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WO2002055690A1 - Nouvelle proteine du recepteur couple a la proteine g et son adn - Google Patents

Nouvelle proteine du recepteur couple a la proteine g et son adn Download PDF

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Publication number
WO2002055690A1
WO2002055690A1 PCT/JP2002/000136 JP0200136W WO02055690A1 WO 2002055690 A1 WO2002055690 A1 WO 2002055690A1 JP 0200136 W JP0200136 W JP 0200136W WO 02055690 A1 WO02055690 A1 WO 02055690A1
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WIPO (PCT)
Prior art keywords
protein
receptor protein
salt
present
coupled receptor
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Ceased
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PCT/JP2002/000136
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English (en)
Japanese (ja)
Inventor
Masanori Miwa
Takashi Ito
Yasushi Shintani
Nobuyuki Miyajima
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Takeda Pharmaceutical Co Ltd
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Takeda Chemical Industries Ltd
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Publication of WO2002055690A1 publication Critical patent/WO2002055690A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • 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
    • 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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Definitions

  • the present invention relates to a novel G protein-coupled receptor protein derived from human kidney or a salt thereof, and a DNA encoding the same.
  • G protein (Hereinafter abbreviated as G protein), and it has intracellular signal transduction and has a common structure with seven transmembrane domains. Therefore, G protein-coupled receptor Yuichi protein Alternatively, it is collectively referred to as 7-transmembrane receptor protein (7TMR).
  • 7TMR 7-transmembrane receptor 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. Receptors transmit signals into cells via binding to biologically active substances, and this signal causes various reactions such as suppression of activation and activation of cells.
  • 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.
  • hormones, neurotransmitters and other physiologically active substances exist in the living body.
  • Many protein structures have not yet been reported.
  • G protein-coupled receptors have been found, and even at present, there are unknown G protein-coupled receptors and so-called orphan receptors whose ligands have not been identified. There is a large number of them, and there is a great need for a search and elucidation of the function of a new G protein-coupled receptor.
  • the G protein-coupled receptor Yuichi is a new It is useful for searching for a suitable ligand (bioactive substance) and for searching for an agonist or antagonist for the receptor.
  • a physiological ligand bioactive substance
  • the agonist or antagonist against the receptor can be analyzed.
  • These ligands, agonists or antagonists to the receptor can be expected to be used as preventive / therapeutic agents or diagnostic agents for diseases associated with G protein-coupled receptor dysfunction.
  • a decrease or enhancement of the function of the receptor in a living body based on a gene mutation of a G protein-coupled receptor often causes some disease.
  • introduction of the receptor gene into a living body (or a specific organ) or introduction of an antisense nucleic acid to the receptor gene can also be applied to gene therapy.
  • the nucleotide sequence of the receptor is indispensable information for examining the presence or absence of a deletion or mutation on the gene. It can also be applied to diagnostics.
  • the present invention provides a novel G protein-coupled receptor protein useful as described above. That is, a novel G protein-coupled receptor protein or a partial peptide thereof or a salt thereof, a polynucleotide (DNA, RA and a derivative thereof) encoding the G protein-coupled receptor protein or a partial peptide thereof can be used.
  • a novel G protein-coupled receptor protein or a partial peptide thereof or a salt thereof a polynucleotide (DNA, RA and a derivative thereof) encoding the G protein-coupled receptor protein or a partial peptide thereof can be used.
  • Polynucleotide (DNA, NA and derivatives thereof), recombinant vector containing the polynucleotide, transformant carrying the recombinant vector, G protein-coupled receptor protein or a salt thereof
  • the present inventors have succeeded in isolating a cDNA encoding a novel G protein-combined receptor Yuichi protein derived from human kidney and analyzing its entire nucleotide sequence. Then, when this nucleotide 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 transduced seven times. It was confirmed that the protein was a G protein conjugated receptor. The present inventors have conducted further studies based on these findings, and have completed the present invention. That is, the present invention
  • a G protein-coupled receptor protein or a salt thereof 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 its salt comprising the amino acid sequence represented by SEQ ID NO: 1;
  • the antibody according to (12) which is a neutralizing antibody that inactivates signal transmission of the G protein-coupled receptor protein according to (1);
  • a pharmaceutical comprising the antibody of (12) above, (16) a G protein-coupled receptor protein of (1) or a partial peptide of claim 3 or a salt thereof.
  • a reagent comprising the G protein-coupled receptor protein described in (1) or the partial peptide described in (3) or a salt thereof.
  • a compound or a salt thereof that alters the binding property between the ligand obtainable by using the screening kit and the G protein-coupled receptor protein or the salt thereof described in (1) above;
  • a pharmaceutical comprising a compound or a salt thereof that alters
  • a pharmaceutical 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 (28).
  • a pharmaceutical comprising a compound or a salt thereof, which alters the amount of the G protein-coupled receptor protein according to claim 1 in a cell membrane obtainable by using the screening method according to (29);
  • the invention further provides:
  • the protein is: (1) an amino acid sequence represented by SEQ ID NO: 1; , More preferably several (1 to 5)) amino acid sequences, and (2) more than two (preferably 1 to 30) amino acid sequences represented by SEQ ID NO: 1 Degree, more preferably about 1 to 10, more preferably An amino acid sequence to which several (1 to 5) amino acids are added; 3 one or more (preferably about 1 to 30, more preferably 1 to 10) amino acids in the amino acid sequence represented by SEQ ID NO: 1
  • the G protein according to (1) above which is a protein containing an amino acid sequence in which several (1 to 5) amino acids have been replaced with other amino acids, or an amino acid sequence obtained by combining them.
  • the ligands are, for example, trace amines such as tyramine, tyramine, beta-phenylethylamine () 3-phenylethylamine), trypamine, octopamine; angiotensin; Pombecin, canapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasopressin, oxoxysin, PACAP (e.g., PACAP27, PACAP38), secretin, glucagon, calcitonin, adorenomejuline Evening Chin, GHRH, CRF, ACTH, GRP, PTH, VIP (Basoactive Intestinal Polypeptide), Somatos Evening Chin, Dopamine, Motilin, Amylin, Bradykinin, CGRP (Calcitonin Gene-Related Peptide), Leukotriene, Pancreatastatin, prostaglandin,
  • a ligand characterized by measuring and comparing the amount of labeled ligand bound to a cell containing the G protein-coupled receptor protein when the cell is contacted with the ligand described in (1) above.
  • a compound that activates the G protein-coupled receptor protein or the salt thereof described in (1) above is added to a cell containing the G protein-coupled receptor protein or protein described in (1) above. And (ii) a compound that activates the G protein-coupled receptor protein described in (1) above or a compound activating the salt thereof, and a test compound, and the G protein-coupled receptor protein described in (1) above.
  • a compound that activates the G protein-coupled receptor protein or its salt according to (1) above is expressed in the cell membrane of the transformant by culturing the transformant according to (8). And a compound that activates the G protein-coupled receptor protein or a salt thereof described in (1) and a test compound described in (8) above. By culturing E. coli, the cell stimulating activity through the G protein-coupled receptor protein was measured when it was brought into contact with the G protein-coupled receptor protein expressed on the cell membrane of the transformant. A compound that alters the binding property between a ligand characterized by comparison and the G protein-coupled receptor protein or a salt thereof according to (1) above.
  • the compound that activates the G protein-coupled receptor protein according to the above (1) is tyramine, tyramine, / 3-plienylethylamine, tryptamine. ), Trace amines such as octopamine; angiotensin, bombesin, kana pinoids, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioids, purines, vasopretsin, oxotosine, PACAP (examples) ⁇ ACAP27, PACAP38), secretin, glucagon, calcitonin, adrenomedullin, somatos, G rat CRF, ACTH, GRP, PTH, VIP (vasoactive intestinal polypeptide), somatos, dopamine, motilin, amylin, Bradykinin, CGRP (calcitoni Gene relayed peptide), oral coturiene
  • Trace amines such
  • the G protein-coupled receptor according to (1) wherein the ratio of the G protein-coupled receptor protein according to (1) or the partial peptide or salt thereof according to (3) is measured.
  • FIG. 1 is a hydrophobicity plot of TGR29. BEST MODE FOR CARRYING OUT THE INVENTION
  • the G protein-coupled receptor protein (hereinafter sometimes abbreviated as receptor protein) of the present invention is a receptor containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. It is a protein.
  • the receptor protein of the present invention may be, for example, a human or non-human mammal (eg, guinea pig, rat, mouse, mouse, egret, bush, sheep, horse, monkey, etc.) in any cell (eg, spleen cell, nerve, etc.).
  • glial cells ligated jS cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fiber cells, muscle cells, fat cells, immune cells (eg, macrophages, T Cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary gland Cells, hepatocytes or stromal cells, or their precursors, stem cells or cancer cells), blood cells, or any group in which these cells are present ,
  • the brain each part of the brain (eg, olfactory bulb, acrosomal nucleus, basal sphere, hippocampus, thalamus, hypothalamus, hypothalamus nucleus, cerebral cortex, medulla, cerebellum
  • the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 1 includes, for example, about 50% or more, preferably about 60% or more, more preferably about 50% or more of the amino acid sequence represented by SEQ ID NO: 1. 70% or more, more preferably about 80% or more, especially Preferably, the amino acid sequence has about 90% or more, and most preferably about 95% or more homology.
  • Examples of the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 of the present invention include, for example, a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 Proteins having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 1 are preferred.
  • substantially the same activity examples include a ligand binding activity and a signal transduction activity.
  • substantially the same means that their activities are the same in nature. Therefore, the activities such as 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).
  • quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
  • the measurement of the activity such as the ligand binding activity and the signal information transmission activity can be performed according to a known method.
  • 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, more preferably about 1 to 10; (1 to 5) amino acids deleted, (2)
  • the amino acid sequence represented by SEQ ID NO: 1 has at least two (preferably about 1 to 30, more preferably 1 to 10) An amino acid sequence to which about several, more preferably several (1 to 5) amino acids have been added, and 3 one or two or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 (preferably about 1 to 30) More preferably about 1 to 10, more preferably several (1 to 5) amino acids in which the amino acid sequence has been substituted with another amino acid, or an amino acid sequence obtained by combining them.
  • SEQ ID NO: 1 preferably about 1 to 30, more preferably about 1 to 10, more preferably about 1 to 10; (1 to 5) amino acids deleted
  • the amino acid sequence represented by SEQ ID NO: 1 has at least two (preferably about 1 to 30, more preferably 1 to 10) An
  • the amino acid sequence of the receptor protein is the N-terminus (amino terminus) at the left end and the C-terminus (capilloxyl terminus) at the right end, according to the convention of peptide labeling.
  • the receptor protein of the present invention including a receptor protein containing the amino acid sequence represented by SEQ ID NO: 1, has a C-terminal lipoxyl group ( -C00H), carboxylate (-COO "), amide (-solid 2 ) or ester (-C00R).
  • R in the ester e.g., methyl, Echiru, n- propyl, _ 6 Arukiru group such as isopropyl or n- butyl, cyclopentyl, cyclohexylene, such as cyclohexyl (; 3 _ 8 cycloalkyl group, for example, phenyl, 0 -!
  • C 7 _ 14 such as _ 12 Ariru group, e.g., benzyl, a one-naphthyl _C Bok 2 alkyl group such as phenylene Lou c 2 ⁇ Le kill group or flying one naphthylmethyl such phenethyl such as naphthyl
  • a Viva yloxymethyl group widely used as an ester for oral use is used.
