[go: up one dir, main page]

WO2002059304A1 - Nouvelle proteine receptrice couplee a une proteine g et adn correspondant - Google Patents

Nouvelle proteine receptrice couplee a une proteine g et adn correspondant Download PDF

Info

Publication number
WO2002059304A1
WO2002059304A1 PCT/JP2002/000405 JP0200405W WO02059304A1 WO 2002059304 A1 WO2002059304 A1 WO 2002059304A1 JP 0200405 W JP0200405 W JP 0200405W WO 02059304 A1 WO02059304 A1 WO 02059304A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
receptor protein
salt
present
coupled receptor
Prior art date
Application number
PCT/JP2002/000405
Other languages
English (en)
Japanese (ja)
Inventor
Masanori Miwa
Takashi Ito
Yasushi Shintani
Nobuyuki Miyajima
Original Assignee
Takeda Chemical Industries, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Takeda Chemical Industries, Ltd. filed Critical Takeda Chemical Industries, Ltd.
Publication of WO2002059304A1 publication Critical patent/WO2002059304A1/fr

Links

Classifications

    • 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
    • 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

Definitions

  • the present invention relates to a novel G protein-coupled receptor protein derived from human brain or a salt thereof, and a DNA encoding the same.
  • G proteins Rereru guanine nucleotide- binding protein
  • 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 many hormones, hormone-like substances, neurotransmitters 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 body, and their receptors Many protein structures have not yet been reported. Furthermore, it is often unknown whether subtypes exist in known receptor proteins.
  • G protein-coupled receptors are useful for searching for new ligands (physiologically active substances), and for searching for agonists or antagonists to the receptor, using the signal transduction action as an index.
  • an agonist or an antagonist for the receptor is produced by analyzing the physiological action of the receptor from an inactivation experiment (knockout animal) of the receptor. It is also possible.
  • ligands, agonists or antagonists to these receptors can be expected to be used as preventive or therapeutic agents for diseases associated with impaired function of G protein-coupled receptors.
  • 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 present invention provides a novel G protein-coupled receptor protein useful as described above. That is, a novel G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, a polynucleotide containing a polynucleotide (DNA, RNA and a derivative thereof) encoding the G protein-coupled receptor protein or a partial peptide thereof (DNA, RNA and derivatives thereof), a recombinant vector containing the polynucleotide, a transformant carrying the recombinant vector, a method for producing the G protein-coupled receptor protein or a salt thereof, An antibody against the G protein-coupled receptor protein or a partial peptide thereof or a salt thereof; a compound that changes the expression level of the G protein-coupled receptor protein; a method for determining a ligand for the G protein-coupled receptor; And the G protein Compounds that alter the binding property between the mold receptor protein (antagonist Agonisuto) or disk of a salt thereof Com
  • the present inventors have succeeded in isolating cDNA encoding a novel G protein-coupled receptor protein derived from human brain and analyzing the entire nucleotide sequence thereof. Then, when this base sequence was translated into an amino acid sequence, the first to seventh transmembrane regions were confirmed on the hydrophobicity plot shown in FIG. 1, and the protein encoded by these cDNAs was transmembrane seven times. It was confirmed that it was a G-protein-coupled receptor protein. The present inventors have conducted further studies based on these findings, and as a result, have completed the present invention.
  • a G protein-coupled receptor protein or a salt thereof which comprises an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1;
  • G protein-coupled receptor protein or a salt thereof, which comprises the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2;
  • a pharmaceutical comprising the G protein-coupled receptor protein according to (1) or the partial peptide according to (4) or a salt thereof;
  • the antibody according to (13) which is a neutralizing antibody that inactivates signal transduction of the G protein-coupled receptor protein according to (1);
  • the G protein-coupled receptor according to (1) which can be obtained by using the G protein-coupled receptor protein according to (1) or the partial peptide according to (4) or a salt thereof.
  • Binding ability of the ligand obtainable by using the screening method according to (20) or the screening kit according to (21) to the G protein-coupled receptor protein or the salt thereof according to (1).
  • a pharmaceutical comprising a compound or a salt thereof that changes
  • a polynucleotide comprising the polynucleotide of (5) or a base sequence complementary to the polynucleotide of (5) or a part thereof under high stringency conditions with the polynucleotide of (5);
  • (32) a compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein according to (1) in a cell membrane obtainable by using the screening method according to (30);
  • a medicament comprising a compound capable of changing the expression level of the G protein-coupled receptor protein according to (1) or a salt thereof, which can be obtained by using the screening method according to (29)!
  • a medicament comprising a compound or a salt thereof that alters the amount of the G protein-coupled receptor protein described in (1) above in a cell membrane obtainable by using the screening method described in (30) above,
  • the present invention relates to the use of the compound according to (22), (31) or (32) or a salt thereof.
  • the invention further provides:
  • the protein is: (1) an amino acid sequence represented by SEQ ID NO: 1;
  • amino acids in the amino acid sequence represented by 1 are deleted
  • One or more (preferably about 1 to 30, more preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 1. Is a protein containing an amino acid sequence in which 1 to 10 amino acids are substituted with another amino acid, and more preferably about 10 amino acids, more preferably several (1 to 5) amino acids.
  • the ligand is, for example, angiotensin, bombesin, cannabinoid, cholecystokinin, gnoletamine, serotonin, melatonin, neuropeptide Y, opioid, purine, pasoprescin, oxotocin, PACAP (e.g., PAC ⁇ 27, P ACAP 3 8), secretin, glucagon, calcitonin, adore nomedulin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (vasoactive intestinal polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcinine) Rated peptide), leukotriene, pancreatastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfamily (eg, IL-18, GRO a GRO, GRO ⁇ NAP —
  • C chemokine subfamily such as lymphotactin
  • CX 3 C chemokine subfamily such as fractalkine
  • endothelin enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (L PA) or sphingosine monophosphate
  • L PA lysophosphatidic acid
  • sphingosine monophosphate the method for determining the ligand according to the above (39), (41) (i) when the ligand is brought into contact with the G protein-coupled receptor protein or the salt thereof described in (1) above or the partial peptide or the salt thereof described in (4) above, and (ii) )
  • the screening method according to the above (20) which is characterized in that:
  • the G protein-coupled receptor protein described in (1) above or a compound that activates the salt thereof and a test compound are cultured in the transformant described in (9) above.
  • a ligand characterized by measuring and comparing cell stimulating activity via a G protein-coupled receptor protein when the transformant is brought into contact with a G protein-coupled receptor protein expressed on the cell membrane of the transformant.
  • the compound that activates the G protein-coupled receptor protein according to the above (1) is angiotensin, bombesin, cannabinoid, cholecystokinin, gnoretamine, serotonin, melatonin, neuropeptide Y, opioid, or primin.
  • Vasoprescin, Oxytocin, PACAP e.g., PACAP27, PCAPAP38
  • Secretin Gourgon, Force ⁇ ⁇ ⁇ Cytonin, Adrenomedullin, Somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal polypeptide), somatostatin, dopamine, motilin, amylin, bradykinin, CGRP (calcitonin gene-related peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokai Superfamily (e.g., IL-8, GROa s GROj3 , GR0 NAP- 2, ENA- 78, GCP- 2, PF4, IP- 10, M ig, CXC chemokine subfamily such as PBSF / SDF- 1; MCAF / MCP -1, MCP-2
  • a compound or a salt thereof that alters the binding property between the ligand obtainable by the screening method according to any of (41) to (48) and the G protein-coupled receptor protein or its salt according to (1).
  • a medicament characterized by containing
  • the antibody of (13) is competitively competent with the test solution and the labeled G protein-coupled receptor protein of (1) or the partial peptide of (4) or a salt thereof. Reacting, and measuring the ratio of the labeled G protein-coupled receptor protein according to (1) or the partial peptide or salt thereof according to (4), which is bound to the antibody. And the method for quantifying the G protein-coupled receptor protein described in the above (1) or the partial peptide or the salt thereof described in the above (4).
  • test solution and the antibody according to (13) insoluble on the carrier and the labeled antibody according to (13) are reacted simultaneously or continuously, and then labeled on the insolubilized carrier.
  • a method for measuring the activity of an agent the method for quantifying the G protein-coupled receptor protein described in (1) above, the partial peptide described in (4) above, or a salt thereof in a test solution.
  • FIG. 1 is a hydrophobicity plot of TGR34L.
  • FIG. 2 is a hydrophobicity plot of TGR34V. BEST MODE FOR CARRYING OUT THE INVENTION
  • the G protein-coupled receptor protein of the present invention is a receptor protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. is there.
  • the receptor protein of the present invention includes, for example, all cells (eg, spleen cells, neurons, glial) of human non-human mammals (eg, guinea pigs, rats, mice, rabbits, pigs, sheep, birds, monkeys, etc.) Cells, germ cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells, fat cells, immune cells (eg, macrophages, ⁇ cells, ⁇ cells) , Natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblast
  • amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 include, for example, about 50% or more, preferably about 60% or more, more preferably about 70% Above, more preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more amino acid sequence having homology.
  • the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 of the present invention include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 A protein having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1 is preferred.
  • an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 more specifically, for example, an amino acid sequence represented by SEQ ID NO: 2, etc.
  • examples thereof include ligand binding activity and signal transduction. Substantially the same means that their activities are the same in nature. Therefore, the activities such as ligand binding activity and signal transduction activity are equivalent (eg, about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times). However, quantitative factors such as the degree of activity and the molecular weight of the protein may be different.
  • the measurement of the activity such as the ligand binding activity and the signal information transmission activity can be performed according to a known method.
  • the activity can be measured according to a ligand determination method described later, ie, a screening method.
  • the receptor protein of the present invention includes (1) one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30, more preferably about 1 to 10, and more preferably about 1 to 10; (1-5) amino acid sequences, and (2) one or more (preferably about 1 to 30, more preferably 1 to 30) amino acids in the amino acid sequence represented by SEQ ID NO: 1.
  • SEQ ID NO: 1 preferably about 1 to 30, more preferably about 1 to 10, and more preferably about 1 to 10; (1-5) amino acid sequences
  • amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence to which about 1 to 10, more preferably several (1 to 5) amino acids have been added; and 3 one or more (preferably 1 or more) amino acids in the amino acid sequence represented by SEQ ID NO: 1 About 30 amino acids, more preferably about 1 to about 10 amino acids, and even more preferably several (1 to 5) amino acids substituted with another amino acid, or a combination thereof.
  • the amino acid sequence of the receptor protein is N-terminal (amino terminal) on the left end and C-terminal (carboxyl terminal) on the right end according to the convention of peptide notation.
  • the receptor protein of the present invention including the receptor protein is as follows.
  • the terminal may be any of a carboxyl group (-C00H), phenolic oleboxylate (—C00—), an amide (one C0NH 2 ) or an ester (—C00R).
  • R in the ester e.g., methyl, Echiru, n- propyl, alkyl groups such as isopropyl, n- butyl, Shikuropen chill, C 3 _ 8 cycloalkyl group such as cyclohexyl, for example, phenyl, alpha-Na C 6 _ 12 Ariru group such Fuchiru, for example, benzyl, phenyl, such as phenethyl - a C 7 _ 14 Ararukiru groups such as C w alpha-Nafuchiru C w alkyl Le group such as an alkyl group or an alpha-naphthylmethyl A pivaloyloxymethyl group commonly used as an ester for oral use and oral use is used.
  • the receptor protein of the present invention When the receptor protein of the present invention has a lipoxyl group (or carboxylate) other than the C-terminus, the receptor protein of the present invention includes a lipoxyl group amide or esterified. .
  • 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, Amino group protecting groups Mechionin residues of N-terminal (e.g., formyl group, etc. C M Ashiru group such as any C 2 _ 6 Arukanoiru group of Asechiru) , The daltamyl group formed by cleavage of the N-terminus in vivo and pyroglutamine oxidation, the substituent on the side chain of the amino acid in the molecule (for example, — 0H, one SH, amino group, imidazole group, indole group, etc.
  • Amino group protecting groups Mechionin residues of N-terminal e.g., formyl group, etc.
  • C M Ashiru group such as any C 2 _ 6 Arukanoiru group of Asechiru
  • Guanijino group appropriate protecting groups (e.g., formyl group, C 2 such Asechiru - 6 such Ashiru groups such Arukanoiru groups) are protected by shall or sugar chain bound Also included are complex proteins such as so-called glycoproteins.
  • receptor protein of the present invention for example, a receptor protein containing an amino acid sequence represented by the sequence No. 1 or SEQ ID NO. 2 is used.
  • the partial peptide of the receptor protein of the present invention may be any of the above partial peptides of the receptor protein of the present invention.
  • the receptor protein of the present invention Among the molecular molecules, those that are exposed outside the cell membrane and have substantially the same activity are used.
  • substantially the same activity indicates, for example, ligand binding activity.
  • the measurement of the ligand binding activity can be performed in the same manner as described above.
  • a partial peptide of the receptor protein having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 the extracellular region (hydrophilicity) in the hydrophobicity plot analysis shown in FIG. 1 or FIG. (Hydrophilic) site).
  • a peptide partially containing a hydrophobic (Hydrophobic) site can also be used.
  • a peptide containing individual domains individually may be used, but a peptide containing a plurality of domains simultaneously may be used.
  • the number of amino acids of the partial peptide of the present invention is at least 20 or more, preferably 50 or more, more preferably 100 or more of the amino acid sequences constituting the receptor protein of the present invention. Peptides and the like are preferred.
  • a substantially identical amino acid sequence is defined as about 50% or more, preferably about 60% or more, more preferably about 70% or more, more preferably about 80% or more, and particularly preferably about 80% or more, of these amino acid sequences.
  • An amino acid sequence having 90% or more, most preferably about 95% or more homology is shown.
  • the partial peptide of the present invention is characterized in that: (1) one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence are deleted; (2) One or more (preferably about 1 to 20, more preferably about 1 to 10, and more preferably several (1 to 5)) amino acids are added to the amino acid sequence, Or 3 One or more (preferably about 1 to 10, more preferably several, more preferably about 1 to 5) amino acids in the above amino acid sequence may be substituted with another amino acid .
  • the C-terminus may be any of a carboxyl group (one C00H), a carboxylate (one C00—), an amide (one C0NH 2 ) or an ester (one C00R) (where R is Show the same significance).
  • the partial peptide of the present invention has a carboxyl group (or carboxylate) other than at the C-terminus, the partial peptide of the present invention includes a carboxyl group amidated or esterified. You.
  • the ester in this case, for example, the above-mentioned C-terminal ester and the like are used.
  • the partial peptide of the present invention has an N-terminal methionine residue in which the amino group of the methionine residue is protected by a protecting group, and is formed by cleavage of the N-terminal side in vivo.
  • a protecting group for protecting the amino group of the methionine residue
  • the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group
  • those in which a sugar chain is bonded such as a so-called glycopeptide.
  • Examples of the salt of the receptor protein of the present invention or its partial peptide include a physiologically acceptable salt with an acid or a base, and a physiologically acceptable acid addition salt is particularly preferable.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumanoleic acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumanoleic acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid
  • the receptor protein of the present invention or a salt thereof can be produced from the above-mentioned human or non-human mammal cell or silk tissue by a known method for purifying a receptor protein, or the present invention described below. It can also be produced by culturing a transformant containing DNA encoding the receptor protein. Also, the protein can be produced by the protein synthesis method described later or according to the method.
  • the tissues or cells of a human non-human mammal are homogenized, extracted with an acid or the like, and the extract is subjected to reverse phase chromatography, Purification and isolation can be achieved by combining chromatography such as ion exchange chromatography.
