, 60 ° C for 30 seconds, 68 ° C for 2 minutes and repeated 35 times, and finally 68 ° C for 5 minutes Was performed.
  • Transfer the PCR reaction product to TA cloning kit (I n V itrogen) was subcloned into a plasmid vector pCR2.1 (Invitrogen) according to the above-mentioned procedure. This was introduced into E. coli TOP10, and clones having cDNA were selected in LB agar medium containing ampicillin.
  • the nucleotide sequence of cDNA (SEQ ID NO: 6) encoding a novel G protein-coupled receptor protein was obtained.
  • a novel G protein-coupled receptor protein having this amino acid sequence (SEQ ID NO: 5) was designated as hTGR39A.
  • the transformant was named Escherichiaco 1 i TOP10 / pCR2.1-hTGR39A.
  • the 9th Asp in the amino acid sequence represented by SEQ ID NO: 1 has been substituted with Glu.
  • the 27th C in the nucleotide sequence represented by SEQ ID NO: 2 has been replaced with A.
  • TGR39 The expression distribution of TGR39 in human tissues was analyzed using TaqMan PCR.
  • the Human Multiple Tissue cDNA Panel (Clontech) is used, and primers 1 (SEQ ID NO: 8) and 2 (SEQ ID NO: 9) as primers for PCR are represented by SEQ ID NO: 10.
  • TaqMan PCR was performed using a probe having a nucleotide sequence.
  • the reaction solution composition for the reaction was 12.5 ⁇ l of TaqMan Universal PCR Master Mix (Applied Biosystems Japan), 0.5 ⁇ l of 10 / ⁇ primer 1 and primer 2 each, 0.5 ⁇ l of 5 ⁇ ⁇ ⁇ ⁇ probe and 2 ⁇ 1 of 5 ⁇ probe.
  • FIG. 3 shows the results calculated as the number of copies per ⁇ l of cDNA based on the obtained results. This indicates that the expression level of TGR39 is high in the heart.
  • the G protein-coupled receptor protein of the present invention or a partial peptide thereof or a salt thereof, and a polynucleotide encoding the receptor protein or a partial peptide thereof are: 2) Obtaining antibodies and antiserum, 3) Constructing a recombinant receptor protein expression system, 4) Developing a receptor binding assay system using the same expression system, and drug candidate compounds (agonists, antagonists) Screening), 5drug design based on comparison with structurally similar ligands receptors, ⁇ ⁇ reagents for preparing probes and PCR primers for genetic diagnosis, ⁇ ⁇ preparation of transgenic animals or 8 It can be used as a drug such as a gene preventive and therapeutic agent.

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Abstract

L'invention concerne une nouvelle protéine s'utilisant par exemple pour cribler un agoniste/un antagoniste, une protéine d'origine humaine ou son sel, un ADN codant cette protéine, un procédé permettant de déterminer un ligand de cette protéine, un procédé/un coffret de criblage pour cribler un composé pouvant modifier les propriétés de liaison du ligand avec cette protéine, des composés obtenus après criblage ou des sels desdits composés, etc. La protéine d'origine humaine mentionnée ci-dessus ou l'ADN la codant peut s'utiliser dans : (1) la détermination d'un ligand de cette protéine ; (2) des agents prophylactiques et/ou thérapeutiques pour des affections liées au dysfonctionnement de cette protéine ; (3) le criblage d'un composé permettant de modifier les propriétés de liaison de la protéine au ligand, etc.
PCT/JP2002/006356 2001-06-26 2002-06-25 Nouvelle proteine a recepteur couple a une proteine g et son adn WO2003000886A1 (fr)

Applications Claiming Priority (4)

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JP2001-193399 2001-06-26
JP2001193399 2001-06-26
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JP2001-311880 2001-10-09

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0885960A2 (fr) * 1997-06-18 1998-12-23 Smithkline Beecham Corporation Récepteur couplé à la protéine g (H7TBA62)
JPH1132770A (ja) * 1997-07-24 1999-02-09 Asahi Chem Ind Co Ltd ７回膜貫通型受容体蛋白質ｊｅｇ６２
WO2000058462A1 (fr) * 1999-03-25 2000-10-05 Banyu Pharmaceutical Co., Ltd. Nouvelles proteines de recepteur couplees aux proteines de liaison a la guanosine triphosphate (gtp), bg3

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0885960A2 (fr) * 1997-06-18 1998-12-23 Smithkline Beecham Corporation Récepteur couplé à la protéine g (H7TBA62)
JPH1132770A (ja) * 1997-07-24 1999-02-09 Asahi Chem Ind Co Ltd ７回膜貫通型受容体蛋白質ｊｅｇ６２
WO2000058462A1 (fr) * 1999-03-25 2000-10-05 Banyu Pharmaceutical Co., Ltd. Nouvelles proteines de recepteur couplees aux proteines de liaison a la guanosine triphosphate (gtp), bg3

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