  • the receptor protein of the present invention has a lipoxyl group (or lipoxylate) other than at the C-terminus
  • a protein in which the carboxyl group is amidated or esterified is also included in the receptor protein of the present invention.
  • the ester in this case for example, the above-mentioned C-terminal ester and the like are used.
  • the receptions evening one protein of the present invention is the protein mentioned above, Amino group protecting groups Mechionin residues of N-terminal (e.g., formyl group, (6 Ashiru group such as any C 2 _ 6 Arukanoiru group of Asechiru ),
  • the N-terminal side is cleaved in vivo, and the darminyl mill group is pyroglutamine-oxidized, the substituent on the side chain of the amino acid in the molecule (for example, —0H, _SH, An amino group, an imidazole group, an indole group, a guanidino group, or the like, which is protected with an appropriate protecting group (for example, a formyl group, an acetyl group or the like, a C 2 _ 6 alkanoyl group, or the like; or) It also includes complex proteins such as so-called glycoproteins to which sugar chains are bonded.
  • Amino group protecting groups Mechionin residues of N
  • the receptor protein of the present invention for example, a receptor protein containing an amino acid sequence represented by SEQ ID NO: 1 or the like is used.
  • the partial peptide of the receptor protein of the present invention (hereinafter sometimes abbreviated as a partial peptide) may be any of the above-mentioned partial peptides of the receptor protein of the present invention.
  • the receptor protein molecules of the present invention 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.
  • the extracellular region (Hydrophilic) in the hydrophobicity plot analysis shown in FIG. ) Site) is a peptide that contains the part that was analyzed.
  • a peptide partially containing a hydrophobic site can also be used.
  • a peptide containing individual domains may be used, but a peptide containing a plurality of domains at the same time may be used.
  • the amino acid number of the partial peptide of the present invention is determined by comparing the amino acid sequence of 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. Are preferred.
  • a substantially identical amino acid sequence is about 50% or more, preferably about 60 or more, more preferably about 70% or more, more preferably about 8 or more, and particularly preferably about 90% or more, of these amino acid sequences. Most preferably, the amino acid sequence has about 95% or more homology.
  • the partial peptide of the present invention has the following features: 1) deletion of one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in the above amino acid sequence; (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 partial peptide of the present invention has a hydroxyl group (or carboxylate) other than the C-terminus
  • the partial peptide of the present invention includes amidation or esterification of the hydroxyl group.
  • 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. It includes those in which Gin is pyroglutamine-oxidized, those in which the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, and those in which a sugar chain is bonded, such as a complex peptide such as a so-called glycopeptide.
  • the salt of the receptor protein or its partial peptide of the present invention includes a physiologically acceptable salt with an acid or a base, and particularly preferably a physiologically acceptable acid addition salt.
  • Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid) , Succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • Succinic acid tartaric acid, citric acid, malic acid, oxalic acid
  • the receptor protein of the present invention or a salt thereof can be produced from the above-mentioned human or non-human mammal cell or tissue by a known method for purifying a receptor protein. Alternatively, the receptor protein of the present invention described later can be prepared.
  • the protein can also be produced by culturing a transformant containing DNA.
  • 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 and then extracted with an acid or the like, and the extract is subjected to reverse phase chromatography or ion exchange chromatography. Purification and isolation can be achieved by combining such chromatographys.
  • a commercially available resin for protein synthesis can be used.
  • a resin include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, and 4-hydroxy resin.
  • an amino acid having an ⁇ -amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods in accordance with the amino acid sequence of a target protein or peptide.
  • the protein or peptide is cleaved from the resin, and at the same time, various protecting groups are removed.
  • an intramolecular disulfide bond formation reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or its amide. To get.
  • the protected amino acid may be added directly to the resin along with a racemization inhibitor (eg, H0Bt, HOOBt), or the protected amino acid may be preformed as a symmetric acid anhydride or HOBt ester or HOOBt ester. It can be added to the resin after activation.
  • a racemization inhibitor eg, H0Bt, HOOBt
  • the protected amino acid may be preformed as a symmetric acid anhydride or HOBt ester or HOOBt ester. It can be added to the resin after activation.
  • the solvent used for activating the protected amino acid or condensing with the resin can be appropriately selected from solvents known to be usable for the protein condensation reaction.
  • solvents known to be usable for the protein condensation reaction for example,
  • Acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol; dimethyl Examples thereof include sulfoxides such as sulfoxide, ethers such as pyridine, dioxane, and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; or an appropriate mixture thereof.
  • the reaction temperature is appropriately selected from the range known to be usable for protein bond formation reactions, and is usually appropriately selected from the range of about -20 ° (: to 50 ° C.
  • Activated amino acid derivative Is usually used in an excess of 1.5 to 4.
  • Examples of the protecting group for the amino group of the starting material include 1, Boc, tertiary-pentyloxycarbonyl, isopolnyloxycarbonyl, 4-methoxybenzyloxycarbonyl, CI—Z, Br—Z, and adamantyloxy. Carbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like are used.
  • the lipoxyl group may be, for example, alkyl esterified (eg, methyl, ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc.) Or cyclic alkyl esterification), aralkyl esterification (e.g., benzyl ester, 4-122 benzyl ester, 4-methoxybenzyl ester, 4-chloro benzyl ester, benzhydryl esterification),
  • the compound can be protected by phenacyl esterification, benzyloxycarbonyl hydrazide, short-butoxycarbonyl hydrazide, trityl hydrazide, or the like.
  • the hydroxyl group of serine can be protected, for example, by esterification or etherification.
  • a group suitable for the esterification for example, a lower alkanol group such as an acetyl group, an aroyl group such as a benzoyl group, a group derived from carbonic acid such as a benzyloxycarbonyl group, an ethoxycarbonyl group and the like are used.
  • a group suitable for etherification include a benzyl group, a tetrahydroviranyl group, a t-butyl group, and the like.
  • a protecting group for the phenolic hydroxyl group of tyrosine for example, Bzl, Cl 2 _Bzl, 2-nitrobenzyl, Br—Z, Yuichi Shari-ptyl and the like are used.
  • protecting group for histidine imidazole examples include, for example, Tos, 4-methoxy-2,
  • activated carboxyl groups of the raw materials include, for example, corresponding acid anhydrides, azides, active esters [alcohols (eg, phenol, 2,2
  • Methods for removing (eliminating) the protecting group include, for example, catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride, methanesulfonic acid, Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia are also used.
  • a catalyst such as Pd-black or Pd-carbon, or hydrogen fluoride anhydride
  • methanesulfonic acid Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof
  • base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., and reduction with sodium in liquid ammonia are also used.
  • the 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 tributofan is replaced with the above-mentioned 1,2-ethanedithiol, 1,4- In addition to deprotection by acid treatment in the presence of butanedithiol, etc., it is also removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia and the like.
  • the protection of the functional group which should not be involved in the reaction of the raw materials, the protecting group, the elimination of the protective group, the activation of the functional group involved in the reaction, and the like can be appropriately selected from known groups or known means.
  • a-hydroxyl-oxyl group of terminal amino acid is protected by amidation, and then a peptide (protein) chain is added to the amino group side of a desired chain.
  • a protein was prepared by removing only the protecting group of the a-amino group at the N-terminus of the peptide chain, and a protein was prepared by removing only the protecting group of the carboxy group at the C-terminus. Condensation in such a mixed solvent. Details of the condensation reaction are the same as described above.
  • all the protecting groups are removed by the above-mentioned method to obtain a desired crude protein. This crude protein is purified by using various known purification means, and the main fraction is freeze-dried to obtain an amide of the desired protein.
  • an ester of a protein for example, one of the carboxy-terminal amino acids
  • an ester of the desired protein can be obtained in the same manner as the amide of a protein.
  • 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, a partial peptide or amino acid capable of constituting the protein of the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting group is eliminated to produce the desired peptide. Wear. Examples of the known condensation method and elimination of the protecting group include 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 such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.
  • the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method, and conversely, when it is obtained as a salt, it is converted to a free form by a known method. be able to.
  • the polynucleotide encoding the receptor protein of the present invention may be any polynucleotide containing the above-described nucleotide sequence (DNA or RNA, preferably DNA) encoding the receptor protein of the present invention. You may.
  • the polynucleotide is an RM such as DNA or mRNA encoding the receptor protein of the present invention, and may be double-stranded or single-stranded. If double stranded, double stranded DNA. Double-stranded RNA or DNA: RNA hybrid may be used. If single-stranded, it may be the sense strand (ie, the coding strand) or the antisense strand (ie, the non-coding strand).
  • the mRNA of the receptor protein of the present invention can be quantified.
  • 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 tissues, and synthetic DNA.
  • the vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.
  • amplification can be performed directly by Reverse Transcriptase Polymerase Chain Reaction (hereinafter abbreviated as RT-PCR method) using a preparation of a total RNA or mRNA fraction from the cells and tissues described above.
  • the DNA encoding the receptor protein of the present invention may be, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 2 or a DNA having the nucleotide sequence represented by SEQ ID NO: 2 It encodes a receptor protein having a DNA that hybridizes under the conditions of a lingent and having substantially the same activity (eg, ligand binding activity, signal transduction activity, etc.) as the receptor protein of the present invention. Any DNA may be used.
  • Examples of the DNA that hybridizes with the DNA having the base sequence represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 70% or more, and preferably about 80% or more of the base sequence represented by SEQ ID NO: 2. % Or more, more preferably about 90% or more, and even more preferably about 95% or more.
  • Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). it can. When using a commercially available library, describe it in the attached instruction manual Can be performed according to the method described in More preferably, it can be performed under high stringent conditions.
  • the high stringency conditions include, for example, conditions in which the sodium concentration is about 19 to 40 ⁇ , 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 preferable.
  • DNA encoding the receptor protein having the amino acid sequence represented by SEQ ID NO: 1 a DNA having the base sequence represented by SEQ ID NO: 2 or the like is used.
  • a polynucleotide comprising a part of the nucleotide sequence of DNA encoding the receptor protein of the present invention or a part of a nucleotide sequence complementary to the DNA is a partial peptide of the present invention described below. It is used to include not only the DNA to encode but also NA.
  • a G protein-coupled receptor is cloned or determined from an antisense polynucleotide (nucleic acid) capable of inhibiting the replication or expression of a protein gene. It can be designed and synthesized based on the nucleotide sequence information of the DNA encoding the type of receptor protein.
  • a polynucleotide (nucleic acid) can hybridize to RNA of a G protein-coupled receptor protein gene; can inhibit the synthesis or function of the RA; or can be a G protein-coupled receptor protein. It can regulate and control the expression of G protein-coupled receptor protein gene through interaction with related RNA.
  • Polynucleotides that are complementary to a 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 and in vivo. It is useful for regulating and controlling the expression of G protein-coupled receptor protein protein outside, and is also useful for treating or diagnosing diseases etc.
  • the drome region and the 3′-end hairpin loop may be selected as preferred regions of interest, but any region within the G protein-coupled receptor protein gene may be selected as a region of interest.
  • Antisense polynucleotides can be polynucleotides containing 2-dexoxy-D-reports, polynucleotides containing D-ribose, or other types of N-glycosides of purine or pyrimidine bases.