  • a commercially available resin for protein synthesis can be usually used.
  • resins include chloromethinole resin, hydroxymethinole resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, and PAM resin.
  • 4-hydroxymethylmethylphenylacetamide methyl resin polyacrylamide tree Luminous, 4- (2 ', 4'-dimethoxyphenylhydroxymethyl) phenoxy tree Luminous, 4- (2', 4, dimethoxyphenyl-mocaminoethyl) phenoxy resin it can.
  • an amino acid having a suitably protected amino group and side chain functional group is condensed on the resin according to various known condensation methods in accordance with the amino acid sequence of the target protein or peptide.
  • the protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed. To get.
  • carbodiimides are particularly preferable.
  • carpimides DCC, N, N'-diisopropylcarpimide, N-ethyl_ ⁇ '-(3-dimethylaminoprolyl) carpimide, and the like are used.
  • Activation by these involves the ability to add the protected amino acid directly to the resin together with a racemization inhibitor additive (eg, HOBt, HOOBt), or as a symmetric anhydride or HOBt ester or HOOBt ester. After the protected amino acid is activated in advance, it can be added to the resin.
  • a racemization inhibitor additive eg, HOBt, HOOBt
  • 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-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride, methylform, and trifluoroethanol Alcohols such as dimethyl sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; and esters such as methyl acetate and ethyl acetate. And the like.
  • the reaction temperature is appropriately selected from a range known to be usable for a protein bond formation reaction, and is usually appropriately selected from a range of about 120 ° C to 50 ° C.
  • the activated amino acid derivative is usually used in a 1.5 to 4-fold excess.
  • Examples of the protecting group for the starting amino group include Z, B oc, tertiary pentoxy carbonyl, isobornyl oxycarbonyl, 4-methoxybenzinol xycanoleponinole, C1-Z, Br_Z, Adamantinoreoxycanoleboninole, trifluoroacetyl, phthaloyl, formyl, 2-ditrophenylsulfenyl, diphenylphosphinothioi, and the like are used.
  • the power boxyl group may be, for example, a linear chain such as an alkyl ester group (eg, methyl, ethyl, propyl, butynole, tertiary butynole, cyclopentinole, cyclohexyl, cyclohepty-cyclooctyl, 2-adamantyl, etc.).
  • alkyl ester group eg, methyl, ethyl, propyl, butynole, tertiary butynole, cyclopentinole, cyclohexyl, cyclohepty-cyclooctyl, 2-adamantyl, etc.
  • Branched or cyclic alkyl esterification branched or cyclic alkyl esterification
  • aralkyl esterification for example, benzyl ester, 412-ester benzyl ester, 4-methoxybenzyl ester, 4-methyl benzyl ester, benzhi) Drill esterification
  • phenacinoleestenolation benzyloxycarbonyl hydrazide, tertiary butoxycarbonyl hydrazide, trityl hydrazide and the like.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for this esterification for example, a lower alkanol group such as an acetyl group, an arylo group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group or an ethoxycarbonyl group, and the like are used.
  • groups suitable for etheric groups include, for example, a benzyl group, a tetrahydrobiranyl group, a t-butyl group, and the like.
  • B z 1, C 1 2 protecting group of the phenolic hydroxyl group of tyrosine - B zl, 2-two Torobenjiru as the B r _ Z, protecting group of the imidazole of histidine such as tertiary butyl is used.
  • B r _ Z protecting group of the imidazole of histidine such as tertiary butyl is used.
  • Tos 4-methoxy-1,2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and the like are used.
  • Examples of the raw material in which the carboxyl group is activated include, for example, a corresponding acid anhydride, azide, active ester [alcohol (for example, pentachlorophenol, 2 , 4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt).
  • active ester for example, pentachlorophenol, 2 , 4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, and esters with HOBt.
  • the corresponding phosphoric amide is used as the raw material of the active amino group.
  • Clicks such as, meth-cresolone / re, saccharesoneole, dimethinoresolenolide, 1,4-butanedithiol, 1,2-ethanedithiol, etc.
  • the addition of an on-capture agent is effective, and the 2,4-dinitrophenyl group used as an imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as an indole protecting group of tryptophan is as described above.
  • it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia, etc.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • a peptide (protein) chain is extended to a desired chain length on the amino group side. Then, a protein from which only the protecting group for the N-terminal a-amino group of the peptide chain was removed and a protein from which only the protecting group for the C-terminal carboxyl group was removed were prepared. Condensation in Details of the condensation reaction are the same as described above. By condensation After purifying the obtained protected protein, all the protecting groups are removed by the above method to obtain a desired crude protein. This crude protein is purified by various known purification means, and the main fraction is lyophilized to obtain an amide of the desired protein.
  • ester of a protein for example, after condensing the ⁇ -carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, the ester of the desired protein can be obtained in the same manner as the protein amide. You can get your body.
  • the partial peptide of the protein of the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein of the present invention with an appropriate peptide.
  • a peptide synthesis method for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used. That is, the desired peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein of the present invention with the remaining portion, and removing the protective group when the product has a protective group. it can.
  • Known condensation methods and elimination of protecting groups include, for example, the methods described in the following 1 to 5.
  • 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 when it is obtained as a salt, it can be converted to a free form by a known method. be able to.
  • the polynucleotide encoding the receptor protein of the present invention may be any polynucleotide as long as it contains the above-described nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention.
  • the polynucleotide is RNA such as DNA or mRNA encoding the receptor protein of the present invention, and may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA or DNA: RNA hybrid. If single stranded, it may be the sense strand (ie, the coding strand) or the antisense strand (ie, the non-coding strand).
  • RNA of the receptor protein of the present invention can be quantified.
  • the DNA encoding the receptor protein of the present invention may be any of genomic DNA, genomic DNA library, cDNA derived from the above-mentioned cells and tissues, cDNA library derived from the above-described cells and itinerary, and synthetic DNA.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Alternatively, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a preparation of total RNA or mRNA fraction from the above-mentioned cell'tissue.
  • 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: 3 or SEQ ID NO: 4, or SEQ ID NO: 3 or SEQ ID NO: 4. It has a DNA that hybridizes under high stringent conditions with DNA having the represented base sequence, and has substantially the same activity as the receptor protein of the present invention (eg, ligand binding activity, signal signaling activity, etc.) Any DNA can be used as long as it encodes a receptor protein.
  • DNA that hybridizes under high stringent conditions with DNA having the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 include, for example, Contains a nucleotide sequence having about 70% or more, preferably about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more homology with the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. DNA or the like is used.
  • Hybridization is performed by a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be carried out according to the method described in the attached instruction manual, and more preferably, can be carried out under high stringent conditions.
  • the high stringent conditions include, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C, preferably about 60 to 65 ° C. Show. In particular, a sodium concentration of about 19 mM and a temperature of about 65 ° C are most preferred.
  • a DNA encoding a receptor protein containing an amino acid sequence represented by SEQ ID NO: 1 a DNA containing a base sequence represented by SEQ ID NO: 3;
  • a DNA encoding a receptor protein containing the amino acid sequence represented by SEQ ID NO: 4 a DNA having the base sequence represented by SEQ ID NO: 4 or the like is used.
  • a polynucleotide comprising a part of the base sequence of DNA encoding the receptor protein of the present invention or a part of a base sequence complementary to the DNA is a partial peptide of the present invention described below. It is used to mean not only DNA to be loaded but also RNA.
  • 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 nucleotide sequence information of the DNA to encode.
  • a polynucleotide can hybridize with RNA of a G protein-coupled receptor protein gene and has the ability to inhibit ⁇ formation or function of the RNA or RNA associated with G protein-coupled receptor protein. With G protein-coupled receptor protein gene expression can be regulated and controlled through interaction.
  • Polynucleotides that are complementary to the selected sequence of G protein-coupled receptor protein-related RNA and polynucleotides that can specifically hybridize with G protein-coupled receptor protein-related RNA are in vivo. It is useful for regulating and controlling the expression of G protein-coupled receptor protein gene in vitro, and is also useful for treating or diagnosing diseases and the like.
  • the G protein-coupled receptor protein gene has 5 terminal hairpin loops, 5 terminal 6-base pair 'repeat, 5' terminal untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon,
  • the 3'-end untranslated region, the 3'-end palindrome region, and the 3'-end hairpin loop can be selected as preferred regions of interest, but any region within the G protein-coupled receptor protein gene can be selected.
  • the relationship between the target nucleic acid and the polynucleotide complementary to at least a part of the target region that is, the relationship between the target nucleic acid and the polynucleotide that can hybridize with the target can be said to be “antisense”.
  • Antisense Polynucleotides are 2-deoxy-D-report-containing polydeoxynucleotides, D-report-containing polynucleotides, and other types of N-glycosides of purine or pyrimidine bases Or other polymers having a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (provided that the polymer is DNA, (Including nucleotides having a configuration that allows base pairing and base attachment as found in RNA).
  • RNA hybrids can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and even DNA: RNA hybrids, and can be unmodified polynucleotides (or unmodified Oligonucleotides), and those with known modifications, for example, those with a label known in the art, capped, methylated, and one or more natural nucleotides.
  • an intramolecular nucleotide such as those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.) Having a bond or a sulfur-containing bond (eg, phosphorothioate, phosphorodithioate, etc.), such as a protein (nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or a sugar ( For example, those having a side chain group such as monosaccharides, etc., those having an interfering compound (eg, acridine, psoralen, etc.), chelating compounds (eg, metal, radioactive metal, boron) , An oxidizing metal, etc.), an alkylating agent, or a compound having a modified bond (for example, ⁇ -anomeric nucleic acid).
  • an uncharged bond eg, methylphosphon
  • nucleoside J may not only contain purine and pyrimidine bases, but may also include those based on other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also have modified sugar moieties, e.g., one or more hydroxyl groups have been replaced with halogens, aliphatic groups, or functional groups such as ethers, amines, etc. It may be converted to a group.
  • the antisense polynucleotide (nucleic acid) of the present invention is an RNA, a DNA or a modified nucleic acid (RNA, DNA).
  • 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 have altered or modified sugars, bases, And may be provided in special forms, such as ribosomes, microspheres, applied by gene therapy, or provided in a cured form.
  • additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes or increase the uptake of nucleic acids ( For example, hydrophobic substances such as phospholipid and cholesterol) can be mentioned.
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • These can be attached to the 3 'end or the 5' end of the nucleic acid, and can be attached via a base, sugar, or intramolecular nucleoside bond.
  • Other groups include capping groups specifically arranged at the 3 'end or 5' end of nucleic acids for preventing degradation by nucleases such as exonuclease and RNase. Examples of such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, such as glycols such as polyethylene glycolone and tetraethyleneglycol.
  • the antisense nucleic acid inhibitory activity can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of a G protein-coupled receptor protein. it can.
  • the nucleic acid can be applied to cells by various known methods.
  • the DNA encoding the partial peptide of the present invention may be any DNA as long as it contains a base sequence encoding the above-described partial peptide of the present invention.
  • any of a genomic DNA, a genomic DNA library, a cDNA derived from the above-described cells and tissues, a cDNA library derived from the above-described cells and tissues, and a synthetic DNA may be used.
  • the vector used for the library may be any of pacteriophage, plasmid, cosmid, and phagemid. Alternatively, it can be directly amplified by the RT-PCR method using an mRNA fraction prepared from the cells and tissues described above.
  • the DNA encoding the partial peptide of the present invention includes, for example, (1) a portion of DNA having a base sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4. (2) having DNA that hybridizes under high stringent conditions with DNA represented by SEQ ID NO: 3 or SEQ ID NO: 4;
  • DNA having a partial base sequence of DNA encoding a protein having the same activity eg, ligand binding activity, signal transduction action, etc.
  • Examples of the DNA that hybridizes with the DNA represented by SEQ ID NO: 3 or SEQ ID NO: 4 under high stringency conditions include, for example, about 70% or more, preferably, about 70% or more of the nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
  • DNA having a nucleotide sequence having homology of about 80% or more, more preferably about 90% or more, and still more preferably about 95% or more is used.
  • the peptide of the present invention may be coded.
  • a DNA fragment labeled with a synthetic DNA fragment or a synthetic DNA can be performed according to, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.
  • the nucleotide sequence of the DNA can be replaced by OD A-LA PCR using PCR or a known kit, for example, Mutan TM —super Express Km (Takara Shuzo), Mutan TM —K (Takara Shuzo), or the like.
  • the method can be carried out according to a known method such as the gapped duplex method, the Kunkel method and the like, or a method analogous thereto.
  • the DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or can be digested with a restriction enzyme if desired, or can be used after adding a linker.
  • the DNA has AT G at the 5 ′ end as a translation initiation codon, and TAA, TGA or TA at the 3 ′ end as a translation stop codon. It may have G. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • the expression vector for the receptor protein of the present invention includes, for example, (a) cutting out a DNA fragment of interest from, for example, cDNA, including DNA encoding the receptor protein of the present invention; It can be produced by ligating downstream of a promoter in an expression vector.
  • Escherichia coli-derived plasmids eg, pCR4, pCR2.1, pBR322, pBR325, pUC12, pUC13
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, pC194
  • yeast-derived plasmids eg, PSH19, pSH15
  • phage such as ⁇ phage
  • animal viruses such as retrovirus, vaccinia virus, and baculovirus
  • pAl-ll pXTl
  • pRc / CMV pRc / RSV
  • pcDNAI / Neo etc.
  • the promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression.
  • SRa promoter when animal cells are used as hosts, SRa promoter, SV40 promoter, LTR open motor, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • the CMV promoter SR ⁇ promoter and the like.
  • the host is Eshierihia genus bacterium, trp promoter, 1 ac flop port motor, r ec A promoter,; LP L promoter, etc.
  • lpp promoter when the host is Bacillus, SPO 1 promoter, SPO2
  • yeast such as a promoter or Pen P promoter, a PHO5 promoter, a PGK promoter, a GAP promoter, an ADH promoter, etc. are preferred.
  • a polyhedrin promoter, a P10 promoter and the like are preferable.
  • expression vectors which contain an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV4 Oori), and the like, if desired, may be used.
  • selectable markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene (methotrexate (MTX) resistance), ampicillin resistance Sex gene (hereinafter sometimes abbreviated as Am p r), neomycin resistant gene (hereinafter sometimes abbreviated as N eo r, G 4 1 8 resistance) and the like.
  • dhfr gene when used as a selection marker using CHO (dhfr-1) cells, the target gene can be selected by using a thymidine-free medium or medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention. If the host is Escherichia, the PhoA signal sequence, OmpA signal sequence, etc., if the host is Bacillus, the amylase signal sequence, subtilisin signal sequence, etc., and the host is yeast In some cases, the MFa 'signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, the insulin signal sequence, a-interferon
  • 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 examples include Escherichia coli K12 ⁇ DH1 [Procedures of the National Academy of Sciences, Ob . Acad. Sci. USA), 60 volumes, 160 (1968)], JM103 [Nucleic Acids Research], 9 volumes, 309 (1981)], JA221 [journal 'ob' molecular 'bio Journal of Molecular Biology], Volume 120, 517 (1978)], HB101 [Journal 'Op' Molecular 'Biology, Volume 41, 459 (1969) 3, C600 [Genetics, 39 , 440 (1954)], DH5a [Inoue, H., Nojima, ⁇ .
  • Bacillus bacteria examples include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Ob'Bioke]
  • yeast such as Saccharomyces cerevisiae AH22, AH22R, NA87-11A, DKD-5D, 20B-12, Zizaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris (Pichia pastoris), etc.