  • Polynucleotides or other polymers with non-nucleotide backbones eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • polymers containing special bonds provided that the polymer is not present in DNA or RNA
  • Pairing of bases as found contains nucleotides having a configuration that allows base attachment
  • RNA DNA: RM octiprids, as well as unmodified polynucleotides (or unmodified oligonucleotides),
  • RM octiprids DNA: RM octiprids, as well as unmodified polynucleotides (or unmodified oligonucleotides),
  • unmodified polynucleotides or unmodified oligonucleotides
  • Those having modified intramolecular nucleotides such as those having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramidate, olebamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphoro Those with thioate, phosphorodithioate, etc., such as evening proteins (nuclease, nuclease-inhibitor, Those having side chain groups such as toxins, antibodies, signal peptides, poly-L-lysine
  • nucleoside As used herein, “nucleoside”, “nucleotide” and “nucleic acid” include not only purine and pyrimidine bases but also other modified heterocyclic bases. May be included. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with a halogen, a force, an aliphatic group, etc., or ethers, amines, etc. It may have been converted to a functional group.
  • the antisense polynucleotide (nucleic acid) of the present invention is RNA, DNA, or a modified nucleic acid (RNA, DNA).
  • modified nucleic acids include, but are not limited to, sulfur derivatives of nucleic acids, thiophosphate derivatives, and polynucleoside amides, which are resistant to degradation of 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 make the antisense nucleic acid more cell permeable, to have a greater affinity for the target sense strand, and if toxic Reduce the toxicity of antisense nucleic acids.
  • the antisense nucleic acids of the present invention may contain altered or modified sugars, bases, or bonds, may be provided in special forms such as ribosomes or microspheres, may be applied by gene therapy, It could be given in additional form.
  • additional forms include polycations, such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids, which enhance interaction with cell membranes and increase nucleic acid uptake (e.g., , Phospholipids, cholesterol, etc.).
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • nucleic acid can be attached to the 3 'or 5' end of the nucleic acid and attached via bases, sugars, or intramolecular nucleoside bonds.
  • Other groups include cap groups specifically arranged at the 3 'end or 5' end of a nucleic acid 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 glycol and tetraethylene glycol.
  • 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 containing the above-described nucleotide sequence encoding the partial peptide of the present invention.
  • cDNA derived from the above-described cells and tissues, cDNA library derived from the above-described cells and tissues, and synthetic DNA may be used.
  • the vectors used for the library are pateriophage, plasmid, cosmid
  • the DNA encoding the partial peptide of the present invention includes, for example, (1) DNA having a partial nucleotide sequence of DNA having the nucleotide sequence represented by SEQ ID NO: 2, or (2) SEQ ID NO: : Having a DNA that hybridizes under high stringent conditions with the DNA represented by 2, and having substantially the same activity as the protein peptide of the present invention.
  • Examples of the DNA that hybridizes with the DNA represented by SEQ ID NO: 2 under high stringent conditions include, for example, about 70% or more, preferably about 80% or more, and more preferably the base sequence represented by SEQ ID NO: 2.
  • DNA containing a nucleotide sequence having a homology of about 90% or more, more preferably about 95% or more is used.
  • a synthetic DNA primer having a partial nucleotide sequence of the nucleotide sequence of the DNA encoding the peptide of the present invention is amplified by the PCR method, or the DNA incorporated into an appropriate vector is used as the receptor of the present invention. Selection can be performed by hybridization with a DNA fragment encoding a part or all of the overnight protein or a DNA fragment labeled with a synthetic DNA. The hybridization method is
  • the DNA base sequence can be replaced by the ODA-LA PCR method using PCR or a known kit, for example, Mutan TM -super Expressions Km (Takara Shuzo Co., Ltd.), Mutan TM -K (Takara Shuzo Co., Ltd.), etc.
  • the method can be performed according to a known method such as the gapped duplex method, the Kimkel method, or the like, or a method analogous thereto.
  • the DNA encoding the cloned receptor protein can be used as it is depending on the purpose, or digested with a restriction enzyme or added with a linker, if desired.
  • the DNA may have ATG as a translation initiation codon at the 5 'end and TAA, TGA or TAG as a translation termination codon at the 3' end. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter.
  • the expression vector of the receptor protein of the present invention can be prepared, for example, by (i) cutting out a DNA fragment of interest from, for example, cDNA containing DNA encoding the receptor protein of the present invention; It can be produced by ligating downstream of a promoter in an expression vector.
  • the vector examples include Escherichia coli-derived plasmids (eg, pCR4, pCR2. PBR322, BR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, UBHO, pTP5, pC194), yeast-derived plasmids (eg, pSH19, PSH15) Bacteriophage such as phage, animal viruses such as retrovirus, vaccinia virus, baculovirus, etc.Al- AlpXTL pRc / CMV, pRc / RSV, pcDNAI / Neo
  • the promoter used in the present invention may be any promoter as long as it is an appropriate promoter corresponding to the host used for gene expression. For example, when animal cells are used as host, SRa promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
  • the CMV promoter, SRo; promoter, etc. If the host is a bacterium belonging to the genus Escherichia, the trp promoter, lac promoter overnight, recA promoter, ⁇ ⁇ promoter, lpp promoter, etc. If the host is Bacillus, SP01 promoter, SP02 promoter, penP promoter, etc. If the host is yeast, PH05 promoter, PGK promoter, GAP promoter, ADH promoter Are preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
  • an expression vector containing, if desired, an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori), etc. should be used.
  • Selection methods include, for example, a dihydrofolate reductase (hereinafter sometimes abbreviated as dMr) gene (methotrexate (MTX) resistance), an ampicillin resistance gene (hereinafter sometimes abbreviated as Am), Neomycin resistance gene (hereinafter sometimes abbreviated as NeoT, G418 resistance) and the like.
  • dMr dihydrofolate reductase
  • MTX metalhotrexate
  • Am ampicillin resistance gene
  • NeoT NeoT, G418 resistance
  • the target gene can be selected using a thymidine-free medium.
  • a signal sequence suitable for the host is added to the N-terminal side of the receptor protein of the present invention.
  • the host is a bacterium belonging to the genus Escherichia, a PhoA * signal sequence, an ⁇ signal sequence, and the like.
  • the host is a bacterium belonging to the genus Bacillus, a single amylase signal sequence, a subtilisin, a signal sequence, and the like.
  • MF o signal sequence, SUC2 signal sequence, etc. If the host is an animal cell, insulin signal sequence, 0! -Interferon signal sequence, antibody molecule, signal sequence, etc. Available.
  • 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 [Processings of the National Academy of Sciences of the United States. Natl. Acad. Sci. USA), 60, 160 (1968)), JM103 (Nucleic Acids Research, 9, 309 (1981) 3, JA221 [Journal of Ob. Journal of Molecular Biology], 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics ics), 39, 440 (1954)], DH5 o; CInoue, H., Nojima, H. and Okayama, H., Gene, 96, 23-28 (1990)], DH10B [Proc. O blanking the National Academy O Breakfast, Saienshiizu, O blanking THE • USA (p ro c. Nat l. Acad. Sci . USA), 87 vol., 4645- 4649 (1990)) and the like.
  • Bacillus bacteria examples include, for example, Bacillus subtilis MI 114 [Gene, 24, 255 (1983)], 207-21 [Journal of Biochemis try] , 95, 87 (1984) 3 and the like, for example, Saccharomyces cerevisiae AH22 AH22R, NA87-11A, DKD-5D, 20B-12, Schizosaccomyces pombe aromyces pombe) NCYC1913, NCYC2036, Pichia pastoris and the like are used.
  • insect cells for example, when the virus is AcNPV, a cell line derived from a larva of Spodoptera (Spodoptera frugiperda cell; Sf cell), an MG1 cell derived from the midgut of Tricoplus ia ni, or an egg derived from Trichoplus ia ni egg High Five TM cells, cells derived from Mames tra brass icae or cells derived from Est igmena acrea are used.
  • Sf cell a silkworm-derived cell line (Bombyx mod N; Bm cell) or the like is used.
  • Sf cell include Sf9 cell (ATCC CRL1711), Sf21 cell (Vaughn, JL et al., In Vivo, 13, 213-217 (1977)) and the like. It is.
  • animal cells examples include monkey cells COS-7, Vero, Chinese hamster cells CH0 (hereinafter abbreviated as CH0 cells), dhir gene deficient Chinese hams Yuichi cells CH0 (hereinafter abbreviated as CHO (d fr) cells), Mouse L cells, mouse AtT-20, mouse myeloma cells, rat GH3, human FL cells, etc. are used.
  • Transformation of a Bacillus bacterium 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).
  • a liquid medium is suitable as a medium used for the cultivation. It contains carbon sources, nitrogen sources, minerals, etc. necessary for growing. Examples of carbon sources include glucose, dextrin, soluble starch, and sucrose. Examples of nitrogen sources include ammonium salts, nitrates, corn chip liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like. Examples of the inorganic or organic substance and the inorganic substance include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.
  • M9 medium containing glucose and casamino acid As a medium for cultivating a bacterium belonging to the genus Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics (Journal of Experiments in Moiety) 1 ecu la Genetics), 431-433, Cold Spring Harbor Laboratory, New York 1972]. If necessary, an agent such as 3] 3-indolylacrylic acid can be added to make the promoter work efficiently.
  • 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.
  • cultivation is usually performed at about 30 to 40 ° C for about 6 to 24 hours, and if necessary, aeration and stirring can be applied.
  • a medium for example, Burkholder's minimum medium [Bostian, KL et al., "Processing's of the National Academy Op.” Proc. Natl. Acad. Sci. USA, 77, 4505 (1980) J and SD medium containing 0.5% casamino acid GBitter, GA et al. Proc. Nat. Acad. Sci. USA, 81, 5330 (1984) J. Procsings of the National Academy of the Socienties of the USA It is preferable to adjust the pH of the medium to about 5 to 8. Culture is usually performed at about 20 ⁇ to 35 ⁇ for about 24 to 72 hours, and aeration and agitation are added as necessary.
  • the culture medium When culturing a transformant whose host is an insect cell or an insect, the culture medium was immobilized to Grace's Insect Medium (Grace, TCC, Nature, 195, 788 (1962)). For example, those to which additives such as% P serum are appropriately added are used.
  • Grace's Insect Medium Grace, TCC, Nature, 195, 788 (1962)
  • those to which additives such as% P serum are appropriately added are used.
  • Culture Preferably, the pH of the ground is adjusted to about 6.2 to 6.4. Culture is usually performed at about 27 ° C for about 3 to 5 days, and if necessary, aeration and / or agitation are added.
  • examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, 122, 501 (1952)], DMEM Medium [Virology, 8, 396 (1959) 3, RPMI 1640 medium
  • the pH is about 6-8.
  • Cultivation is usually performed at about 30 ° C to 40t for about 15 to 60 hours, and aeration and stirring are added as necessary.
  • the G protein-coupled receptor overnight protein of the present invention can be produced in the cells, in the cell membrane, or outside the cells of the transformant.
  • the receptor protein of the present invention can be separated and purified from the culture by, for example, the following method.
  • 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 subjected to ultrasonic wave, lysozyme and / or freezing. After the cells or cells are ruptured by thawing or the like, a method of obtaining a crude extract of the receptor protein by centrifugation or filtration is appropriately used.
  • the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM.
  • Purification of the receptor protein contained in the thus obtained culture supernatant or extract 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. Mainly using difference in molecular weight, Method using charge difference such as ion-exchange chromatography, affinity mouth chromatography Methods that utilize specific affinity such as —, methods that use differences in hydrophobicity such as reversed-phase high-performance liquid chromatography, and methods that use differences in isoelectric points such as isoelectric focusing .
  • the receptor protein thus obtained when it is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when it is obtained in a salt, a known method Alternatively, it can be converted into a free form or another salt by a method analogous thereto.
  • the receptor protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by the action of an appropriate protein-modifying enzyme before or after purification.
  • an appropriate protein-modifying enzyme for example, trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like are used.
  • the activity of the receptor protein or its salt of the present invention thus produced can be measured by a binding experiment with a labeled ligand, an enzyme immunoassay using a specific antibody, or the like.
  • An antibody against the receptor protein of the present invention or its partial peptide or a salt thereof may be an antibody capable of recognizing the receptor protein of the present invention or its partial peptide or a salt thereof, such as a polyclonal antibody or a monoclonal antibody. Any of the antibodies may be used.
  • An antibody against the receptor protein of the present invention, its partial peptide, or a salt thereof may be a known antibody or a known antibody using the receptor protein of the present invention as an antigen. It can be produced according to the production method of antiserum. [Preparation of monoclonal antibody]
  • the receptor protein of the present invention or the like is administered to a mammal at a site where the antibody can be produced by administration, itself or together with a carrier or a diluent.
  • complete Freund's adjuvant and incomplete Freund's Subuvant may be administered. 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, egrets, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats 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.
  • a monoclonal antibody-producing hybridoma can be prepared.
  • the measurement of the antibody titer in the antiserum can be performed, for example, by reacting a labeled receptor protein and the like described below with the antiserum, and then measuring the activity of the labeling agent bound to the antibody.
  • the fusion operation can be carried out according to a known method, for example, the method of Koehler and Millsin [Nature, 256, 495 (1975)].
  • the fusion promoter include polyethylene glycol (PEG) and Sendai virus, but PEG is preferably used.
  • myeloma cells examples include NS-1, P3UL SP2 / 0 and the like, but ⁇ 3 ⁇ 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 PEG600) is used at a concentration of about 10 to 80%.
  • Cell fusion can be carried out efficiently by adding the mixture and incubating at about 20 to 40 ° C., preferably about 30 to 37 ° C. for about 1 to 10 minutes.
  • hybridomas can be cultured on a solid phase (eg, microplate) onto which an antigen such as receptor protein has been adsorbed directly or together with a carrier.
  • a solid phase eg, microplate
  • Anti-immunoglobulin antibody labeled with a radioactive substance or enzyme anti-mouse immunoglobulin antibody is used if the cells used for cell fusion are mice or protein A, and solid phase is added.
  • a method for detecting a monoclonal antibody bound to a protein adding a hybridoma culture supernatant to a solid phase to which an anti-immunoglobulin antibody or protein A has been adsorbed, and adding a receptor protein or the like labeled with a radioactive substance, an enzyme, or the like;
  • a method for detecting a monoclonal antibody bound to a phase is exemplified.
  • Monoclonal antibodies is performed according to a known method or a method equivalent thereto. Usually, it can be performed 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 medium Serum medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) or the like can be used.
  • the culture temperature is usually 20 to 40 ° C, preferably about 37 ° C.
  • the culture time is generally 5 days to 3 weeks, preferably 1 week to 2 weeks.
  • the culture can be usually performed under 5% carbon dioxide.
  • the antibody titer of the hybridoma culture supernatant can be measured in the same manner as the measurement of the antibody titer in the antiserum described above.
  • Monoclonal antibodies can be separated and purified in the same manner as normal polyclonal antibodies. [Examples: salting out, alcohol precipitation, isoelectric focusing, electrophoresis, ion exchangers (ex. , DEAE), ultracentrifugation, gel filtration, antigen-binding solid phase or specific adsorbent using an active adsorbent such as protein A or protein G to dissociate the bond to obtain the antibody. Purification method].
  • the polyclonal antibody of the present invention can be produced by 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 method for producing a monoclonal antibody described above. From the receptor protein of the present invention and the like, and the antibody can be separated and purified.
  • the type of carrier protein and the mixture ratio of carrier and hapten depend on the hapten immunized by cross-linking the carrier. Any antibody may be cross-linked at any ratio as long as it can be efficiently produced.
  • a method is used in which albumin, thyroglobulin, keyhole, limpet, and hemocyanin are coupled in a weight ratio of about 0.1 to 20, preferably about 1 to 5, with respect to one hapten.
  • various condensing agents can be used for force coupling between the hapten and the carrier.
  • an active ester reagent containing a daltaraldehyde-carpoimide, a maleimide active ester, a thiol group, or a dithioviridyl group is used.
  • the condensation product is administered to a warm-blooded animal itself or together with a carrier or diluent at a site where antibody production is possible.
  • Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability upon administration.
  • the administration can usually be made once every about 2 to 6 weeks, for a total of about 3 to 10 times.
  • the polyclonal antibody can be collected from blood, ascites, etc., preferably from blood, of the mammal immunized by the above method.
  • the measurement of the polyclonal antibody titer in the antiserum can be performed in the same manner as the measurement of the antibody titer in the serum described above. Separation and purification of the polyclonal antibody can be carried out according to the same immunoglobulin separation and purification method as the above-mentioned separation and purification of the monoclonal antibody.
  • the receptor protein of the present invention or its salt, its partial peptide or its salt
  • DNA encoding the receptor protein or a partial peptide thereof include (1) determination of a ligand (agonist) for the G protein-coupled receptor protein of the present invention, (2) G protein-coupled receptor of the present invention A prophylactic and / or therapeutic agent for a disease associated with protein dysfunction, (3) a genetic diagnostic agent, (4) a method for screening a compound that alters the expression level of the receptor protein of the present invention or a partial peptide thereof, (5 ) A preventive and / or therapeutic agent for various diseases containing a compound that alters the expression level of the receptor protein of the present invention or its partial peptide; (6) a ligand for the G protein-coupled receptor protein of the present invention (7) Changes in binding between ligand and G protein-coupled receptor protein of the present invention To compound (Agonisu Bok, antagonist, etc.) screening method, the (8) G protein-coupled receptions evening compounds that alter the binding property between one protein and a ligand of the present invention ( ⁇ Gore two strike, Ann evening agonist)
  • (11) a method for preventing and / or treating various diseases containing a compound that alters the amount of the receptor protein or its partial peptide of the present invention in a cell membrane; Neutralization by an antibody against the receptor protein of the present invention or its partial peptide or a salt thereof, (13) Creation of a non-human transgenic animal having DNA encoding the G protein-coupled receptor protein of the present invention, etc. Can be used.
  • ligand binding to a G protein-coupled receptor specific to human / non-human mammals can be achieved.
  • Compounds that alter the sex eg, agonist, angyu gonist, etc.
  • the agonist or angyu gonist can be used as an agent for preventing or treating various diseases.
  • DNA encoding the receptor protein or partial peptide of the present invention or a salt thereof hereinafter sometimes abbreviated as the receptor protein of the present invention, etc.
  • the receptor protein of the present invention or a DNA encoding the partial peptide thereof (hereinafter referred to as the present invention).
  • the use of an antibody against the receptor protein or the like of the present invention hereinafter sometimes abbreviated as the antibody of the present invention will be specifically described below.
  • the receptor protein of the present invention or a salt thereof or the partial peptide of the present invention or a salt thereof is used for searching for or determining a ligand (agonist) for the receptor protein of the present invention or a salt thereof.
  • Test compounds include known ligands (for example, tyramine, tylamine, j3-phenylethylamine, tryptamine, octopamine).
  • Reisamine (trace amine); angiotensin, bombesin, canapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, purine, vasoprescin, oxytocin, PACAP (eg, PACAP27, PACAP38), Secretin, dal gongon, calcitonin, adrenomedullin, somatosintin, GHRH, CRF, ACTH, GRP, PTH, VIP (passoactive intestinal and related polypeptide), somatosintin, dopamine, motilin, Amylin, bradykinin, CGR P (calcitonin gene Peptide, leukotriene, pancreastatin, prostaglandin, tropoxane, adenosine, adrenaline, chemokine superfamily (eg, IL-8, GR0 o !, GR0 / 3, GROr, NAP
  • the ligand determination method of the present invention uses the receptor protein of the present invention or a partial peptide thereof or a salt thereof, or constructs an expression system for a recombinant receptor protein, and By using the receptor binding system using the receptor system, it is possible to bind to the receptor protein of the present invention and to stimulate cell stimulation (for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production).
  • cell stimulation for example, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production.
  • the amount of the test compound bound to the receptor protein or the partial peptide is characterized by measuring cell stimulating activity and the like.
  • the present invention provides
  • the labeled test compound When the labeled test compound is brought into contact with the receptor protein expressed on the cell membrane by culturing a transformant containing the DNA encoding the receptor protein of the present invention, the labeled test compound A method for determining a ligand for the receptor protein of the present invention, which comprises measuring the amount of binding to the receptor protein or a salt thereof;
  • Receptor protein-mediated cell stimulating activity e.g., arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular
  • ⁇ Receptor protein-mediated cell stimulating activity e.g., arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular
  • cAMP production e.g., arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular
  • the receptor protein of the present invention or a salt thereof A method for determining a ligand, and
  • Stimulatory activity e.g., arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-ios
  • a method for determining a ligand to the receptor protein or a salt thereof of the present invention which comprises measuring the activity of promoting or suppressing the activation of pH, lowering the pH, and the like.
  • the receptor protein used in the method for determining a ligand may be any one as long as it contains the above-described receptor protein of the present invention or the partial peptide of the present invention. Expressed receptor proteins are suitable.
  • the expression method described above is used to produce the receptor protein of the present invention, but it is preferably carried out by expressing the DNA encoding the receptor protein in mammalian cells or insect cells.
  • Complementary DNA is usually used as the DNA fragment encoding the protein portion of interest, but is not necessarily limited to this.
  • gene fragments or 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 to express them efficiently, the DNA fragment must be obtained by using the nucleopolyhedropathy virus (Baculovirus) belonging to an insect host.
  • NPV polyhedrin promoter of nuclear poly edros
  • virus SV40-derived promoter
  • retrovirus promoter meta-mouth thionine promoter
  • human heat shock promoter cytomegalovirus mouth motor
  • SRa promoter etc.
  • it is incorporated into Examination of the quantity and quality of the expressed receptor can be performed by a known method. For example, it can be carried out according to the method described in the literature [Namb i, P. et al., The 'Journal' of 'Pyological' Chemistry (J. Biol. Chem.), 267, 19555-19559, 1992]. so Wear.
  • the receptor protein of the present invention or a partial peptide thereof or a salt thereof may be a receptor protein or a partial peptide thereof or a salt thereof purified according to a known method.
  • a cell containing the receptor protein or a cell membrane fraction thereof may be used.
  • the cell When a cell containing the receptor protein of the present invention is used in the ligand determination method of the present invention, the cell may be immobilized with glutaraldehyde, formalin, or the like.
  • the immobilization method can be performed according to a known method.
  • the cell containing the receptor protein of the present invention refers to a host cell expressing the receptor protein of the present invention.
  • a host cell Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like are used. .
  • 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 disrupted by crushing the cells with a PoUer-Elveheni type homogenizer, ⁇ One Ring Plender ⁇ Crushing with a polytron (Kinema Uca), crushing with an ultrasonic wave, pressing the 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 (typically, about 1-10 minutes), and the supernatant is further centrifuged at a high speed (15000-30000 rpm), usually 30 minutes-2 hours.
  • the resulting precipitate is used as the 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 receptions evening Isseki lymph click cytoplasm and in the membrane fraction containing the receptions evening Isseki protein is preferably 1 cell per ⁇ 10 8 molecules, is 10 5 -10 7 molecules Is preferred.