  • insect cells for example, when the virus is Ac NPV, the cell line derived from the larvae of night moth (Spodoptera frugiperda cell; Sf cell), MG1 cells derived from the midgut of Trichoplusia ni, and eggs derived from eggs of Trichoplusia ni High Five TM cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used.
  • Sf cells for example, Sf9 cells (ATCC CRL1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217 (1977)) and the like are used.
  • insects for example, silkworm larvae are used [Maeda et al., Nature, 31 ⁇ , 5 (1985)].
  • animal cells examples include monkey cells COS_7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter abbreviated as CHO (dhfr-) cell), mouse L Cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc. are used.
  • CHO cell Chinese hamster cell CHO
  • dhfr gene-deficient Chinese hamster cell CHO hereinafter abbreviated as CHO (dhfr-) cell
  • mouse L Cells mouse AtT-20
  • mouse myeloma cells rat GH3, human FL cells, etc.
  • Transformation of Bacillus spp. Can be performed, for example, according to the method described in Molecular & General Genetics, Vol. 168, 111 (1979).
  • Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
  • Transformation of animal cells can be performed, for example, by the methods described in Cell Engineering Annex 8 New Cell Engineering Experimental Protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973). It can be performed according to the method.
  • a liquid medium is suitable as the medium used for the culturing, and a carbon source necessary for the growth of the transformant is contained therein.
  • the carbon source include glucose, dextrin, soluble starch, and sucrose.
  • the nitrogen source include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like.
  • 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.
  • M9 medium containing glucose and casamino acid M9 medium containing glucose and casamino acid (Miller, Journal 'Ob' Experimen'in ',' Journal of Experiments in Molecular Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972].
  • an agent such as 3j3-indolyl atalilic acid can be added in order to make the promoter work efficiently.
  • the cultivation is usually performed at about 15 to 43 ° C for about 3 to 24 hours, and if necessary, aeration and stirring can be added.
  • the cultivation is usually carried out at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.
  • the culture medium is Grace's Insect Medium (Grace, TCCC, Nature, 195, 788 (1962)).
  • Suitable additives such as immobilized 10% serum are used.
  • the pH of the culture medium is preferably adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and aeration and agitation are added as necessary.
  • examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], DMEM Medium [Virology, 8 volumes, 396 (1959)], RPMI 1640 medium [Journal of the American Medical Association (1992), 519 (1967) 3, 199 medium [Proceding of the Society for the Biological Medicine], 73, 1 (1950)], etc.
  • the culture is preferably about 6 to 8. Culture is usually performed at about 30 ° C to 40 ° C for about 15 to 60 hours, and aeration and agitation are used as necessary.
  • the G protein-coupled receptor protein of the present invention can be produced in the transformant, in 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 destroyed by thawing or the like, a method of obtaining a crude extract of the receptor protein by centrifugation or filtration is appropriately used.
  • the buffer may contain a protein denaturing agent such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM.
  • Purification of the receptor protein contained in the culture supernatant or extract thus obtained can be carried out by appropriately combining known separation and purification methods.
  • These known separation and purification methods include methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method using difference in molecular weight, Method using charge difference such as ion exchange chromatography, Method using specific affinity such as affinity mouth chromatography, Reverse phase high-performance liquid mouth chromatography, etc.
  • a method utilizing the difference in the hydrophobicity of the polymer, a method utilizing the difference in the isoelectric point such as isoelectric focusing, and the like are used.
  • the receptor protein thus obtained When the receptor protein thus obtained is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto. It can be converted into a free form or another salt by an analogous method.
  • the receptor protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein-modifying enzyme before or after purification.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, proteinase, glycosidase and the like are used.
  • the activity of the receptor protein of the present invention or a salt thereof thus produced can be determined by a binding experiment with a labeled ligand and an enzyme antibody using a specific antibody. It can be measured by means such as a sail.
  • the antibody against the receptor protein of the present invention or its partial peptide or its salt may be any of a polyclonal antibody and a monoclonal antibody as long as it can recognize the receptor protein or its partial peptide or its salt of the present invention. I ’m sorry.
  • the receptor protein of the present invention or its partial peptide or its salt (hereinafter referred to as
  • the antibody against (there may be abbreviated as the receptor protein or the like of the present invention) can be produced according to a known antibody or antiserum production method using the receptor protein or the like of the present invention as an antigen.
  • 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.
  • 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 / Incomplete Freund's adjuvant may be administered to enhance antibody production ability.
  • the administration is usually performed once every 2 to 6 weeks, for a total of about 2 to 10 times. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats, and mouse and rat are preferably used.
  • a warm-blooded animal immunized with the antigen for example, a mouse with an antibody titer is selected from a mouse, and the spleen or lymph node is collected 2 to 5 days after the final immunization.
  • monoclonal antibody-producing hybridomas can be prepared.
  • the antibody titer in the antiserum can be measured, for example, by reacting a labeled receptor protein or the like described below with the antiserum, and then measuring the activity of a labeling agent bound to the antibody.
  • the fusion operation can be carried out according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • Myeloma cells include, for example, NS-1, P3U1, SP2 / 0, etc. i P 3 U 1 is preferably used.
  • the preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG1000 to PEG6000) is added at a concentration of about 10 to 80%.
  • the cell fusion can be carried out efficiently by incubating at about 20 to 40 ° C, preferably about 30 to 37 ° C for about 1 to 10 minutes.
  • the hybridoma culture supernatant is collected on a solid phase (eg, a microplate) to which antigens such as receptor proteins are directly or adsorbed together with a carrier. Then, an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice) or protein A labeled with a radioactive substance or an enzyme, and then added to the solid phase
  • a method for detecting a monoclonal antibody is exemplified.
  • the selection of the monoclonal antibody can be carried out according to a known method or a method analogous thereto. Usually, it can be carried out in a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) is added.
  • HAT hyperxanthine, aminopterin, thymidine
  • any medium can be used as long as it can grow a hybridoma.
  • RPMI 1640 medium containing 1-20%, preferably 10-20% fetal bovine serum, GIT medium containing 1-10% fetal bovine serum (Wako Pure Chemical Industries, Ltd.) or hybridoma culture A serum-free medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) can be used.
  • the culturing temperature is usually 20 to 40 ° (preferably about 37 ° C.)
  • the culturing time is usually 5 days to 3 weeks, preferably 1 to 2 weeks.
  • the antibody titer of the culture supernatant of the hybridoma can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies. , DEAE) adsorption / desorption method, ultracentrifugation method, Filtration, an antigen-binding solid phase or a specific purification method in which an antibody alone is collected using an active adsorbent such as protein A or protein G, and the bond is dissociated to obtain the antibody.
  • DEAE adsorption / desorption method
  • ultracentrifugation method Ultracentrifugation method
  • Filtration an antigen-binding solid phase
  • an antigen-binding solid phase or a specific purification method in which an antibody alone is collected using an active adsorbent such as protein A or protein G, and the bond is dissociated to obtain the antibody.
  • the polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (antigen such as the protein of the present invention) and a carrier protein is formed, and a mammal is immunized in the same manner as in the above-described method for producing a monoclonal antibody. It can be produced by collecting an antibody-containing substance against a protein or the like and separating and purifying the antibody.
  • the mixing ratio of the carrier protein and the hapten is different from that of the hapten immunized by cross-linking the carrier.
  • Any antibody may be cross-linked at any ratio as long as it can be efficiently used.
  • the weight ratio of serum albumin, thyroglobulin, key hornore limpet, hemocyanin, etc. A method of coupling about 0.1 to 20, preferably about 1 to 5 with respect to 1 hapten is used.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • glutaraldehyde ⁇ carpoimide, a maleimide active ester, an active ester reagent containing a thiol group or a dithioviridyl group, or the like is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant / incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration can usually be performed once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from blood, ascites, etc., preferably from blood, of the mammal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. Separation and purification of polyclonal antibodies can be performed according to the same immunoglobulin separation and purification method as described above for monoclonal antibodies. You.
  • the receptor protein or its salt, its partial peptide or its salt, and the DNA encoding the receptor protein or its partial peptide of the present invention are as follows: (1) The ligand (for the G protein-coupled receptor protein of the present invention) (2) prophylactic and / or therapeutic agents for diseases associated with dysfunction of the G protein-coupled receptor protein of the present invention, (3) genetic diagnostic agents,
  • a screening method for a compound that changes the expression level of the receptor protein or its partial peptide of the present invention (5) prevention of various diseases containing a compound that changes the expression level of the receptor protein or its partial peptide of the present invention.
  • Z or a therapeutic agent (6) a method for quantifying a ligand for the G protein-coupled receptor protein of the present invention, (7) a compound that changes the binding property between the ligand and the G protein-coupled receptor protein of the present invention ( (8)
  • Various methods containing compounds (agonist, antagonist) that change the binding property between the G protein-coupled receptor protein and the ligand of the present invention Various methods containing compounds (agonist, antagonist) that change the binding property between the G protein-coupled receptor protein and the ligand of the present invention.
  • a preventive and / or therapeutic agent for a disease (9) the receptor protein of the present invention or a portion thereof; (10) a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane, (11) the amount of the receptor protein of the present invention or its partial peptide in the cell membrane
  • a prophylactic and / or therapeutic agent for various diseases containing a compound that alters the following: (12) neutralization by an antibody against the receptor protein of the present invention or its partial peptide or a salt thereof; (13) the G protein-conjugated type of the present invention It can be used for producing non-human transgenic animals having DNA encoding the receptor protein.
  • ligands for G protein-coupled receptors specific to human and non-human mammals can be obtained.
  • Compounds that alter the binding properties eg, agonists, antagonists, etc.
  • the receptor protein or partial peptide of the present invention or a salt thereof hereinafter, may be abbreviated as the receptor protein of the present invention, etc.
  • the DNA encoding the receptor protein of the present invention or the partial peptide thereof hereinafter, referred to as the present invention.
  • the use of an antibody against the receptor protein or the like of the present invention may be specifically described below.
  • the receptor protein of the present invention or its salt or the partial peptide or its salt of the present invention is useful as a reagent for searching for or determining a ligand (agonist) for the receptor protein of the present invention or its salt. It is.
  • the present invention provides a method for determining a ligand to the receptor protein of the present invention, which comprises contacting the receptor protein of the present invention or a salt thereof or a partial peptide of the present invention or a salt thereof with a test compound. .
  • Test compounds include known ligands (eg, angiotensin, bombesin, cannabinoid, cholecystokinin, gnoletamine, serotonin, melatonin, neuropeptide Y, opioid, purine, pasoprescin, oxytocin, ⁇ ACAP (eg, PACAP 27 , PACAP 38), secretin, glucagon, calciton, adrenomedullin, somatostatin, GHRH, CRF, ACTH, GRP, PTH, VIP (Pasoactive Intestinal and Rerated Polypeptide), somatostatin, dopamine, motilin, amiline, Bradykinin, CGRP (calcitonin gene relayed peptide), leukotriene, pancreastatin, prostaglandin, thromboxane, adenosine, adrenaline, chemokine superfiber Millie (eg, CXC chemokine subfamily such as IL
  • CC chemokine subfamily CC chemokine subfamily
  • C chemokine subfamily such as lymphotactin
  • CX 3 chemokine such as fractalkine Subfamily, etc.
  • endoselin enterogastrin, histamine, neurotensin, TRH, pancreatic polypeptide, galanin, lysophosphatidic acid (LPA), sphingosine monomonophosphate, etc.
  • Tissue extracts of human or non-human mammals eg, mouse, rat, pig, porcine, sheep, monkey, etc.
  • cell culture supernatant eg, the tissue extract, cell culture supernatant and the like are added to the receptor protein of the present invention, and fractionation is performed while measuring cell stimulating activity and the like, so that a single ligand can be finally obtained.
  • the method for determining a ligand of the present invention comprises constructing an expression system for a mosquito or recombinant receptor protein using the receptor protein of the present invention or a partial peptide thereof or a salt thereof, and using the expression system.
  • the receptor-binding Atsei system which binds to the receptor protein of the present invention, it stimulates cell stimulating activity (eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production, intracellular c A compound having an activity of promoting or suppressing GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc. (for example, peptides, Protein, non-peptidic compound, synthetic compound, fermentation product, etc.) or a salt thereof.
  • cell stimulating activity eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production,
  • 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 When the labeled test compound is brought into contact with the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof, the labeled test compound or the salt thereof, or the salt thereof.
  • the receptor protein of the present invention which is characterized by measuring the amount of binding to a partial peptide or a salt thereof. Is the method of determining the ligand for the salt,
  • the labeled test compound When a labeled test compound is brought into contact with a receptor protein expressed on a cell membrane by culturing a transformant containing DNA encoding the receptor protein of the present invention, the labeled test compound A method for determining a ligand for a receptor protein of the present invention, which comprises measuring an amount of a substance bound to the receptor protein or a salt thereof;
  • cell stimulating activity via the receptor protein eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular c AMP production, intracellular c GMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, pH reduction, etc.
  • a method for determining a ligand for the receptor protein or a salt thereof of the present invention which comprises measuring
  • Receptor protein-mediated cell stimulating activity when a test compound is brought into contact with a receptor protein expressed on a cell membrane by culturing a transformant containing DNA encoding the receptor protein of the present invention.
  • a receptor protein expressed on a cell membrane by culturing a transformant containing DNA encoding the receptor protein of the present invention.
  • a method for determining a ligand for the receptor protein or a salt thereof of the present invention which comprises measuring the activity of promoting or suppressing fos activation, reduction of H, and the like.
  • the receptor protein used in the method for determining a ligand may be any containing the above-described receptor protein of the present invention or the partial peptide of the present invention. Any receptor protein may be used, but a receptor protein expressed in large amounts using animal cells is suitable.
  • the receptor protein of the present invention is preferably produced by expressing DNA encoding the 1S receptor protein, which is used in the above expression method, in mammalian cells or insect cells.
  • DNA fragment encoding the protein portion of interest cDNA is usually used, but is not necessarily limited thereto.
  • 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 / less promoter metamouth thionine promoter
  • human heat shock promoter cytomegaloy / less promoter
  • SR ⁇ promoter SR ⁇ promoter
  • the receptor protein of the present invention or a partial peptide thereof or a salt thereof includes a receptor protein or a partial peptide thereof or a salt thereof purified according to a known method.
  • a cell containing the receptor protein or a cell membrane fraction thereof may be used.
  • the cells containing the receptor protein of the present invention When cells containing the receptor protein of the present invention are used in the method for determining a ligand of the present invention, the cells may be fixed with daltaraldehyde, formalin, or the like.
  • the fixing method can be performed according to a known method.
  • the cell containing the receptor protein of the present invention refers to a host cell expressing the receptor protein of the present invention.