  • the receptor protein fraction is preferably a natural receptor protein fraction or a recombinant receptor protein fraction having an activity equivalent thereto.
  • equivalent activity refers to equivalent ligand binding activity, signal transduction activity and the like.
  • the labeled test compound [3 ⁇ 4], [125 1], [14 c], [] labeled tyramine, etc.
  • Teyramine beta-phenylene milled by wet tyramine (j3-phenyl ethylamin e), tryptamine (tryptamine), Trace amines such as octopamine; angiotensin, bombesin, canapinoid, cholecystokinin, glutamine, serotonin, melatonin, neuropeptide Y, opioid, pudding, vasopletcin, oxytocin, PACAP (eg, PACAP27) , PACAP38), secretin, glucagon, calcitonin, 7-drenomedullin, somatos, GHRH, CRF, ACTH, GRP, PTH, VIP (Vasoactive Intestinal and Reited Polypeptide), somatostin, dopamine, motilin, Amylin
  • a cell or a cell containing the receptor protein of the present invention is first used.
  • a buffer appropriate for the method of determination. Any buffer may be used as long as it does not inhibit the binding between the ligand and the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a tris-HCl buffer.
  • various proteins such as surfactants such as CHAPS, Tween-80 TM (Kao-Atlas), digitonin, deoxycholate, serum albumin, and gelatin are buffered. Can also be added.
  • a protease inhibitor such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and peptide suptin can be added for the purpose of suppressing the degradation of the receptor and ligand by the protease.
  • a certain amount of (5,000 to 500,000 cpm) [3 ⁇ 4], [125 1] coexist test compound labeled with a [1 0, C 35 S] .
  • reaction tube containing a large excess of unlabeled test compound to determine the amount of non-specific binding (NS B).
  • the reaction is carried out at about 0T: 5050 ° C., preferably at about 4 ° C.-37 ° C., for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
  • the reaction solution is filtered through a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and the radioactivity remaining on the glass fiber filter is measured using a liquid scintillation filter or a ⁇ 1 counter.
  • a test compound in which the count (B-NSB) obtained by subtracting the non-specific binding amount (NSB) from the total binding amount (B) exceeds 0 cpm is defined as a ligand (agonist) for the receptor protein of the present invention or a salt thereof. You can choose.
  • cell stimulating activity via the receptor protein for example, arachidonic acid release, Acetylcholine release, intracellular Ca2 + release, intracellular cAMP generation, intracellular cGMP generation, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of c-ios, decrease in pH, etc.
  • Activity or activity to be suppressed can be measured by a known method or a commercially available measurement kit. Specifically, first, cells containing the receptor protein are cultured on a multiwell plate or the like.
  • the ligand When determining the ligand, replace the medium with a fresh medium or an appropriate buffer that is not toxic to cells in advance, add the test compound, etc., incubate for a certain period of time, then extract the cells or recycle the supernatant. And quantitate the product produced according to each method. If the production of a substance (for example, arachidonic acid) as an indicator of cell stimulating activity is difficult due to a degrading enzyme contained in a cell, an assay may be performed by adding an inhibitor to the degrading enzyme. . In addition, activities such as cAMP production suppression can be detected as a production suppression effect on cells whose basic production has been increased by forskolin or the like.
  • a substance for example, arachidonic acid
  • the kit for determining a ligand that binds to the receptor protein of the present invention or a salt thereof includes a receptor protein of the present invention or a salt thereof, a partial peptide or a salt thereof of the present invention, a cell containing the receptor protein of the present invention, Alternatively, it contains a membrane fraction of cells containing the receptor protein of the present invention.
  • Examples of the ligand determination kit of the present invention include the following.
  • CH0 cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at 5 ⁇ 10 5 Z-wells, and cultured at 37 ° C., 5% CO 2 , 95% air for 2 days.
  • Measurement method Wash CHO cells expressing the receptor protein of the present invention cultured in a 12-well tissue culture plate twice with 1 ml of the measurement buffer, and add 490 / x1 measurement buffer to each well.
  • Examples of the ligand that can bind to the receptor protein or its salt of the present invention include, for example, substances specifically present in the hypothalamus, cerebral cortex, colon cancer, lung cancer, heart, placenta, lung, and the like. Specific examples include tyramine, beta-phenylethylamine () 3-phenylethylamine), tryptamine (tryptamine), octupamine (traceamine such as octopamine); and angiotensin.
  • the receptor protein or the receptor protein of the present invention can be used in accordance with the action of the ligand.
  • the encoded DNA can be used as a medicament such as an agent for preventing and / or treating a disease associated with dysfunction of the receptor protein of the present invention.
  • the receptor protein of the present invention when there is a patient who cannot expect the physiological action of the ligand due to a decrease in the receptor protein of the present invention in a living body (deficiency of the receptor protein), there are the following cases: 1) the receptor protein of the present invention; Is administered to the patient to replenish the amount of the receptor protein, or (ii) the DNA encoding the receptor protein of the present invention is administered to the patient and expressed, or After the DNA encoding the receptor protein of the present invention is introduced into cells and expressed, the amount of the protein in the affected body is increased by transplanting the cells into the patient or the like. Thus, the effect of the ligand can be sufficiently exerted. That is, the DNA encoding the receptor protein of the present invention is useful as a safe and low-toxic agent for preventing and / or treating diseases associated with dysfunction of the receptor protein of the present invention.
  • the receptor protein of the present invention is a novel seven-transmembrane receptor protein having about 43% homology at the amino acid sequence level in the G protein-coupled receptor protein neurotransmitter receptor. It's all night long.
  • D ⁇ encoding the receptor protein of the present invention or the receptor protein may be, for example, a central disease (eg, Alzheimer's disease, dementia, eating disorder, depression, epilepsy, anxiety, etc.), an endocrine disease (eg, Addison's disease, Cushing's syndrome, pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonadal dysfunction , Thyroid dysfunction, pituitary dysfunction, etc.), metabolic disorders (eg, diabetes, lipid metabolism disorders, diabetic complications, obesity, gout, cataract, hyperlipidemia, etc.), cancer (eg, non-small cell lung cancer, ovary) Cancer, prostate cancer, stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc., inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia) ), Cardiovascular disease (eg, hypertension, cardiac
  • the receptor protein of the present invention When used as the above-mentioned prophylactic / therapeutic agent, it can be formulated according to a conventional method.
  • 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 retroviral vector. 1. 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 auxiliary agent for promoting uptake, using a gene gun or a catheter such as a hide mouth gel catheter.
  • the DNA encoding the receptor protein of the present invention or (2) the receptor protein is orally administered as tablets, capsules, elixirs, microcapsules, etc., if necessary, or coated with water or water. It can be used parenterally in the form of an injectable preparation such as a sterile solution with other pharmaceutically acceptable liquids or a suspension.
  • an injectable preparation such as a sterile solution with other pharmaceutically acceptable liquids or a suspension.
  • 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 normal pharmaceutical practice, such as dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil, etc. .
  • a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil, etc.
  • aqueous liquid for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
  • Auxiliaries such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, Polysorbate 80 TM, HCO-50) .
  • oily liquid for example, sesame oil, soybean oil, and the like are used, and may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • the prophylactic and therapeutic agents include, for example, buffers (for example, phosphate buffer and sodium acetate buffer), soothing agents (for example, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (for example, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the preparations obtained in this way are safe and low toxic, so they can be used, for example, in humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, horses, cats, dogs, monkeys, etc.). Can be administered.
  • the dosage of the receptor protein of the present invention may vary depending on the administration subject, target organ, symptoms, administration method, and the like. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, More preferably, the dose is about 1. (! To 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, and the like. In general, for example, in a cancer patient (assuming 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day Conveniently, it is administered by intravenous injection In the case of other animals, the dose can be administered in terms of 60 kg.
  • the dosage 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, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the administration target, target organ, symptoms, administration method, etc.
  • it is usually used, for example, in a cancer patient (60 kg).
  • the dose can be administered in terms of 60 kg.
  • the DNA of the present invention can be used as a probe to produce the receptor protein of the present invention in a human or non-human mammal (eg, rat, mouse, rabbit, sheep, pig, pig, cat, dog, sal, etc.).
  • a human or non-human mammal eg, rat, mouse, rabbit, sheep, pig, pig, cat, dog, sal, etc.
  • abnormalities in the DNA or mRNA encoding the partial peptide thereof, for example, damage, mutation or reduced expression of the DNA or mRNA, and increased or expressed DNA or mRNA. It is useful as an agent for genetic diagnosis of excess or the like.
  • the above-described genetic diagnosis using the DNA of the present invention can be performed, for example, by the well-known Northern hybridization or PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Processing's ob. The National Academy of Sciences of the United States, Vol. 86, pp. 2766-2770 (1989) It can be implemented by such as. (4) A method for screening a compound that changes the expression level of the receptor protein or a partial peptide thereof of the present invention
  • the DNA of the present invention can be used for screening for a compound that changes the expression level of the receptor protein of the present invention or its partial peptide.
  • the present invention relates to, for example, (i) a non-human mammal's (2) blood, (2) a specific organ, (3) a tissue or cell isolated from an organ, or (ii) a transformant of the present invention.
  • the measurement of the mRNA amount of the receptor protein of the present invention or its partial peptide is specifically performed as follows.
  • Obese mice arteriosclerosis, egrets, cancer-bearing mice, etc.
  • drugs eg, anti-dementia drugs, blood pressure lowering drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light and dark
  • blood or specific organs eg, brain, lung, colon, etc.
  • tissues or cells isolated from the organs are obtained.
  • the mRNA of the receptor protein of the present invention or a partial peptide thereof contained in the obtained cells can be obtained, for example, by extracting mRNA from cells or the like by a conventional method, and using, for example, 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 its partial peptide is prepared according to the above method, and the mRNA of the receptor protein of the present invention or its partial peptide contained in the transformant is similarly determined. Quantification and analysis can be performed.
  • 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 before to 6 hours before) or after a certain time (after 30 minutes to 3 days, preferably after 1 hour to 2 days, more preferably after 1 hour to 24 hours), or 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 protein of the present invention contained in the cells Alternatively, it can be performed by quantifying and analyzing the mRNA amount of the partial peptide,
  • the test compound When culturing the transformant according to a conventional method, the test compound is mixed in a medium, and after culturing for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to Three days later), the amount can be determined by quantifying and analyzing the mRNA amount of the receptor protein of the present invention or its partial peptide contained in the transformant.
  • the compound obtained by using the screening method of the present invention or a salt thereof is a compound having an action of changing the expression level of the receptor protein or the partial peptide thereof of the present invention.
  • G protein-coupled receptor e.g., arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular cAMP production, Compounds that enhance or inhibit cGMP production, inositol phosphate production, cell membrane potential fluctuations, phosphorylation of intracellular proteins, activation of C-fos, pH reduction, etc.
  • G protein-coupled receptor e.g., arachidonic acid release, acetylcholine release, intracellular Ca2 + release, intracellular cAMP production, Compounds that enhance or inhibit cGMP production, inositol phosphate production, cell membrane potential fluctuations, phosphorylation of intracellular proteins, activation of C-fos, 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. 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 produced by the production of the receptor protein or the like of the present invention. It is useful as a safe and low toxic drug for reducing the physiological activity.
  • a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned drug containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and low toxic, and thus include, for example, human and non-human mammals (eg, rat, mouse, rabbit, sheep, bush, horse, cat, dog, monkey, etc.). Can be administered.
  • human and non-human mammals eg, rat, mouse, rabbit, sheep, bush, horse, cat, dog, monkey, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • oral 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.