  • a host cell Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like are used.
  • cell membrane fractions are obtained by crushing cells and then using known methods. Refers to the fraction included.
  • the cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Warlinda blender-Polytron (Kinema tica), crushing by ultrasonic waves, pressing the cells while pressing with a French press, etc. There is a broken frame by ejecting from a thin nozzle.
  • a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used.
  • the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1 minute to 10 minutes), and the supernatant is further spun at a higher speed (15000 to 30000 rm) for 30 minutes to 2 hours. After centrifugation, the obtained precipitate is used as a membrane fraction.
  • the membrane fraction is rich in the expressed receptor protein and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of the receptor protein in the cells containing the receptor protein or in the membrane fraction thereof is preferably 10 3 to 10 8 molecules, and more preferably 10 5 to 10 7 molecules per cell.
  • 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.
  • chemokine one-perfamily eg, IL-8, GROa, GRO] 3, GROy s NAP—2, ENA— 7 8
  • CXC chemokine subfamily such as GC P-2, PF 4, IP-10, Mig, PBSF / SDF-1; MCAF / MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, RANTES, ⁇ ⁇ _1 ⁇ , MIP—lj3, HCC—1, MIP-3a / LARC, MIP-3 ⁇ / ELC, 1—309, TARC, MIPF—1, MIPF-2 / eot axin-2, MDC, DC-CK1 / PARC, CCLC chemokines such as SLC, etc .; 1 Cmpokine subfamily such as ymphotactin; CX3 chemo such as
  • a cell or a membrane fraction of the cell containing the receptor protein of the present invention is suspended in a buffer suitable for the determination method.
  • a buffer suitable for the determination method.
  • the buffer may be any buffer such as a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8) or a buffer such as Tris-HCl buffer which does not inhibit the binding between the ligand and the receptor protein.
  • various proteins such as surfactants such as CHAPS, Tween-80 TM (Kao Ichi Atlas), digitonin, and deoxycholate, serum albumin, and gelatin are used as buffers. Can be added. Furthermore, a protease inhibitor such as PMSF, leptin, E-64 (manufactured by Peptide Research Institute), or pepstatin can be added for the purpose of suppressing the degradation of the receptor and ligand by the protease. 0.01: to the 10 m 1 of the receptor solution, a certain amount (5,000 to 500,000 c pm) of [3 H], [125 I], [14 C], coexist test compounds labeled with a [35 S] Let it.
  • test compound having a count (B-NSB) of less than 0 cpm obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) is used as a ligand (agonist) for the receptor protein of the present invention or a salt thereof.
  • B-NSB count of less than 0 cpm obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B)
  • a cell stimulating activity via the receptor protein for example, arachidonic acid release, acetylcholine release, intracellular Ca 2 + Release, promotes intracellular cAMP production, intracellular cGMP production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c-fos, decrease in pH, etc.
  • Activity or inhibitory activity can be measured using a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured in a multiwell plate or the like.
  • a substance for example, arachidonic acid
  • an inhibitor for the degrading enzyme may be added to perform the assay.
  • activities such as suppression of cAMP production can be detected for production suppression of cells whose basic production has been increased by forskolin or the like.
  • the kit for determining a ligand that binds to the receptor protein of the present invention or a salt thereof includes the receptor protein of the present invention or a salt thereof, a partial peptide or a salt thereof of the present invention, a cell containing the receptor protein of the present invention, or the present invention. And a membrane fraction of cells containing the receptor protein.
  • kits for determining a ligand of the present invention include the following.
  • CHO cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at 5 ⁇ 10 5 holes and cultured for 2 days at 37 ° C., 5% CO 2 and 95% air.
  • the ligand capable of binding to the receptor protein of the present invention or a salt thereof includes, for example, substances specifically present in the hypothalamus, cerebral cortex, colon cancer, lung cancer, heart, placenta, lung, and the like.
  • the receptor protein of the present invention becomes clear, depending on the action of the ligand, (1) the receptor protein of the present invention or (2) DNA encoding the receptor protein may be replaced with the present invention. It can be used as a medicament such as a preventive and / or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention.
  • DNA encoding the receptor protein of the present invention is useful as a safe and low-toxic agent for the prevention and / or treatment of diseases associated with dysfunction of the receptor protein of the present invention.
  • the receptor protein of the present invention is a novel seven-transmembrane receptor protein in which the orphan receptor RE2 (GenBank accession number AF091890) has about 32% homology at the amino acid sequence level.
  • the receptor protein of the present invention or the DNA encoding the receptor protein may be a central disease (eg, Alzheimer's disease, dementia, eating disorder, depression, epilepsy, etc.), an endocrine disease (eg, Addison's disease, Cushing's syndrome) , Brown cell types, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic diseases (eg, diabetes, dyslipidemia, diabetic complications, obesity , Gout, cataract, hyperlipidemia, etc., cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), inflammatory Diseases (e.g., allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.), cardiovascular diseases (e.g., hypertension, heart Hypertrophy, angina, myo
  • the receptor protein of the present invention When used as the above-mentioned prophylactic / therapeutic agent, It can be formulated according to conventional methods.
  • the DNA of the present invention when used as the above-mentioned prophylactic or therapeutic agent, the DNA of the present invention may be used alone or in a retrovirus vector. After insertion into an appropriate vector such as an adenovirus vector or an adenovirus-associated virus vector, it can be carried out according to a conventional method.
  • the DNA of the present invention can be administered as it is or together with an adjuvant for promoting ingestion, using a gene gun or a catheter such as Hyde-mouth gel force table.
  • the receptor protein of the present invention or (2) DNA encoding the receptor protein may be orally administered as tablets, capsules, elixirs, microcapsenoles, or the like, or coated with water, if necessary. It can be used parenterally in the form of an injectable solution such as a sterile solution with a pharmaceutically acceptable liquid or a suspension.
  • an injectable solution such as a sterile solution with a pharmaceutically acceptable liquid or a suspension.
  • (1) the receptor protein of the present invention or (2) DNA encoding the receptor protein is generally recognized together with known physiologically recognized carriers, flavors, excipients, vehicles, preservatives, stabilizers, binders, and the like. It can be manufactured by mixing in the unit dosage form required for practice. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated according to standard pharmaceutical practice, such as dissolving or suspending the active substance in vehicles such as water for injection, and naturally occurring vegetable oils such as sesame oil and coconut oil. it can.
  • Aqueous liquids for injection include, for example, physiological saline, isotonic solutions containing pudose and other adjuvants (eg, D-sorbitol, D-mannitol, Thorium, etc.), and suitable solubilizers, for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, Polysorbate 80 TM, HCO — 50) may be used in combination.
  • As the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • prophylactic and therapeutic agents include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), anti-acidic agents and the like.
  • buffers for example, phosphate buffer and sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers for example, human serum Albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • anti-acidic agents e.g, anti-acidic agents and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals
  • 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)
  • the dose is usually 1 day.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • it is usually used, for example, in a cancer patient (as 60 kg).
  • the dose can be administered in terms of 60 kg.
  • the dose of the DNA of the present invention varies depending on the administration subject, target organ, symptoms, administration method, and the like. About 0.1 to 100 mg per day, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the subject to be administered, target organ, symptoms, administration method, and the like. Is about 0.01 to 3 per day It is convenient to administer about 0 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, a dose equivalent to 60 kg can be administered. (3) Gene diagnostic agent
  • the DNA of the present invention can be used as a probe to produce the receptor of the present invention in human or non-human mammals (eg, rat, mouse, rabbit, sheep, pig, pig, cat, dog, monkey, etc.).
  • Abnormality (genetic abnormality) of DNA or mRNA encoding a protein or a partial peptide thereof can be detected. For example, damage, mutation or reduced expression of the DNA or mRNA, or decrease of the DNA or It is also useful as a gene diagnostic agent for increasing or overexpressing mRNA.
  • the above-described genetic diagnosis using the DNA of the present invention includes, for example, known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Prosessing Ops. The National Academy of Sciences of the United States of America (Proceedings of the National Academy of Sciences of the United States of America), Vol. 86, pp. 2766-2770 (1989)) be able to.
  • 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, and specific organs.
  • Receptor protein of the present invention contained in a transformant or the like.
  • Receptor protein of the present invention or a portion thereof by measuring the mRNA amount of peptide.
  • a method for screening a compound that changes the expression level of a peptide is provided. The measurement of the mRNA amount of the receptor protein of the present invention or its partial peptide is specifically performed as follows.
  • non-human mammals eg, mice, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerotic rabbits
  • Drugs eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
  • blood or tissue or cells isolated from a specific organ eg, brain, lung, colon, etc.
  • the mRNA of the receptor protein of the present invention or its partial peptide contained in the obtained cells can be obtained, for example, by extracting mRNA from cells or the like by a conventional method, for example, by using a technique such as TaqMan PCR. It can be quantified and can also be analyzed by performing Northern blots by known means.
  • a transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method, and the mRNA of the receptor protein of the present invention or the partial peptide thereof contained in the transformant is prepared. It can be quantified and analyzed in the same way.
  • Screening of a compound that changes the expression level of the receptor protein or a partial peptide thereof according to the present invention includes:
  • a predetermined time before giving a drug or physical stress to a normal or disease model non-human mammal (30 minutes to 24 hours before, preferably 30 minutes to 12 hours before, more preferably 1 hour to 12 hours before Hours to 6 hours before) or after a certain time (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), or simultaneously with drug or physical stress
  • the test compound is administered, and after a certain period of time after administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), the receptor of the present invention contained in cells Quantification and analysis of the amount of mRNA: of the protein or its partial peptide,
  • 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 to 7 days, preferably 1 to 3 days, more preferably Two to three days later), it can be performed by quantifying and analyzing the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the transformant.
  • the compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the expression level of the receptor protein or a partial peptide thereof of the present invention.
  • the cell stimulating activity via the G protein-coupled receptor eg, arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular c) AMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity to promote or suppress pH reduction, etc.
  • the G protein-coupled receptor eg, arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular c) AMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, activity to promote or suppress pH reduction, etc.
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like, and these compounds may be novel compounds or known compounds.
  • the compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein or the like of the present invention.
  • the compound that attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein of the present invention.
  • 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-described pharmaceuticals containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.
  • non-human mammals eg, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.
  • the dose of the compound or its salt depends on the subject of administration, target organ, symptoms, administration method, etc.
  • oral administration in general, for example, in cancer patients (as 60 kg), about 0 .; to 100 mg per day, preferably about 1.0 to 50 mg per day. More preferably about 1.0-20 mg.
  • parenteral administration the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc. It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • a preventive and / or therapeutic agent for various diseases containing a compound that alters the expression level of the receptor protein or its partial peptide of the present invention.
  • the receptor protein of the present invention is considered to play some important role in vivo such as central function. Therefore, the compound of the present invention that alters the expression level of the receptor protein or its partial peptide can be used as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention.
  • Examples of the disease associated with dysfunction of the receptor protein of the present invention include central diseases (eg, Alzheimer's disease, dementia, eating disorders, depression, epilepsy, etc.), endocrine diseases (eg, Addison's disease, Cushing's syndrome, brown) Cell types, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic disorders (eg, diabetes, dyslipidemia, diabetic complications, obesity, Gout, cataract, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), inflammatory disease ( For example, allergies, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc., cardiovascular diseases (eg, hypertension, cardiac hypertrophy, stenosis) Diseases, myocardial infarction
  • 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 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 can be used together with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in a unit dosage form generally required for the practice of preparations. It can be manufactured by mixing with. The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. And sucrose, lactose or saccharine, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry.
  • 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 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), non-ionic surfactants (eg, Polysorbate 80 TM, HCO-50) Is also good.
  • solubilizers such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), non-ionic surfactants (eg, Polysorbate 80 TM, HCO-50) Is also good.
  • the oily liquid for example, sesame oil,
  • prophylactic and therapeutic agents examples include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffers for example, phosphate buffer and sodium acetate buffer
  • soothing agents for example, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers for example, human serum Albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep,
  • 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.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like.
  • it is usually used, for example, in patients with cancer (60 kg).
  • the amount converted per 60 kg can be administered.
  • 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 for such compounds.
  • G protein-coupled receptors eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential Fluctuation, phosphorylation of intracellular proteins, activation of c-fos, reduction of pH, etc.
  • a so-called agonist against the receptor protein of the present invention (mouth) a compound not having the cell stimulating activity (a so-called antagonist against the receptor protein of the present invention), (no) Includes compounds that enhance the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (2) compounds that decrease the binding force between the ligand and the G protein-coupled receptor protein of the present invention.
  • the compound (a) is preferably screened by the ligand determination method described above).
  • the present invention relates to (i) a case where the receptor protein of the present invention or its partial peptide or a salt thereof is brought into contact with a ligand; and (ii) a receptor protein of the present invention or its partial peptide or a salt thereof, Ligand and test A method for screening a compound or a salt thereof that alters the binding property between a ligand and a receptor protein or a partial peptide thereof or a salt thereof of the present invention, which is compared with the case of contacting with a compound.
  • the screening method of the present invention is characterized in that in the cases (i) and (i), for example, the amount of a ligand bound to the receptor protein or the like, the cell stimulating activity, and the like are measured and compared.
  • the present invention provides
  • a method for screening a compound or a salt thereof that changes the binding property between a ligand and a receptor protein or the like of the present invention which is characterized by measuring and comparing the amount of the ligand; When the cells are brought into contact with the cells containing them or the membrane fraction of the cells, and when the labeled ligand and the test compound are brought into contact with the cells containing the receptor protein of the present invention or the membrane fraction of the cells. The amount of the labeled ligand bound to the cell or the membrane fraction is measured and compared.
  • Ligand and method of screening a compound or its salt that alters the binding property between the receptor protein or the like of the present invention characterized in that,
  • a compound that activates the receptor protein of the present invention eg, a ligand for the receptor protein of the present invention
  • a cell containing the receptor protein of the present invention Receptor-mediated cell stimulating activity (e.g., arachidonic acid release, acetylcholine release, intracellular C a 2+ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein And the activity of promoting or suppressing c-fos, c-fos reduction, pH reduction, etc.
  • a 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 test compound were expressed on the cell membrane by culturing a transformant containing the DNA of the present invention when the compound was brought into contact with the receptor protein or the like, and when the transformant containing the DNA of the present invention was cultured.
  • Receptor-mediated cell stimulating activity for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular c when brought into contact with the receptor protein or the like of the present invention
  • GMP production inositolonoleic acid production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of c_fos, decrease in pH
  • the present invention provides a method for screening a compound or a salt thereof, which alters the binding property between a ligand and a receptor protein of the present invention, which is characterized by measuring and comparing the activity of promoting or suppressing the thrombosis.
  • the receptor protein or the like of the present invention Prior to obtaining the receptor protein or the like of the present invention, when screening for G protein-coupled receptor agonists or antagonists, first, cells, such as rats, containing G protein-coupled receptor protein, mitogen, or a cell membrane fraction thereof are used. To obtain candidate compounds (primary screening), and then to test whether the candidate compounds actually inhibit the binding between human G protein-coupled receptor protein and ligand (secondary screening) ) was required. If the cell, tissue or cell membrane fraction is used as it is, other receptor proteins will also be present, so it has been difficult to directly screen an agonist or an antagonist for the target receptor protein in practice.
  • cells such as rats, containing G protein-coupled receptor protein, mitogen, or a cell membrane fraction thereof are used.
  • candidate compounds primary screening
  • secondary screening to test whether the candidate compounds actually inhibit the binding between human G protein-coupled receptor protein and ligand
  • 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.
  • the compound screened is an agonist or antago It is possible to easily evaluate whether it is a nest.
  • 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-derived organs are particularly difficult to obtain, it is suitable to use human-derived receptor proteins and the like, which are expressed in large amounts using recombinants, for screening.
  • the above method is used for producing the receptor protein and the like of the present invention, but it is preferable to carry out the expression by expressing the DNA of the present invention in mammalian cells and insect cells.
  • CDNA is used as the DNA fragment encoding the protein portion of interest, but is not necessarily limited thereto.
  • a gene fragment or a synthetic DNA may be used.
  • the DNA fragment In order to introduce the DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, the DNA fragment must be prepared by transferring the DNA fragment to a nuclear polyhedrosis virus (nuclear polyhedrosis virus belonging to baculovirus using an insect as a host).
  • Hedrosis virus (NPV) polyhedrin promoter Hedrosis virus (NPV) polyhedrin promoter, SV40-derived promoter, retrovirus / less promoter, meta-mouth thionine promoter, human heat shock promoter, cytomegaloinores promoter, downstream of SRa promoter, etc. Incorporation is preferred.
  • the amount and quality of the expressed receptor can be examined by a known method. For example, the method can be carried out according to the method described in the literature [Nambi, P. et al., The 'Journal of Ob, Biological' Chem., 267, 19555-19559, 1992]. it can.
  • the protein containing the receptor protein or the like of the present invention may be a receptor protein or the like purified according to a known method, or a cell containing the receptor protein or the like. May be used, or a membrane fraction of cells containing the receptor protein or the like may be used. Fixed with aldehyde, formalin, etc. You can do it.
  • the immobilization method can be performed according to a known method.
  • the cell containing the receptor protein or the like of the present invention refers to a host cell that expresses the receptor protein or the like, and the host cell is preferably Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, or the like.
  • the cell membrane fraction refers to a cell membrane-rich fraction obtained by disrupting cells and then obtained by a known method.
  • Cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Warlinda blender-Polytron (manufactured by Kinema tica), crushing by ultrasonic waves, pressing cells with a French press, etc. And crushing by ejecting from a thin nozzle.
  • a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used.
  • the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short time (usually about 1-10 minutes), and the supernatant is further centrifuged at a high speed (15000-30000 rpm) for 30 minutes to 2 hours.
  • the resulting precipitate is used as the membrane fraction.
  • the membrane fraction is rich in expressed components such as receptor protein and cell-derived phospholipids and membrane proteins.
  • the amount of the receptor protein in the cell or membrane fraction containing the receptor protein or the like is preferably 10 3 to 10 8 molecules per cell, and more preferably 10 5 to 10 7 molecules per cell.
  • 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 labeled ligand a labeled ligand, a labeled ligand analog compound and the like are used.
  • a cell containing the receptor protein of the present invention or a membrane fraction of the cell is first screened.
  • Prepare the receptor protein preparation by suspending it in a suitable buffer.
  • the buffer may be any buffer that does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer of pH 4 to 10 (preferably pH 6 to 8) and a buffer of tris-hydrochloride.
  • a surfactant such as CHAPS, Tween-80 TM (Kao Ichi Atlas), digitonin, and dexcholate can be added to the buffer for the purpose of reducing non-specific binding.
  • a protease inhibitor such as PMS F, leptin, E-64 (manufactured by Peptide Research Laboratories), or papstatin can be added for the purpose of suppressing the degradation of the receptor or ligand by the protease.
  • the reaction is performed for 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours
  • the mixture is filtered with a glass fiber filter paper and the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter paper is measured by a liquid scintillation counter or ⁇ -
  • NS. non-specific binding amount
  • SB non-specific binding amount
  • the specific binding amount (B— NSB) Power It can be selected as a powerful candidate substance.
  • a compound which changes the binding property between a ligand and the receptor protein of the present invention for example, cell stimulating activity via the receptor protein (eg, arachidonic acid release, acetylcholine Release, intracellular Ca2 + release, intracellular cAMP generation, intracellular cGMP generation, inositol monophosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, decrease in pH, etc. Activity to promote or inhibit)
  • the measurement can be performed using a commercially available measurement kit.
  • cells containing the receptor protein or the like of the present invention are cultured on a multi-well plate or the like. Before conducting screening, replace the cells with a fresh medium or an appropriate buffer that is not toxic to cells, add test compounds, etc., incubate for a certain period of time, extract the cells or collect the supernatant. The products produced are quantified according to the respective method. If the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult due to the presence of a degrading enzyme contained in the cells, an inhibitor for the degrading enzyme is added to the mixture to perform an assay. Well ,. In addition, an activity such as cAMP production suppression can be detected as a production suppression effect on cells whose basal production has been increased with forskolin or the like.
  • a substance for example, arachidonic acid
  • cells expressing an appropriate receptor protein are required.
  • a cell expressing the receptor protein of the present invention a cell line having the natural type receptor protein of the present invention, a cell line expressing the above-mentioned recombinant receptor protein or the like is desirable.
  • test compounds for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal thread and tissue extracts, and the like are used. Or a known compound.
  • a screening kit for a compound or a salt thereof that alters the binding between a ligand and the receptor protein of the present invention or the like includes a receptor protein of the present invention, a cell containing the receptor protein of the present invention, or a receptor of the present invention. And those containing a membrane fraction of cells containing proteins and the like.
  • 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 with 5 ⁇ 10 5 Z-wells, and cultured for 2 days at 37 ° C., 5% CO 2 and 95% air.
  • the compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is a compound having an action of changing the binding property between a ligand and the receptor protein or the like of the present invention.
  • Cell stimulating activity via G protein-coupled receptors eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production,
  • G protein-coupled receptors eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production,
  • a compound having no cell stimulating activity (so-called antagoni against the receptor protein of the present invention)
  • C a compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (2) reduces the binding force between the ligand and the G protein-coupled receptor protein of the present invention. It is a compound.
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like, and these compounds may be novel compounds or known compounds.
  • the agonist against the receptor protein or the like of the present invention has the same action as the physiological activity of the ligand for the receptor protein or the like of the present invention
  • the antagonist to the receptor protein or the like of the present invention can suppress the physiological activity of the ligand to the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug for suppressing the ligand activity.
  • the compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention is useful as a safe and low-toxic drug for enhancing the physiological activity of the ligand for the receptor protein or the like of the present invention. .
  • the compound that decreases the binding force between the ligand and the G protein-coupled receptor protein of the present invention is useful as a safe and low-toxic drug for reducing the physiological activity of the ligand for the receptor protein of the present invention.
  • the compound or a salt thereof is used as the above-mentioned 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 in the above-described drug containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals (eg, rats, egrets, sheep, pigs, pigs, cats, dogs, monkeys, etc.). Can be administered.
  • non-human mammals eg, rats, egrets, sheep, pigs, pigs, cats, dogs, monkeys, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • microbial administration in general, for example, in a cancer patient (60 kg), It is about 0.1-100 mg per day, preferably about 1.0-50 mg, more preferably about 1.0-20 mg.
  • parenteral administration the single dose varies depending on the subject, target organ, symptoms, administration method, and the like.
  • it is usually used, for example, in cancer patients (60 kg).
  • the amount converted per 60 kg can be administered.
  • 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 role in vivo, such as central function, circulatory function, digestive function, and cardiac function. Accordingly, compounds (agonists, antagonists) that alter the binding property between the receptor protein of the present invention and the ligand, and ligands for the receptor protein of the present invention, are useful for diseases associated with dysfunction of the receptor protein of the present invention. It can be used as a prophylactic or therapeutic agent.
  • diseases associated with the dysfunction of the receptor protein of the present invention include central diseases (eg, Alzheimer's disease, dementia, eating disorders, depression, epilepsy, etc.), endocrine diseases (eg, Addison's disease, Cushing's syndrome, brown) Cell type, primary Rudosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic diseases (for example, diabetes, dyslipidemia, diabetic complications, obesity, gout, cataract, high fat) ), Cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), inflammatory disease (eg, allergy, asthma) , Rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc., cardiovascular diseases (eg, hypertension, cardiac hypertrophy, angina, myocardial infarction, arteriosclerosis, etc.), cardiovascular
  • the compound or ligand 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 conventional means.
  • the compound or ligand can be aseptically mixed with tablets, capsules, elixirs ij, microcapsules and the like, if necessary, which are sugar-coated, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections such as aqueous solutions or suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be incorporated into tablets, capsules, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid, etc. Swelling agents such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry. Which is used.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile preparations for injection are formulated according to standard pharmaceutical practice, such as by dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil, and the like.
  • a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil, and the like.
  • aqueous solution for injection include physiological saline, isotonic solution containing glucose and other auxiliary agents (eg, D-sorbitol, D-mannitol, sodium salt, etc.) and the like.
  • Solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80 TM, HCO-50) May be.
  • the oily liquid include sesame oil and soybean oil.
  • solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • the prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • buffers eg, phosphate buffer, sodium acetate buffer
  • soothing agents eg, benzalkonium chloride, procaine hydrochloride, etc.
  • stabilizers eg, human Serum albumin, polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants e.g, antioxidants and the like.
  • 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 D
  • the preparations obtained in this way are safe and have low toxicity, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, rabbits, cats, dogs, dogs ) Can be administered.
  • non-human mammals eg, rats, mice, rabbits, sheep, pigs, rabbits, cats, dogs, dogs
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like. About 0.:! To 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. Is about 0 per day It is convenient to administer about 01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0 :! to about 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg. (9) Quantification of the receptor protein of the present invention or its partial peptide or its salt
  • 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,
  • 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 monoclonal antibody of the present invention In addition to measuring the receptor protein and the like of the present invention using a monoclonal antibody against the receptor protein and the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining and the like is also possible. it can.
  • the antibody molecule itself may be used, or F (ab,) 2 , Fab ′, or Fab fraction of the antibody molecule may be used.
  • the assay method using an antibody against the receptor protein or the like of the present invention is not particularly limited, and may be an antibody, an antigen, or an antibody-antigen complex corresponding to the amount of antigen (eg, the amount of receptor protein) in the test solution.
  • Any method that can be used to detect the amount of ⁇ by chemical or physical means and calculate this from a standard curve prepared using a standard solution containing a known amount of antigen may be used.
  • nephelometry, a competitive method, an immunometric method and a sandwich method are preferably used, but it is particularly preferable to use a sandwich method described later in terms of sensitivity and specificity.
  • a labeling agent used in a measuring method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like are used.
  • the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • the enzyme a stable enzyme having a specific activity of 1 "is preferable.
  • monogalactosidase, j3-dalcosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • the luminescent substance for example, luminol, luminol derivative, luciferin, lucigenin, etc. Further, binding of antibody or antigen to labeling agent Alternatively, a biotin-avidin system can be used.
  • insolubilization of the antigen or antibody physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used.
  • the carrier for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.
  • a test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction).
  • 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 and the method of insolubilization can be based on those 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 an antibody having a different binding site such as a receptor protein is preferably used. That is, the antibody used in the primary reaction and the secondary reaction is, for example, when the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is preferably other than the C-terminal. For example, an antibody that recognizes the N-terminal is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry.
  • the competition method after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated ( (B / F separation) Measure the amount of any of B, F, and label and quantify the amount of antigen in the test solution.
  • a soluble antibody was used as the antibody, a liquid phase method using polyethylene glycol for B / F separation, a second antibody against the above antibody, and a solid-phased antibody as the first antibody.
  • a solid-phase 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 fixed amount of the labeled antibody, and then the force for separating the solid phase and the liquid phase, or The antigen in the medium is allowed to react with an excessive amount of the labeled antibody, and then the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase. Then, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