  • the single dose varies depending on the subject of administration, target organ, symptoms, administration method, and the like.
  • it is usually used, for example, in cancer patients (60 kg).
  • the dose can be administered in terms of 60 kg.
  • 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 a partial peptide thereof may be used as a preventive agent for a disease associated with dysfunction of the receptor protein of the present invention and as a Z or therapeutic agent. it can.
  • Diseases associated with dysfunction of the receptor protein of the present invention include, for example, central diseases (eg, Alzheimer's disease, dementia, eating disorders, depression, epilepsy, anxiety, etc.), endocrine diseases (eg, Addison's disease) , Cushing's syndrome, pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic diseases (e.g., diabetes, dyslipidemia, diabetes) Complications, obesity, gout, cataract, hyperlipidemia, etc., cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.) ), Inflammatory diseases (eg, allergy, asthma, rheum, bronchiectivitis, etc.), bronchiectivitis, etc.
  • Inflammatory diseases eg, allergy, asthma, rheum
  • the compound can be used as a sugar-coated tablet, capsule, elixir, microcapsule or the like as needed, orally, or aseptic solution with water or another pharmaceutically acceptable liquid. It can be used parenterally or in the form of injections such as suspensions.
  • the compound is mixed with known physiologically acceptable carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like in a unit dosage form generally required for the practice of pharmaceutical preparations. It can be manufactured by The 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.
  • a liquid carrier such as an oil or fat may be contained.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice, such as dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil, etc. .
  • aqueous liquids for injection include physiological saline, isotonic solutions containing glucose and other adjuvants (for example, D-Sorbi! ⁇ -I, D-mannitol, sodium chloride, etc.).
  • Suitable solubilizers such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene daricol), nonionic surfactants (eg, Polysorbate 80 TM , HCO-50) May be.
  • the oily liquid for example, sesame oil, soybean oil, and the like are used, and may be used in combination with a dissolution aid such as benzyl benzoate or benzyl alcohol.
  • the prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human serum Albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • 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 dosage of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • the daily dose is generally as follows. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc., for example, usually in the form of injection, for example, in cancer patients (60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the receptor protein and the like of the present invention have a binding property to a ligand, the ligand concentration in a 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 according to the method described in the following (1) or (2) or a method analogous thereto.
  • Such compounds include (a) cell stimulating activity via G protein-coupled receptors (eg, Arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production , A compound that has an activity to promote or inhibit intracellular cGMP production, inositol phosphate production, fluctuations in cell membrane potential, phosphorylation of intracellular proteins, activation of C-fos, lowering of pH, etc.
  • G protein-coupled receptors eg, Arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production
  • the present invention relates to (i) the case where the receptor protein of the present invention or its partial peptide or a salt thereof is brought into contact with a ligand; and (ii) the receptor protein of the present invention or its partial peptide or its partial peptide.
  • a compound or a salt thereof that changes the binding property between a ligand and a receptor protein of the present invention or a partial peptide thereof or a salt thereof, which is compared with a case where a salt is brought into contact with a ligand and a test compound. is provided.
  • the screening method of the present invention is characterized in that, in the cases (i) and (ii), for example, the amount of a ligand bound to the receptor protein or the like, the cell stimulating activity, and the like are measured and compared.
  • the present invention provides
  • the receptor protein of the labeled ligand is brought into contact with the receptor protein and the like.
  • a method for screening a compound or a salt thereof that changes the binding between a ligand and a receptor protein or the like of the present invention which comprises measuring and comparing the amount of binding to
  • the labeled ligand and the test compound are compared with those of the present invention.
  • the transformant containing DNA is cultured and brought into contact with the receptor protein of the present invention expressed on the cell membrane, the amount of the labeled ligand bound to the receptor protein is measured and compared.
  • 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
  • cell stimulating activity via the receptor eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of C-fos, activity to promote or suppress activities such as lowering pH
  • the binding between the ligand and the receptor protein of the present invention is characterized.
  • a compound of the present invention in which a compound that activates the receptor protein or the like of the present invention eg, a ligand for the receptor protein or the like of the present invention
  • 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
  • the receptor of the present invention expressed on a cell membrane by culturing a transformant containing the DNA of the present invention with a compound that activates the receptor protein or the like of the present invention and a test compound when the sample is brought into contact with a receptor protein or the like;
  • Receptor-mediated cell stimulating activity e.g., arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, intracellular cGMP generation, wild boar
  • Etc. activity or inhibiting activity was measured, provides a comparison ligand and a test compound to the binding property between the receptor protein or the like Ru varying according to the invention, characterized in that.
  • a cell, tissue or cell thereof containing a G protein-coupled receptor receptor protein such as a rat is used prior to obtaining the receptor protein of the present invention.
  • a candidate compound is obtained using the membrane fraction (primary screening), and then whether or not the candidate compound actually inhibits the binding of human G protein-coupled receptor protein to ligand is determined.
  • a confirmatory test was required. If the cell, tissue or cell membrane fraction is used as is, other receptor proteins may be mixed. As a result, it has been difficult to directly screen an agonist or an antagonist for the desired receptor protein.
  • the human receptor protein of the present invention by using the human 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. Further, it is possible to easily evaluate whether the screened compound is an agonist or an antagonist.
  • the receptor protein of the present invention used in the screening method of the present invention may be any as long as it contains the above-described receptor protein of the present invention.
  • Cell membrane fractions of mammalian organs containing proteins and the like are preferred.
  • human-derived organs are particularly difficult to obtain, human-derived receptor proteins and the like which are expressed in large amounts using recombinants are suitable for screening.
  • 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 a DNA fragment encoding the receptor protein of the present invention into host animal cells and express them efficiently, the DNA fragment must be expressed in a nuclear polyhedrosis virus belonging to a baculovirus using an insect as a host.
  • NMV virus
  • SV40-derived promoter Downstream of nuclear polyhedros is virus (NPV) polyhedrin promoter, SV40-derived promoter, retroviral promoter, meta-oral thionine promoter, human heat shock promoter, cytomegalovirus promoter, SRa promoter, etc. It is preferable to incorporate them into The amount and quality of the expressed receptor can be detected by a known method. For example, the method described in the literature [Nambi, P. et al., The Journal of Biological, Chemistry, 267, 19555-19559, 1992]. Can be.
  • the receptor protein of the present invention may be a receptor protein or the like purified according to a known method, or may be a cell containing the receptor protein or the like.
  • the cells when cells containing the receptor protein or the like of the present invention are used, the cells may be immobilized with daltaraldehyde, formalin, or the like. .
  • the immobilization method can be performed according to a known method.
  • Cells containing the receptor protein or the like of the present invention include host cells expressing the receptor protein and the like.
  • Examples of the host cells include Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, and the like. preferable.
  • 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-Elvehj em homogenizer, crushing with a Warlinda blender ⁇ polytron (Kinema tica), crushing by ultrasonic waves, or pressing with a French press. 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 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 contains a large amount of expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.
  • the amount of receptor protein in cells or membrane fractions 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. is there.
  • an appropriate receptor protein fraction and a labeled ligand are required.
  • the receptor protein fraction is preferably a natural receptor protein fraction or a recombinant receptor protein fraction having the same activity as that of the receptor protein fraction.
  • 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.
  • ligands labeled with [3 ⁇ 4], c 125 n, [ I4 c], c 35 s) and the like are used.
  • cells containing the receptor protein or the like of the present invention or a membrane fraction of the cell are used.
  • a buffer suitable for screening to prepare a receptor protein preparation Any buffer may be used as long as it does not inhibit the binding of the ligand to the receptor protein, such as a phosphate buffer having a pH of 4 to 10 (preferably pH 6 to 8) or a tris-hydrochloride buffer.
  • surfactants such as CHAPS, Tween-80 TM (Kaoichi Atlas), digitonin, and dexcholate can be added to the buffer for the purpose of reducing non-specific binding.
  • a protease inhibitor such as PMSF, leptin, E-64 (manufactured by Peptide Research Laboratories), and peptide suptin can be added for the purpose of suppressing receptor degradation and ligand degradation by proteases.
  • the reaction is carried out at about o ° c to 50 ° (:, preferably at about 4 to 37 ° C, for about 20 minutes to 24 hours, preferably for about 30 minutes to 3 hours.
  • filtration with a glass fiber filter paper or the like After washing with an appropriate amount of the same buffer, measure the radioactivity remaining on the glass fiber filter paper using a liquid scintillation counter or an counter ..
  • Count when there is no antagonist (Bo) to non-specific binding (NSB) Assuming that the count ( BQ -NSB) minus 100 is 100%, the test compound with a specific binding amount (B-NSB) of, for example, 50% or less can be selected as a candidate substance with competitive inhibition ability. it can.
  • Compounds that alter the binding between ligand and receptor protein of the present invention In order to carry out the above-mentioned methods (1) to (4) for screening, for example, cell stimulating activity via receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, Intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of C-ios, activity to promote or suppress pH reduction, etc.) by known methods or commercially available assays. It can be measured using a kit for measurement.
  • receptor protein eg, arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP generation, Intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, activation of C-ios, activity to promote or suppress pH reduction, etc.
  • cells containing the receptor protein or the like of the present invention are cultured on a multi-well plate or the like.
  • 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 as a production suppression effect on cells whose basic production has been increased by forskolin or the like.
  • Cells expressing an appropriate receptor protein are required.
  • Cells expressing the receptor protein of the present invention and the like are preferably cell lines having the natural receptor protein of the present invention and the like, and cell lines expressing the above-described recombinant receptor protein and the like.
  • test compounds for example, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. are used, and these compounds are novel compounds. Or a known compound.
  • a screening kit for a compound or a salt thereof that alters the binding between the ligand and the receptor protein or the like of the present invention is a cell containing the receptor or protein of the present invention, the protein of the present invention, or And those containing the membrane fraction of cells containing the protein of the present invention.
  • Examples of the screening kit of the present invention include the following.
  • It may be sterilized by filtration with a 0.45 im filter and stored at 4 t: or may be prepared at the time of use.
  • CH0 cells expressing the receptor protein of the present invention were subcultured on a 12-well plate at 5 ⁇ 10 5 cells and cultured for 2 days in 37, 5% CO 2 , 95 ° air.
  • the compound obtained by using the screening method or the screening kit of the present invention or a salt thereof is a compound having an action of changing the binding property between the ligand and the receptor protein of the present invention or the like.
  • the G protein co auditors receptor cell stimulating activity e.g., Arakidon acid release, Asechiruko phosphorus release, intracellular Ca w release, intracellular cAMP production, intracellular cGMP production, inositol Li (A so-called agonist to the receptor protein of the present invention) having an activity of promoting or inhibiting acid production, fluctuation of cell membrane potential, phosphorylation of intracellular protein, activation of C-fos, decrease of pH, etc.
  • (Mouth) a compound not having the cell stimulating activity (so-called compound for the receptor protein of the present invention) (8) a compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention, or (2) the binding between the ligand and the G protein-coupled receptor protein of the present invention A compound that reduces force.
  • Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, a fermentation product, and the like. These compounds may be novel compounds or known compounds.
  • the agonist against the receptor protein or the like of the present invention has the same activity as the physiological activity of the ligand for the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug according to the ligand activity. It is.
  • the antagonist to the receptor protein or the like of the present invention can suppress the physiological activity of the ligand for the receptor protein or the like of the present invention, it is useful as a safe and low-toxic drug for suppressing the ligand activity.
  • the compound that enhances the binding force between the ligand and the G protein-coupled receptor protein of the present invention is useful as a safe and low-toxic drug for enhancing the physiological activity of the ligand for the receptor protein of the present invention and the like. It is.
  • 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 or the like.
  • a compound or a salt thereof obtained by using the screening method or the screening kit of the present invention is used as the above-mentioned pharmaceutical composition, it can be carried out according to conventional means.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as in the above-described medicine containing the receptor protein of the present invention.
  • the preparations obtained in this way are safe and have low toxicity, so they can be used, for example, in humans and non-human mammals (eg, rats, puppies, sheep, bush, pus, cats, dogs, monkeys, etc.) Can be administered.
  • non-human mammals eg, rats, puppies, sheep, bush, pus, cats, dogs, monkeys, etc.
  • the dosage of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • the daily dose is generally as follows. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc., for example, usually in the form of injection, for example, in cancer patients (60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • a preventive and / or therapeutic agent for various diseases containing a compound (agonist, angonist) that alters the binding between a G protein-coupled receptor protein and a ligand of the present invention.
  • the receptor protein of the present invention is considered to play some important roles in vivo, such as central functions, circulatory functions, digestive functions, and cardiac functions. Therefore, compounds that alter the binding property between the receptor protein of the present invention and the ligand (agonist, angelic gonist) and ligands for the receptor protein of the present invention are related to dysfunction of the receptor protein of the present invention. It can be used as a prophylactic and / or therapeutic agent for diseases that occur.
  • 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, anxiety, etc.), endocrine diseases (eg, Addison's disease, Cushing's syndrome, Pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic disorders (e.g.
  • 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, , Allergies, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.
  • cardiovascular diseases eg, hypertension, heart Hypertrophy, angina, myocardial infarction, arteriosclerosis, etc.
  • respiratory diseases eg, cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis, pulmonary thrombosis, etc.
  • digestive system diseases eg, , Gastric ulcer, duodenal ulcer, gastritis, reflux esophagitis, knee inflammation, etc.
  • immune system diseases eg, autoimmune diseases, etc.
  • infectious diseases eg, immune dysfunction, pneumonia
  • 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 sterilized with tablets or capsules, elixirs, microcapsules, etc., as required, or sugar-coated, or with water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of injections, such as 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 like Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharine, flavoring agents such as peppermint, cocoa oil or cellulose are used.
  • the unit dosage form is a capsule, the above type of material can further contain a liquid carrier such as an oil or fat.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice, such as dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil, etc. .
  • a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil, etc.
  • aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.) and the like are used.
  • Auxiliaries such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene daricol), non-ionic surfactants (eg, Polysorbate 80 TM, HC0-50) .
  • the oily liquid for example, sesame oil, soybean oil and the like are used, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol.
  • the prophylactic / therapeutic agents include, for example, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • the prepared injection solution is usually filled in a suitable ampoule.
  • the above-mentioned prophylactic / therapeutic agent can be used in combination with an appropriate drug, for example, as a DDS preparation specifically targeting an organ or tissue in which the receptor protein of the present invention is highly expressed.
  • the preparations obtained in this way are safe and low toxic, such as, for example, human and non-human mammals (eg, rat, mouse, rabbit, sheep, bush, horse, cat, dog, monkey, etc.) Can be administered.
  • human and non-human mammals eg, rat, mouse, rabbit, sheep, bush, horse, cat, dog, monkey, etc.
  • the dose of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, administration method, etc. Usually, for example, in a cancer patient (as 60 kg), about 0.01 to 30 mg per day, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day It is convenient to administer by injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the antibody of the present invention can specifically recognize the receptor protein of the present invention and the like, quantification of the protein of the present invention in a test solution, particularly quantification by a sandwich immunoassay, etc. Can be used for 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. Is preferred.
  • the receptor protein and the like of the present invention can be measured using a monoclonal antibody against the receptor protein and the like of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention). Can also be performed.
  • 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 of the present invention is not particularly limited, and may be an antibody, an antigen or an antibody corresponding to the amount of an antigen (eg, the amount of the receptor protein) in the test solution.
  • the amount of one antigen complex is detected by chemical or physical means, and this is Any measurement method may be used as long as it is a measurement method calculated from a standard curve prepared using a standard solution containing For example, nephelometry, a competition method, an immunometric method and a sandwich method are suitably used, but in terms of sensitivity and specificity, it is particularly preferable to use a sandwich method described later.
  • 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. Radioisotopes, if example embodiment, [1 1], [131 1], [3 ⁇ 4], C 14 C) are used. As the above enzyme
  • Stable and large specific activities are preferred.
  • galactosidase ⁇ -darcosidase, alkaline phosphatase, passoxidase, malate dehydrogenase and the like are used.
  • fluorescent substance for example, fluorescamine, fluorescein isothiosinate and the like are used.
  • luminescent substance for example, luminol, luminol derivative, reluciferin, lucigenin and the like are used.
  • a biotin-avidin system may be used for binding the antibody or antigen to the labeling agent.
  • insolubilization of the antigen or antibody physical adsorption may be used, or a method using a chemical bond usually used for insolubilizing or immobilizing a protein or enzyme may be used.
  • the carrier for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, and glass are used.
  • the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further reacted with the labeled monoclonal antibody of the present invention (secondary reaction).
  • primary reaction the insolubilized monoclonal antibody of the present invention
  • secondary reaction the labeled monoclonal antibody of the present invention
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at staggered times.
  • the labeling agent 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 method for measuring receptor protein and the like by the sandwich method of the present invention 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.
  • an antibody having a different binding site such as a receptor protein is preferably used. That is, when the antibody used in the primary reaction and the secondary reaction is, for example, the antibody used in the secondary reaction recognizes the C-terminal of the receptor protein, the antibody used in the primary reaction is preferably An antibody that recognizes other than the C-terminal, for example, the N-terminal, is used.
  • the monoclonal antibody of the present invention can be used in a measurement system other than the sandwich method, for example, a competition method, an immunometric method, or a nephrometry.
  • a competition method after the antigen in the test solution and the labeled antigen are allowed to react competitively with the antibody, the unreacted labeled antigen is separated from (F) and the labeled antigen (B) bound to the antibody. (B / F separation) The amount of labeling in any of B and F is measured, and the amount of antigen in the test solution is quantified.
  • a soluble antibody is used as an antibody
  • B / F separation is performed using a polyethylene glycol
  • a liquid phase method using a second antibody against the above antibody or an immobilized antibody is used as the first antibody.
  • An immobilization method using a soluble first antibody and an immobilized antibody as the second antibody is used.
  • the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated. After reacting the antigen with an excess amount of the labeled antibody, the immobilized antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to determine the amount of antigen in the test solution.
  • the amount of insoluble sediment generated as a result of the antigen-antibody reaction in a gel or in a solution is measured. Even when the amount of antigen in the test solution is small and only a small amount of sediment is obtained, laser nephrometry utilizing scattering by laser 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 present in a subject such as a body fluid or a tissue.
  • the antibody of the present invention can specifically recognize the receptor protein of the present invention or its partial peptide or its salt, the amount of the receptor protein of the present invention or its partial peptide in the cell membrane can be increased. It can be used for screening of compounds that change the value. That is, the present invention, for example,
  • Non-human mammal 1) Blood, 2) Specific organs, 3) Tissues or cells isolated from the organs are destroyed, the cell membrane fraction is isolated, and the receptor of the present invention contained in the cell membrane fraction
  • the cell membrane fraction is isolated, and the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction 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,
  • Sections of (1) blood, (2) specific organs, and (3) tissues or cells isolated from organs of non-human mammals, and then using immunostaining to obtain the receptor at the cell surface Provided is a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in a cell membrane by confirming the protein on the cell membrane by quantifying the degree of protein staining. You.
  • the present invention provides a method for screening a compound that changes the amount of the receptor protein of the present invention or its partial peptide in a cell membrane by confirming the protein on the cell membrane by quantifying the degree of staining of the cell.
  • the quantitative determination of the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction is specifically performed as follows.
  • non-human mammals for example, mice, rats, rabbits, sheep, hidge, puta, puppies, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, arteriosclerosis ⁇ A heron, a tumor-bearing mouse, etc.
  • a drug eg, an anti-dementia drug, a blood pressure lowering drug, an anti-cancer drug, an anti-obesity drug, etc.
  • a physical stress eg, After a certain period of time, the blood or specific organs (eg, brain, lungs, large intestine, etc.), or tissues isolated from the organs, Alternatively, obtain cells.
  • the obtained organ, tissue or cell is suspended in, for example, an appropriate buffer (for example, Tris-HCl buffer, phosphate buffer, or Hase buffer) to destroy the organ, tissue or cell.
  • 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 technique 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-Elvehem em homogenizer, crushing with a Waring Blender or a polytron (Kinema tica), crushing by ultrasonic waves, or pressurizing with a French press. Crushing by ejecting cells 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 contains a large amount of expressed receptor proteins and membrane components such as cell-derived phospholipids and membrane proteins.
  • the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified by, for example, a sandwich immunoassay using the antibody of the present invention, Western blot analysis, or the like.
  • Such a sandwich immunoassay can be performed in the same manner as described above, and the Western plot can be performed by known means.
  • a transformant expressing the receptor protein of the present invention or its partial peptide can be prepared according to the above method, and the receptor protein of the present invention or its partial peptide contained in the cell membrane fraction can be quantified. it can.
  • 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:
  • Drugs or physical drugs against normal or disease model non-human mammals A certain time before giving stress etc. (30 minutes to 24 hours before, preferably 30 minutes to 12 hours before, more preferably before to 6 hours before) or after a certain time (30 minutes to 3 days after, preferably Is 1 hour to 2 days, more preferably 1 hour to 24 hours), or a test compound is administered simultaneously with a drug or physical stress, and after a certain time after administration (30 minutes to 3 days, Preferably after 1 hour to 2 days, more preferably after 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 When culturing the transformant according to a conventional method, the test compound is mixed in the medium, and after culturing for a certain period of time (1 day to 7 days, preferably 1 day to 3 days, more preferably 2 days to Three days later), it can be carried out by quantifying the amount of the receptor protein of the present invention or its partial peptide in the cell membrane.
  • non-human mammals eg, mice, rats, egrets, higgs, bushus, mice, cats, dogs, monkeys, etc., more specifically, dementia rats, obese mice, Drugs (eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.) or physical stress (eg, flooding stress, electric shock, light / dark, low temperature, etc.)
  • Drugs eg, anti-dementia drugs, anti-hypertensive drugs, anti-cancer drugs, anti-obesity drugs, etc.
  • physical stress eg, flooding stress, electric shock, light / dark, low temperature, etc.
  • blood or specific organs eg, heart, placenta, lung, etc.
  • tissues or cells isolated from the organs are obtained.
  • the obtained organ, tissue or cell is cut into a tissue section according to a conventional method, and immunostained with the antibody of the present invention.
  • the receptor protein of the present invention or its protein on the cell membrane can be quantitatively or qualitatively determined.
  • the amount of the partial peptide can be confirmed.
  • the compound or a salt thereof obtained by using the screening method of the present invention changes the amount of the receptor protein of the present invention or its partial peptide in the cell membrane. Specifically, (a) increasing the amount of the receptor protein of the present invention or its partial peptide in the cell membrane to thereby increase the G protein-coupled receptor receptor; Cell stimulating activity (e.g., arachidonic acid release, acetylcholine release, intracellular Ca 2+ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, C- a compound that enhances the activity of promoting or suppressing fOS, such as the activation or suppression of pH, etc.). It is a compound that reduces the cell stimulating activity. Examples of the compound include a peptide, a protein, a non-peptidic compound, a synthetic compound, and a fermentation product. These compounds may be a novel compound or a known compound.