  • nephelometry the amount of insoluble sediment generated as a result of an antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and a small amount of sediment cannot be obtained, laser nephrometry utilizing laser scattering is preferably used.
  • 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.
  • the antibody of the present invention can be used for specifically detecting the receptor protein of the present invention or the like present in a subject such as a body fluid or a tissue. Further, preparation of an antibody column used for purifying the receptor protein of the present invention, detection of the receptor protein of the present invention in each fraction at the time of purification, behavior of the receptor protein of the present invention in test cells It can be used for analysis and the like.
  • the antibody of the present invention can specifically recognize the receptor protein of the present invention or its partial peptide or a salt thereof, a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane It can be used for screening.
  • the present invention for example, (i) After rupture of (1) blood, (2) specific organ, or (3) tissue or cells isolated from the organ of the non-human mammal, the cell membrane fraction is isolated, and the cell membrane fraction of the present invention contained in the cell membrane fraction is isolated.
  • 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.
  • Non-human mammal's (1) blood, (2) specific organ, (3) tissue or cells isolated from the organ, sectioned and immunostaining is used to obtain the receptor protein on the cell surface.
  • a method of screening for a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane by quantifying the degree of staining of the cell membrane.
  • the present invention provides a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane by confirming the protein on the cell membrane.
  • the quantification of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically performed as follows.
  • non-human mammals for example, mice, rats, rabbits, higgs, stags, puppies, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, atherosclerotic puppies
  • Drugs eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, waterlogging stress, electric shock, light / dark, low temperature, etc.
  • the obtained organ, tissue or cell is suspended in, for example, an appropriate buffer (for example, Tris-HCl buffer, phosphate buffer, or Hase buffer) to destroy the organ, tissue or cell.
  • an appropriate buffer for example, Tris-HCl buffer, phosphate buffer, or Hase buffer
  • a cell membrane fraction is obtained by using a surfactant (eg, Triton X100 TM, Tween-20 TM, etc.), and further using a method such as centrifugation, filtration, or column fractionation.
  • a surfactant eg, Triton X100 TM, Tween-20 TM, etc.
  • the cell membrane fraction refers to a cell membrane-rich fraction obtained by disrupting cells and then obtained by a known method.
  • the cells can be crushed by crushing the cells with a Potter-Elvehjem homogenizer, crushing with a Perling Blender ⁇ Polytron (manufactured by Kinema tica), crushing by ultrasonic waves, pressing with a French press, etc. Crushing by ejecting cells from a thin nozzle may be mentioned.
  • a fractionation method by centrifugal force such as a fractionation centrifugation method or a density gradient centrifugation method is mainly used.
  • the cell lysate is centrifuged at a low speed (500-3000 rpm) for a short period of time (usually about 1-10 minutes), and the supernatant is further centrifuged at a high speed (15000-30000 rpm) for 30 minutes to 2 hours.
  • the resulting precipitate is used as the membrane fraction.
  • the membrane fraction is rich in expressed receptor proteins and membrane components such as cell-derived phospholipid / membrane proteins.
  • the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified by, for example, sandwich immunoassay using the antibody of the present invention, Western blot analysis, or the like.
  • Such a sandwich immunoassay can be performed in the same manner as described above, and the Western plot can be performed by known means.
  • a transformant expressing the receptor protein of the present invention or a partial peptide thereof is prepared according to the above method, and the receptor protein of the present invention or a partial peptide thereof contained in the cell membrane fraction can be quantified. .
  • Screening for a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane is performed by:
  • the administration preferably 1 hour to 2 days, more preferably 1 hour to 24 hours
  • the test compound is administered simultaneously with the drug or physical stress, and after a certain time after the administration (30 minutes to 3 days, preferably 1 hour to 2 days, more preferably 1 hour to 24 hours), by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane,
  • test compound is mixed with the medium when culturing the transformant according to a conventional method, and after culturing for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days).
  • a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to 3 days).
  • After 3 days can be carried out by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane.
  • non-human mammals for example, mice, rats, rabbits, sheep, pigs, pigs, cats, dogs, monkeys, etc .; more specifically, dementia rats, obese mice, arteriosclerotic rabbits
  • Drugs eg, anti-dementia drugs, antihypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
  • blood or a specific organ eg, heart, placenta, lung, etc.
  • tissue or cells isolated from the organ is obtained.
  • the obtained organ, tissue, cell, or the like is used as a thread and tissue section according to a conventional method, and immunostaining is performed using the antibody of the present invention.
  • immunostaining is performed using the antibody of the present invention.
  • the compound or a salt thereof obtained by using the screening method of the present invention is a compound having an action of changing the amount of the receptor protein of the present invention or a partial peptide thereof in a cell membrane.
  • protein-coupled receptors eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential Fluctuations, phosphorylation of intracellular proteins, activation of c-fos, activity of promoting or suppressing pH reduction, etc.
  • (mouth) receptor protein of the present invention or its portion in cell membrane It is a compound that reduces the cell stimulating activity by reducing the amount of the peptide.
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like, and these compounds may be novel compounds or known compounds.
  • the compound that enhances the cell stimulating activity is useful as a safe and low-toxic drug for enhancing the physiological activity of the receptor protein or the like of the present invention.
  • the compound that attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein of the present invention.
  • 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-described pharmaceuticals containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.
  • non-human mammals eg, rats, mice, rabbits, sheep, pigs, horses, 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. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg. When administered parenterally, the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like. Is about 0.01-30 mg per day, preferably about 0.1-20 mg, more preferably about 0.1-10 mg per day. Conveniently, about mg is administered by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • a prophylactic and / or therapeutic agent for various diseases containing a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane
  • the receptor protein of the present invention is considered to play some important role in a living body such as, for example, heart or central function. Therefore, a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be used as a prophylactic or therapeutic agent for diseases associated with dysfunction of the receptor protein of the present invention.
  • Examples of the disease associated with dysfunction of the receptor protein of the present invention include central diseases (eg, Alzheimer's disease, dementia, eating disorders, depression, epilepsy, etc.), endocrine diseases (eg, Addison's disease, Cushing's syndrome, brown) Cell types, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic disorders (eg, diabetes, dyslipidemia, diabetic complications, obesity, Gout, cataract, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), inflammatory disease ( For example, allergies, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc., cardiovascular diseases (eg, hypertension, cardiac hypertrophy, stenosis) Diseases, myocardial infarction
  • 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 conventional means.
  • the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule, etc., orally, or aseptic solution with water or another pharmaceutically acceptable liquid, if necessary. It can be used parenterally or in the form of injections, such as suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by The amount of the active ingredient in these preparations is such that a suitable dosage in the specified range can be obtained.
  • Additives that can be mixed with Tablet IJ, Capsenole, etc. include, for example, binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, Swelling agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used.
  • binders such as gelatin, corn starch, tragacanth, gum arabic
  • excipients such as crystalline cellulose, corn starch, gelatin
  • Swelling agents such as alginic acid
  • lubricants such as magnesium stearate
  • sweeteners such as sucrose, lactose or saccharin
  • flavoring agents such as peppermint, cocoa oil or cherry are used.
  • the unit dosage form is a capsule
  • the above type of material can further contain a liquid carrier such
  • 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 for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium salt, etc.) and the like are used.
  • dissolution aids such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, Polysorbate 80 TM, HCO-50) You may.
  • 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 / therapeutic agents examples include buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants, etc. You may mix it.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic and can be used, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptom, administration method, and the like.
  • the daily dose is generally one day.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • it is usually used, for example, in cancer patients (60 kg).
  • the amount converted per 60 kg can be administered.
  • the neutralizing activity of an antibody against the receptor protein of the present invention or a partial peptide thereof or a salt thereof against the receptor protein or the like means an activity of inactivating a signal transduction function involving the receptor protein. . Therefore, when the antibody has a neutralizing activity, signal transduction associated with the receptor protein, for example, cell stimulating activity via the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release) Activating or suppressing intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-fos, decrease in pH, etc. Activity, etc.) can be inactivated. Therefore, it can be used for prevention and / or treatment of diseases caused by overexpression of the receptor protein and the like.
  • signal transduction associated with the receptor protein for example, cell stimulating activity via the receptor protein (eg, arachidonic acid release, acetylcholine release,
  • diseases caused by overexpression of the receptor protein include central diseases (eg, Alzheimer's disease, dementia, eating disorders, depression, epilepsy, etc.), Endocrine disorders (eg, Addison's disease, Cushing's syndrome, pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonad dysfunction, thyroid dysfunction, pituitary dysfunction, etc.), metabolic disorders (eg, diabetes) , Dyslipidemia, diabetic complications, obesity, gout, cataract, hyperlipidemia, etc., cancer (eg, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon) Cancer, rectal cancer, etc., inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.), cardiovascular diseases (eg, hypertension, cardiac hypertrophy, angina) Diseases, myocardial in
  • transgenic animals expressing the receptor protein and the like of the present invention can be produced.
  • animals include mammals (for example, rats, mice, egrets, sheep, stags, puppies, cats, cats, dogs, monkeys, etc.) (hereinafter sometimes abbreviated as animals). And egrets are preferred.
  • 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 expressing in animal cells.
  • a promoter capable of expressing in animal cells For example, when introducing the DNA of the present invention derived from egret, the homology is high, and a gene construct linked downstream of various promoters capable of expressing the DNA of the present invention derived from animals in animal cells is used.
  • a DNA-transferred animal that highly produces the receptor protein or the like of the present invention can be produced by injecting into a rabbit fertilized egg by mouth opening.
  • ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ A ubiquitous expression promoter such as a virus-derived promoter or meta-mouth thionine may be used, but a promoter of a gene specifically expressed in the heart is preferably used.
  • the presence of the receptor protein or the like of the present invention in the germinal cells of the produced animal after the DNA transfer means that all the offspring of the produced animal have the receptor protein or the like of the present invention in all of the germ cells and somatic cells.
  • Means The progeny of this type of animal that has inherited the gene has the receptor protein of the present invention in all of its germinal and somatic cells.
  • the animal having the DNA can be reared in a normal breeding environment as the DNA-bearing animal. Furthermore, by crossing male and female animals having the target DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by crossing the male and female animals, all the offspring will have the DNA Breeding to have The animal into which the DNA of the present invention has been introduced has high expression of the receptor protein or the like of the present invention, and thus is useful as an animal for screening for an agonist or antagonist against the receptor protein or the like of the present invention.
  • the DNA-introduced animal of the present invention can also be used as a cell source for tissue culture.
  • the present invention can be carried out by directly analyzing DNA or RNA in the tissue of the DNA-introduced mouse of the present invention, or by analyzing the ligament containing the receptor protein of the present invention expressed by a gene. Can be analyzed.
  • the cells of a tissue having the receptor protein of the present invention are cultured by standard tissue culture techniques, and these are used to study the function of cells from a generally difficult tissue such as brain or peripheral tissue. be able to.
  • 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.
  • Trp Trip, Tojuan
  • TC thiazolidine-1 (R) -carboxamide group
  • substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
  • FIG 1 shows the amino acid sequence of human-derived novel G protein-coupled receptor protein TGR 34 L of the present invention.
  • SEQ ID NO: 3 shows the amino acid sequence of human-derived novel G protein-coupled receptor protein TGR34V of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding the novel human-derived G protein-coupled receptor protein TGR34L of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding the novel human-derived G protein-coupled receptor protein TGR34V of the present invention.
  • the transformant Escherichia coli TOPlO / pC R2.1-hTGR34L obtained in Example 1 below was prepared on March 5, 2001 at Tsukuba East, Ibaraki, Japan. 1-chome 1 At the Central Organ No. 6 (zip code 305-8566), National Institute of Advanced Industrial Science and Technology (AIST) (formerly National Institute of Advanced Industrial Science and Technology (NIBH)) Deposit number F ERM BP-7490, from February 20, 2001 (Heisei 13) 2-17-85, Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka (postal code 532-8686) (IFO) as deposit number IFO 16577.
  • AIST National Institute of Advanced Industrial Science and Technology
  • NIBH National Institute of Advanced Industrial Science and Technology
  • the transformant Escherichia coli TOPlO / pC R2.1-hTGR34V obtained in the following Example 1 was obtained from March 5 S, 2001, Tsukuba, Ibaraki, Japan, East 1 Deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (former National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology (NIBH)) at Central No. 6 (Zip code 305-8566) No.
  • a PCR reaction was performed using two primers, Primer 1 (SEQ ID NO: 5) and Primer 2 (SEQ ID NO: 6).
  • the composition of the reaction solution used in the reaction was 1/50 volume of the above cDNA, and 1/50 volume of Advantage-GC2 Polymerase Mix (CL0NTECH), primer 1 (SEQ ID NO: 5) and primer 2 (SEQ ID NO: 6) was added to each of 0.5 juM, dNTPs 200 ⁇ , 1/5 volume of the buffer attached to the enzyme, and 1/5 volume of GC Melt to obtain a volume of 20 ⁇ l.
  • the PCR reaction is performed at 94 ° C for 5 minutes, followed by a cycle of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 2 minutes 35 times, and finally at 68 ° C for 5 minutes.
  • An extension reaction was performed.
  • the PCR reaction product was subcloned into a plasmid vector pCR2.1 (Invitrogen) according to the procedure of a TA cloning kit (Invitrogen). This was introduced into Escherichia coli TOP10, and clones having cDNA were selected on LB agar medium containing ampicillin.
  • nucleotide sequence (SEQ ID NO: 3 and 4) of cDNA encoding a novel G protein-coupled receptor protein was obtained. These two types of sequences differ by one base at residue 370, and the derived amino acid sequences are SEQ ID NOs: 1 and 2 in which the amino acid at residue 124 is leucine or palin.
  • Novel G protein-coupled receptor containing sequence One protein was named TGR34L and TGR34V. The two types of transformants E.
  • the G protein-coupled receptor protein of the present invention or a partial peptide thereof or a salt thereof, a polynucleotide encoding the receptor protein or a partial peptide thereof can be obtained by the following methods: (1) ligand (agonist) 2) Obtaining antibodies and antiserum, 3) Constructing a recombinant receptor protein expression system, 4) Developing a receptor binding assay system using the expression system and screening drug candidate compounds, 4) Structure Implementation of drug design based on comparison with chemically similar ligands and receptors 6Problems in gene diagnosis ⁇ ⁇ Reagents for creating PCR primers 7Generation of transgenic animals or 8Gene therapy agents And the like