  • the compound that enhances the cell stimulating activity is useful as a safe and low toxic drug for enhancing the physiological activity of the receptor protein of the present invention or the like.
  • the compound that attenuates the cell stimulating activity is useful as a safe and low toxic drug for decreasing the physiological activity of the receptor protein of the present invention.
  • a compound or a salt thereof obtained by using the screening method of the present invention is used as a pharmaceutical composition, it can be carried out according to a conventional method.
  • tablets, capsules, elixirs, microcapsules, sterile solutions, suspensions, and the like can be prepared in the same manner as the above-mentioned 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, 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, symptoms, administration method, and the like. However, in the case of oral administration, for example, in a patient with cancer (as 60 kg), the dose is usually 1 day. The amount is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the subject of administration, target organ, symptoms, administration method, etc., for example, usually in the form of injection, for example, in cancer patients (60 kg). , About 0.01 to 30 mg per day It is convenient to administer the drug by intravenous injection, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg. In the case of other animals, the dose can be administered in terms of 60 kg. (11) A preventive and / or therapeutic agent for various diseases containing a compound that alters the amount of the receptor protein of the present invention or its partial peptide in the cell membrane
  • Receptor protein of the present invention as described above, for example, 9 considered to play some important roles in the body such as the heart or central functional Thus, receptions evening Isseki protein or portions thereof of the present invention in cell membrane
  • the compound that alters the amount of the 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.
  • 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, anxiety, etc.) and endocrine diseases (eg, Addison's disease, Cushing Syndrome, pheochromocytoma, primary aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic disorders (e.g., diabetes, dyslipidemia, diabetes mellitus) Disease, obesity, gout, cataract, hyperlipidemia, etc.), cancer (for example, non-small cell lung cancer, ovarian cancer, prostate cancer, gastric cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.), Inflammatory diseases (eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.), cardiovascular diseases (eg, hypertension, Cardiac hypertrophy,
  • 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 also be used parenterally in the form of injections, such as suspensions or night drops.
  • 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.
  • 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 normal pharmaceutical practice, such as dissolving or suspending the active substance in a vehicle such as water for injection, or naturally occurring vegetable oils such as sesame oil, coconut oil, etc. .
  • aqueous solution for injection include physiological saline, isotonic solution containing pudose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • solubilizers for example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene daricol), nonionic surfactant (eg, Polysorbate 80 TM, HC0-50) May be.
  • 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.), 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 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 dosage of the compound or a salt thereof varies depending on the administration subject, target organ, symptoms, administration method, and the like.
  • the daily dose is generally as follows. About 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the administration subject, target organ, symptoms, administration method, etc., for example, usually in the form of injection, for example, in cancer patients (60 kg). It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day by intravenous injection. In the case of other animals, the dose can be administered in terms of 60 kg.
  • the neutralizing activity of the antibody against the receptor protein of the present invention or its partial peptide or a salt thereof against the receptor protein or the like means that the activity of inactivating the signal transduction function involving the receptor protein. To taste. Therefore, when the antibody has neutralizing activity, signal transduction involving the receptor protein, for example, cell stimulating activity through the receptor protein
  • diseases caused by overexpression of the receptor protein include central diseases (e.g., Alzheimer's disease, dementia, eating disorders, depression, epilepsy, anxiety, etc.), endocrine diseases (e.g., Addison's disease, Cushing's syndrome, Pheochromocytoma, primary sexual aldosteronism, menopause, endometriosis, gonadal dysfunction, thyroid dysfunction, pituitary dysfunction, etc., metabolic disorders (eg, diabetes, dyslipidemia, diabetic complications, obesity, gout, cataract, high fat Blood cancer, etc. (eg, non-small cell lung cancer, ovarian cancer
  • Prostate cancer stomach cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, etc.
  • inflammatory diseases eg, allergy, asthma, rheumatism, arthritis, nephritis, hepatitis, retinopathy, cystitis, pneumonia, etc.
  • Cardiovascular diseases eg, hypertension, cardiac hypertrophy, angina, myocardial infarction, arteriosclerosis, etc.
  • respiratory diseases eg, cold syndrome, asthma, pneumonia, pulmonary hypertension, pulmonary thrombosis, Pulmonary emboli, etc.
  • gastrointestinal diseases eg, gastric ulcer, duodenal ulcer, gastritis, reflux esophagitis, ⁇ inflammation, etc.
  • immune system diseases eg, autoimmune diseases, etc.
  • infectious diseases eg, immune dysfunction, Pneumonia, cytomegal virus, influenza virus, herpes virus, and other viral infections, rickettsial infections, bacterial infections, etc.
  • migraines eg, migraines.
  • transgenic animals expressing the receptor protein of the present invention and the like can be produced.
  • Animals include mammals (for example, rats, mice, egrets, sheep, pigs, pigs, cats, dogs, monkeys, etc.) (hereinafter sometimes abbreviated as animals). In particular, mice, egrets and the like 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 the DNA in animal cells.
  • a promoter capable of expressing the DNA in animal cells For example, when the DNA of the present invention derived from Pergum is introduced, a gene construct in which the DNA of the present invention derived from an animal having high homology to this is linked to the downstream of various promoters capable of expressing in animal cells, for example, a fertilized egg of Pergum
  • a DNA-transferred animal that highly produces the receptor protein of the present invention can be produced.
  • a ubiquitous expression promoter such as a promoter derived from a virus, meta-thionine, or the like may be used, but a promoter of a gene specifically expressed in the heart is preferably used.
  • Introduction of the DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target animal.
  • the presence of the receptor protein or the like of the present invention in the germinal cells of the produced animal after the DNA transfer indicates that all the offspring of the produced animal have the receptor protein or the like of the present invention in all of their germ cells and somatic cells. It means having.
  • the progeny of this type of animal that has inherited the gene has the receptor protein of the present invention in all of its germ cells and somatic cells.
  • the animal After confirming that the DNA-transferred animal of the present invention stably retains the gene by mating, the animal can be subcultured in a normal breeding environment as the DNA-bearing animal. Further, by crossing male and female animals having the DNA of interest, homozygous animals having the transgene in both homologous chromosomes are obtained, and by crossing the male and female animals, all progeny have the DNA. Breeding can be subcultured.
  • the animal into which the DNA of the present invention has been introduced has high expression of the receptor protein of the present invention, it is useful as an animal for screening 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 ability to directly analyze DNA or RNA in the tissue of the DNA-introduced mouse of the present invention, or the analysis of the tissue in which the receptor protein of the present invention expressed by a gene is present can be used to analyze the receptor protein of the present invention. Etc. can be analyzed.
  • the cells of a tissue having the receptor protein of the present invention etc. are cultured by standard tissue culture techniques, and these are used to study the function of cells from generally difficult-to-cultivate tissues such as brain and peripheral tissues. can do.
  • the receptor protein of the present invention can be isolated and purified therefrom.
  • bases, amino acids, and the like are indicated by abbreviations, the indications are based on the abbreviations by IUP C-IUB Commission on Biochemical Nomenclature or the abbreviations commonly used in the art. An example is shown below. Also When there is an optical isomer for an amino acid, the L-form is indicated unless otherwise specified.
  • Trt Trityl
  • H0NB 1-Hydroxy-5-norpolene-2,3-Dicarbo
  • sequence numbers in the sequence listing in the present specification indicate the following sequences.
  • FIG 1 shows the amino acid sequence of the novel human-derived G protein-coupled receptor protein TGR29 of the present invention.
  • FIG. 1 shows the nucleotide sequence of cDNA encoding the novel human-derived G protein-coupled receptor protein TGR29 of the present invention.
  • SEQ ID NO: 3 1 shows the base sequence of primer 1 used in the PCR reaction in Example 1 below.
  • the base sequence of primer 2 used in the PCR reaction in Example 1 below is shown.
  • L-hTGR29, obtained in Example 1 below was used in Tsukuba East, Ibaraki Prefecture from March 5, 2001 (Heisei 13). Chome No. 1 1 Chuo No.
  • PCR reaction was performed using two primers, Primer 1 (SEQ ID NO: 3) and Primer 2 (SEQ ID NO: 4).
  • the composition of the reaction solution used in the reaction was 1/50 volume of the above cDNA, and 1/50 volume of Advantage-GC 2 Polymerase Mix (CL0NTECH), primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 3) 4) 0.5 / M each, 200 ⁇ of dNTPs, 1/5 volume of the buffer attached to the enzyme, 1/5 volume of GC Melt, to give a solution volume of 20 ⁇ .
  • the PCR reaction is performed at 94 ° C for 5 minutes, followed by 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 68 ° C for 4 minutes 35 times, and finally at 68 ° C for 5 minutes.
  • An extension reaction was performed.
  • the PCR reaction product was subcloned into plasmid vector pCR2.1 (Invitrogen) according to the procedure of TA Cloning Kit (Iitrotrogen). Loaned. This was introduced into E. coli TOP10, and clones having cDNA were selected on LB agar medium containing ampicillin.
  • a cDNA sequence (SEQ ID NO: 2) encoding a novel G protein-coupled receptor Yuichi protein was obtained. Further, a novel G protein-coupled receptor Yuichi protein containing this amino acid sequence (SEQ ID NO: 1) was named TGR29. The transformant was named Escherichia coli T0P10 / pCR2.1-hTGR29. Industrial applicability
  • the G protein-coupled receptor protein of the present invention or a partial peptide thereof or a salt thereof, a polynucleotide encoding the receptor protein or a partial peptide thereof includes the following: Gonist); 2 acquisition of antibodies and antisera; 3 construction of a recombinant receptor protein expression system; ⁇ ⁇ development of a receptor binding assay system using the expression system; and screening of drug candidate compounds; 5 Conduct drug design based on comparison with structurally similar ligands / receptors, ⁇ ⁇ Reagents for the preparation of probes and PCR primers in gene diagnosis, ⁇ Production of transgenic animals or ⁇ Gene therapeutic agents, etc. And the like.

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Abstract

L'invention vise à rechercher une nouvelle protéine de récepteur couplé à la protéine G et d'éclaircir sa fonction. La nouvelle protéine du récepteur couplé à la protéine G de cette invention possède une séquence d'acides aminés qui est semblable ou sensiblement semblable à la séquence d'acides aminés représentée par SEQ ID No1 ou son sel. L'invention concerne en outre un polynucléotide codant ladite protéine et ses applications sous forme médicinale.
PCT/JP2002/000136 2001-01-15 2002-01-11 Nouvelle proteine du recepteur couple a la proteine g et son adn Ceased WO2002055690A1 (fr)

Applications Claiming Priority (4)

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JP2001006464 2001-01-15
JP2001-102483 2001-03-30
JP2001102483 2001-03-30
JP2001-6464 2001-03-30

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117990A (en) * 1999-04-06 2000-09-12 Synaptic Pharmaceutical Corporation DNA encoding SNORF1 receptor
WO2001062797A2 (fr) * 2000-02-23 2001-08-30 Pharmacia & Upjohn Company Nouveaux recepteurs couples a la proteine g

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117990A (en) * 1999-04-06 2000-09-12 Synaptic Pharmaceutical Corporation DNA encoding SNORF1 receptor
WO2001062797A2 (fr) * 2000-02-23 2001-08-30 Pharmacia & Upjohn Company Nouveaux recepteurs couples a la proteine g

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