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pain & Pain Management (AREA)
  • Obesity (AREA)
  • Cardiology (AREA)
  • Rheumatology (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une nouvelle protéine utilisée dans le criblage d'un agoniste/d'un antagoniste, etc. Plus spécifiquement, cette invention a trait à une protéine d'origine humaine ou son sel, à l'ADN codant cette protéine, à une méthode de détermination d'un ligand se liant à la protéine, à une méthode/un kit de criblage d'un composé modifiant les propriétés de liaison du ligand se liant à la protéine, à un composé obtenu par criblage ou son sel. On utilise la protéine d'origine humaine susmentionnée et l'ADN codant celle-ci, par exemple, (1) dans la détermination d'un ligand se liant à la protéine, (2) dans des agents prophylactiques et/ou des remèdes de maladies associées à l'hypofonction de la protéine, et (3) dans le criblage d'un composé (un agoniste, un antagoniste etc.) modifiant les propriétés de liaison de la protéine au ligand.
PCT/JP2002/000405 2001-01-23 2002-01-22 Nouvelle proteine receptrice couplee a une proteine g et adn correspondant WO2002059304A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2001015050 2001-01-23
JP2001-15050 2001-01-23
JP2001-102560 2001-03-30
JP2001102560 2001-03-30

Publications (1)

Publication Number Publication Date
WO2002059304A1 true WO2002059304A1 (fr) 2002-08-01

Family

ID=26608157

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/000405 WO2002059304A1 (fr) 2001-01-23 2002-01-22 Nouvelle proteine receptrice couplee a une proteine g et adn correspondant

Country Status (1)

Country Link
WO (1) WO2002059304A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001009184A1 (fr) * 1999-07-15 2001-02-08 Solvay Pharmaceuticals B.V. Recepteur humain couple a une proteine g
WO2001036471A2 (fr) * 1999-11-17 2001-05-25 Arena Pharmaceuticals, Inc. Versions endogenes et non-endogenes de recepteurs couples a la proteine g humaine
WO2001048188A1 (fr) * 1999-12-28 2001-07-05 Helix Research Institute Nouveaux recepteurs couples a la proteine de liaison a guanosine triphosphate, leurs genes, leur preparation et leur utilisation
WO2001062797A2 (fr) * 2000-02-23 2001-08-30 Pharmacia & Upjohn Company Nouveaux recepteurs couples a la proteine g
WO2001088126A2 (fr) * 2000-05-15 2001-11-22 Bayer Aktiengesellschaft Regulation du recepteur couple aux proteines g du type recepteur adrenergique $g(a)1a humain

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001009184A1 (fr) * 1999-07-15 2001-02-08 Solvay Pharmaceuticals B.V. Recepteur humain couple a une proteine g
WO2001036471A2 (fr) * 1999-11-17 2001-05-25 Arena Pharmaceuticals, Inc. Versions endogenes et non-endogenes de recepteurs couples a la proteine g humaine
WO2001048188A1 (fr) * 1999-12-28 2001-07-05 Helix Research Institute Nouveaux recepteurs couples a la proteine de liaison a guanosine triphosphate, leurs genes, leur preparation et leur utilisation
WO2001062797A2 (fr) * 2000-02-23 2001-08-30 Pharmacia & Upjohn Company Nouveaux recepteurs couples a la proteine g
WO2001088126A2 (fr) * 2000-05-15 2001-11-22 Bayer Aktiengesellschaft Regulation du recepteur couple aux proteines g du type recepteur adrenergique $g(a)1a humain

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MOMBAERTS P.: "Seven-transmembrane proteins as odorant and chemosensory receptors", SCIENCE, vol. 286, 1999, pages 707 - 711, XP002193533 *

Similar Documents

Publication Publication Date Title
WO2001077325A1 (fr) Nouvelle proteine de recepteur couple aux proteines g et adn de celle-ci
WO2000049046A1 (fr) Nouvelle proteine de recepteur couplee a la proteine g et adn
WO2001094582A1 (fr) Nouvelle proteine de recepteur couple a la proteine g et adn pour cette proteine de recepteur
WO2001083748A1 (fr) Proteine de recepteur couple a la proteine g et adn correspondant
WO2001059106A1 (fr) Nouvelles proteines receptrices couplees a des proteines g et adn associes
WO2002004640A1 (fr) Nouvelle proteine du type recepteur couple aux proteines g et adn correspondant
WO2002006466A1 (fr) Nouvelle proteine gr et son adn
WO2000035953A1 (fr) Nouvelle proteine recepteur couplee a une proteine g et son adn
WO2001096567A1 (fr) Nouvelle proteine receptrice couplee a la proteine g et adn de ce recepteur
WO2002057441A1 (fr) Nouvelle proteine receptrice couplee a la proteine g et adn de cette proteine
WO2002059304A1 (fr) Nouvelle proteine receptrice couplee a une proteine g et adn correspondant
WO2002053593A1 (fr) Nouvelle proteine de recepteur couple a la proteine g et adn de celle-ci
WO2002057309A1 (fr) Nouvelle proteine receptrice couplee a la proteine g et adn de cette proteine
JP2000166576A (ja) 新規ｇ蛋白質共役型レセプタ―蛋白質およびそのｄｎａ
WO2002088182A1 (fr) Nouvelle proteine des recepteurs couples aux proteines-g et adn associe
WO2002002767A1 (fr) Proteine de recepteur couple a la proteine g et adn correspondant
JP2002355052A (ja) 新規ｇ蛋白質共役型レセプター蛋白質およびそのｄｎａ
WO2002016607A1 (fr) Proteine de recepteur couple a la proteine g et adn correspondant
WO2002002770A1 (fr) Nouvelle proteine receptrice couplee a une proteine g et adn associe
WO2001077326A1 (fr) Proteine receptrice couplee a la proteine g et son adn
WO2002004624A1 (fr) Nouvelle proteine receptrice couplee a la proteine g et adn destine a celle-ci
WO2002046394A1 (fr) Nouvelle proteine des recepteurs lies aux proteines g et adn associe
WO2002004639A1 (fr) Nouvelle proteine receptrice couplee a la proteine g et adn de celle-ci
JP2002355050A (ja) 新規ｇ蛋白質共役型レセプター蛋白質およびそのｄｎａ
JP2003024088A (ja) 新規ｇタンパク質共役型レセプタータンパク質およびそのｄｎａ

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP