WO2015128894A1 - Procédé de détection de transduction de signal d'un récepteur couplé à une protéine g - Google Patents

Procédé de détection de transduction de signal d'un récepteur couplé à une protéine g Download PDF

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WO2015128894A1
WO2015128894A1 PCT/JP2014/000992 JP2014000992W WO2015128894A1 WO 2015128894 A1 WO2015128894 A1 WO 2015128894A1 JP 2014000992 W JP2014000992 W JP 2014000992W WO 2015128894 A1 WO2015128894 A1 WO 2015128894A1
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nucleotide sequence
polynucleotide
seq
protein
sequence represented
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PCT/JP2014/000992
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Japanese (ja)
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青木　淳賢
飛鳥 井上
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国立大学法人東北大学
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Priority to JP2014513383A priority Critical patent/JP6093946B2/ja
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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
    • G01N33/53—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention is a method for detecting signal transduction activation of G protein induced by binding between a test substance (ligand) and a G protein-coupled receptor (GPCR), and is induced by binding between a ligand and GPCR.
  • the present invention relates to a kit for detecting signal transduction activation of G protein.
  • GPCR The G protein-coupled receptor
  • the G protein-coupled receptor (GPCR) is a membrane-bound receptor having a characteristic structure of 7-transmembrane type, and about 800 kinds of proteins are known in humans. GPCRs accept a wide variety of ligands such as neurotransmitters and hormones and are involved in various signal transduction in cells. In addition, since about 50% of drugs that are currently clinically applied are considered to act on GPCRs, GPCRs are regarded as important target proteins for drug development.
  • G ⁇ a G ⁇ subunit constituting a trimeric ( ⁇ ) G protein plays a particularly important role.
  • G ⁇ a G ⁇ subunit constituting a trimeric ( ⁇ ) G protein plays a particularly important role.
  • the three-dimensional structure of the protein changes from an inactive GPCR to an active GPCR.
  • the active GPCR binds to G ⁇ of the trimeric G protein, thereby changing the three-dimensional structure of the protein from the non-active (GDP-binding) G ⁇ to the active (GTP-binding) G ⁇ .
  • the active G ⁇ is dissociated from the trimeric G protein by the change.
  • the dissociated activated G ⁇ activates the effector protein, and a signal is transmitted through the effector protein.
  • the G ⁇ subunit is composed of four types of families (Gs, Gi, Gq, and G12), and the signal transduction pathway by active G ⁇ is roughly divided to some extent by the types of these families.
  • Gs family regulates cAMP increase through adenylate cyclase activation
  • Gi family regulates cAMP reduction through adenylate cyclase inhibition
  • Gq family through phospholipase C.
  • RhoGEF Rho's activation protein
  • LPA6 also referred to as “P2Y5”
  • TGF ⁇ Transforming growth factor alpha
  • TGF ⁇ cleavage assay TGF ⁇ ⁇ ⁇ shedding assay
  • Non-patent Document 3 gene targeting methods using guide RNA (sgRNA; single-guide RNA) and Cas9 endonuclease have been developed as a technique for simply and effectively knocking out genes in mammalian cells.
  • the subject of the present invention is [1] activation of endogenous Gq or G12 family signal transduction induced by binding of a test substance (ligand) and a G protein-coupled receptor (GPCR) in a mammalian cell.
  • a test substance ligand
  • GPCR G protein-coupled receptor
  • the present inventors have found that Gq family signal transduction and G12 family signal transduction are involved in the cleavage of TGF ⁇ .
  • Gq family signal transduction and G12 family signal transduction are involved in the cleavage of TGF ⁇ .
  • GNAQ and GNA11 genes genes included in the Gq family
  • ⁇ GNAQ / 11 strains of such genes are used as a guide RNA. It was prepared by gene targeting method using (sgRNA; single-guide RNA) and Cas9 endonuclease, and TGF ⁇ cleavage assay was performed using ⁇ GNAQ / 11 strain. As a result, it was induced by binding of ligand and its GPCR. We found that the signal transduction activation of the family can be specifically detected.
  • a knockout cell ( ⁇ GNA12 / 13) strain of two types of genes (GNA12 and GNA13 genes) constituting the G12 family was prepared by the above gene targeting method, and a TGF ⁇ cleavage assay was performed using the ⁇ GNA12 / 13 strain. It has been found that Gq family signaling activation induced by the binding of a ligand and its GPCR can be specifically detected.
  • a cell ( ⁇ GNAQ / 11/12/13) strain was prepared by the above gene targeting method, and a G ⁇ chimeric protein having the Gq family as a skeleton was expressed in the ⁇ GNAQ / 11/12/13 strain and examined by a TGF ⁇ cleavage assay.
  • the present invention provides (1) signal transduction induced by the binding between a test substance and a G protein-coupled receptor (GPCR), comprising the following steps (a) to (c):
  • GPCR G protein-coupled receptor
  • the present invention relates to a method for detecting activation.
  • A mammalian cells knocked out of GNAQ and GNA11 genes or GNA12 and GNA13 genes, A GPCR gene or a protein encoding the gene; Introducing a labeling substance-bound membrane-bound TGF ⁇ gene having a labeling substance bound to the amino terminus of membrane-bound TGF ⁇ or a protein encoding the gene;
  • B a step of culturing the mammalian cell introduced in step (a) in the presence of a test substance and collecting a culture supernatant;
  • C a step of detecting the presence or absence of a labeling substance in the culture supernatant collected in step (b);
  • the present invention also includes (2) signal transduction activity induced by the binding of a test substance and a G protein-coupled receptor (GPCR), characterized by comprising the following steps (A) to (C):
  • GPCR G protein-coupled receptor
  • A mammalian cells knocked out of GNAQ and GNA11 genes and GNA12 and GNA13 genes, A GPCR gene or a protein encoding the gene;
  • a labeling substance-bound membrane-bound TGF ⁇ gene in which a labeling substance is bound to the amino terminus of membrane-bound TGF ⁇ or a protein encoding the gene Introducing a gene comprising a polynucleotide of any one of the following (p-1) to (p-3) or a protein encoding the gene;
  • B a step of culturing the mammalian cell introduced in step (A) in the presence of a test substance and collecting a culture supernatant;
  • C a step of detecting the presence or absence of a labeling substance in the culture supernatant collected in step (B);
  • P-1) a polynucleotide selected from the following [Gq family gene group] and [G12 family gene group];
  • P-2) an amino terminal domain of a protein encoded by a
  • the present invention is also characterized in that (3) the amino terminal domain of the chimeric protein is the amino terminal domain of the protein encoded by any one of the polynucleotides (GNAQ-1) to (GNAQ-3) ( (2) or (3), wherein the method according to 2) or (4) the chimeric protein is selected from any one of the following proteins (q-1) to (q-3): Relates to the method described in.
  • (Q-1) a protein encoded by the nucleotide sequence represented by SEQ ID NO: 95, 97, 103, or 104
  • (Q-2) consisting of a protein encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added in the nucleotide sequence represented by SEQ ID NO: 95, 97, 103 or 104, and GPCR A protein having signal transduction activity between TACE and TACE
  • (Q-3) consists of a protein encoded by a nucleotide sequence having at least 80% identity with the nucleotide sequence represented by SEQ ID NO: 95, 97, 103, or 104, and has signaling activity between GPCR and TACE protein;
  • the present invention also provides (5) the method according to any one of (1) to (4) above, wherein the labeling substance is alkaline phosphatase, and (6) the mammalian cell is a human cell.
  • R-1 a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 117
  • R-2 a protein comprising a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added in the nucleotide sequence represented by SEQ ID NO: 117, and having a TACE cleavage site and membrane-bound activity
  • R-3 a polynucleotide comprising a nucleotide sequence having at least 80% identity to the nucleotide sequence represented by SEQ ID NO: 117 and encoding a protein having a TACE cleavage site and a membrane-binding activity
  • the present invention also includes (8) a mammalian cell in which GNAQ and GNA11 genes or GNA12 and GNA13 genes are knocked out; A test substance and a G protein-coupled receptor (GPCR), comprising: a labeling substance-bound membrane-bound TGF ⁇ gene having a labeling substance bound to the amino terminus of the membrane-bound TGF ⁇ ; or a protein encoding the gene.
  • GPCR G protein-coupled receptor
  • the present invention relates to a kit for detecting signal transduction activation induced by the binding of.
  • the present invention also includes (9) a mammalian cell in which GNAQ and GNA11 genes, and GNA12 and GNA13 genes are knocked out; A labeling substance-bound membrane-bound TGF ⁇ gene in which a labeling substance is bound to the amino terminus of membrane-bound TGF ⁇ or a protein encoding the gene A test substance and a G protein-coupled receptor (GPCR) comprising a gene comprising a polynucleotide of any one of the following (p-1) to (p-3) or a protein encoding the gene: It is related with the kit for detecting the signal transduction activation induced by the coupling
  • GPCR G protein-coupled receptor
  • (P-1) a polynucleotide selected from the following [Gq family gene group] and [G12 family gene group]
  • (P-2) an amino terminal domain of a protein encoded by a polynucleotide selected from the following [Gq family gene group], and the following [G12 family gene group], [Gs family gene group], and [Gi family gene]
  • (P-3) an amino terminal domain of a protein encoded by a polynucleotide selected from the following [G12 family gene group], and the following [Gq family gene group], [Gs family gene group], and [Gi family gene]
  • the amino terminal domain of the chimeric protein is the amino terminal domain of the protein encoded by any one of the polynucleotides (GNAQ-1) to (GNAQ-3) ( (9) or (10), wherein the kit according to 9) or (11) the chimeric protein is selected from the following proteins (q-1) to (q-3): It relates to the kit described in 1.
  • (Q-1) a protein encoded by the nucleotide sequence represented by SEQ ID NO: 95, 97, 103, or 104
  • (Q-2) consisting of a protein encoded by a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added in the nucleotide sequence represented by SEQ ID NO: 95, 97, 103 or 104, and GPCR A protein having signal transduction activity between TACE and TACE
  • (Q-3) consists of a protein encoded by a nucleotide sequence having at least 80% identity with the nucleotide sequence represented by SEQ ID NO: 95, 97, 103, or 104, and has signaling activity between GPCR and TACE protein;
  • the present invention provides (12) the kit according to any one of (8) to (11) above, wherein the labeling substance is alkaline phosphatase, and (13) the mammalian cell is a human cell.
  • the kit according to any one of (8) to (12) above, or (14) the membrane-bound TGF ⁇ gene is derived from any one of the following polynucleotides (r-1) to (r-3): The kit according to any one of (8) to (12) above, which is selected.
  • R-1 a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 117
  • R-2 a protein comprising a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added in the nucleotide sequence represented by SEQ ID NO: 117, and having a TACE cleavage site and membrane-bound activity
  • R-3 a polynucleotide comprising a nucleotide sequence having at least 80% identity to the nucleotide sequence represented by SEQ ID NO: 117 and encoding a protein having a TACE cleavage site and a membrane-binding activity
  • the present detection method 1 it is possible to highly sensitively, specifically and quantitatively detect endogenous Gq or G12 family signaling activation induced by binding of a ligand and its GPCR in a mammalian cell, Since it is not necessary to use exogenous Gq or G12 family protein, it is excellent in convenience.
  • G ⁇ signal transduction activation induced by binding of a ligand and its GPCR can be detected with high sensitivity, specific and quantitative, and signal transduction activation of many G ⁇ families Can be evaluated with a single assay system, so that the signal transduction activation level between different G ⁇ s can be relatively evaluated.
  • FIG. 1A shows a model diagram of membrane-bound alkaline phosphatase (AP) fusion TGF ⁇ (AP-TGF ⁇ ).
  • FIG. 1B shows a model diagram of G protein signal transduction induced by binding between a test substance (ligand) and its receptor (GPCR).
  • FIG. 2A shows a model diagram of G12 family signal transduction induced by binding between a test substance (ligand) and its receptor (GPCR) in a Gq family gene knockout cell line.
  • FIG. 2B shows a model diagram of Gq family signaling induced by binding of a ligand to its GPCR in a G12 family gene knockout cell line.
  • FIG. 1A shows a model diagram of membrane-bound alkaline phosphatase (AP) fusion TGF ⁇ (AP-TGF ⁇ ).
  • FIG. 1B shows a model diagram of G protein signal transduction induced by binding between a test substance (ligand) and its receptor (GPCR).
  • FIG. 2A shows a model diagram of G
  • FIG. 2C shows a model diagram of G ⁇ chimeric protein signaling induced by binding of a ligand to its GPCR in Gq and G12 family gene knockout cell lines expressing G ⁇ chimeric protein.
  • FIG. 3A shows 16 genes of G ⁇ (Gs family genes 2 [GNAS and GNAL genes], Gi family genes 8 [GNAI1, GNAI2, GNAI3, GNAO1, GNAZ, GNAT1, GNAT2, and GNAT3 genes], Gq family genes It is a figure which shows the evolutionary phylogenetic tree of 4 types [GNAQ, GNA11, GNA14, and GNA15 genes] and 2 types of G12 family genes [GNA12 and GNA13 genes]).
  • FIG. 3A shows 16 genes of G ⁇ (Gs family genes 2 [GNAS and GNAL genes], Gi family genes 8 [GNAI1, GNAI2, GNAI3, GNAO1, GNAZ, GNAT1, GNAT2, and GNAT3 genes], Gq family genes It is a figure which shows the evolutionary phylog
  • 3B shows the amino acid sequence at the carboxyl (C) terminus of the protein encoded by the 16 genes of G ⁇ . “-” Indicates that the amino acid is the same as the top protein among the proteins encoded by the genes of each family. It is a figure which shows the result of having analyzed the expression level of mRNA of 4 types of Gq family genes (GNAQ, GNA11, GNA14, and GNA15 genes) and 2 types of G12 family genes (GNA12 and GNA13 genes) in HEK293 cells. The vertical axis represents the relative value to the expression level of mRNA of ⁇ -actin (ACTB) gene (copy number per cell [1000]).
  • ACTB ⁇ -actin
  • HEK293 wild type strain knockout HEK293 cells ( ⁇ GNAQ / 11, ⁇ GNA12 / 13, and ⁇ GNAQ / 11/12 /) of two Gq family genes (GNAQ and GNA11 genes) and / or two G12 family genes (GNA12 and GNA13 genes) 13)
  • TGF alpha
  • G ⁇ chimeric proteins (G ⁇ q / s, G ⁇ 11 / s, G ⁇ 14 / s, and G ⁇ 16 / s) having four Gq family proteins (G ⁇ q, G ⁇ 11, G ⁇ 14, and G ⁇ 16) and two G12 family proteins (G ⁇ 12 and G ⁇ 13) as the backbone ,
  • G ⁇ 12 / s and G ⁇ 13 / s are graphs showing the results of analysis by TGF ⁇ cleavage assay using HEK293 wild-type strains.
  • Gq and G12 family gene knockout cells ⁇ GNAQ / 11 /
  • G ⁇ q family protein G ⁇ q
  • GNAQ and GNA11, and GNA12 and GNA13 genes Four genes (GNAQ and GNA11, and GNA12 and GNA13 genes) in HEK293 cells treated with siRNA targeting the mRNAs of two Gq family genes (GNAQ and GNA11 genes) and two G12 family genes (GNA12 and GNA13 genes) It is a figure which shows the result of having analyzed the expression of mRNA.
  • the vertical axis represents the relative value when the expression level of ACTB gene mRNA is 1.
  • Knockdown HEK293 cells (GNAQ / 11 siRNA, GNA12 / 13 siRNA, and GNAQ / 11/12/13 siRNA of two Gq family genes (GNAQ and GNA11 genes) and / or two G12 family genes (GNA12 and GNA13 genes) It is a figure which shows the result analyzed by the TGF (alpha) cutting
  • Gq family genes Two Gq family genes (GNAQ and GNA11 genes) and G12 expressing G ⁇ chimeric proteins (G ⁇ q / s, G ⁇ q / i1, G ⁇ q / 12, and G ⁇ q / 13) with one Gq family protein (G ⁇ q) as the backbone
  • stock of two family genes (GNA12 and GNA13 gene).
  • the detection method of the present invention includes GNAQ gene and GNA11 gene (hereinafter collectively referred to as “the present Gq family gene”), or GNA12 gene and GNA13 gene (hereinafter collectively referred to as “the present G12 family gene”).
  • the method of detecting the presence or absence of a labeling substance in the collected culture supernatant (c); the steps (a) to (c) in this order (hereinafter referred to as “the present detection method 1”) is particularly limited.
  • the GPCR gene or protein, the labeling substance-bound membrane-bound TGF ⁇ gene or protein, and the above (p-1) to (p-) are added to mammalian cells knocked out of the Gq family gene and the G12 family gene.
  • the cultured mammalian cells are cultured in the presence of a test substance, and the culture supernatant is collected (B); the presence of the labeling substance in the culture supernatant collected in the step (B) Is not particularly limited as long as it is a method comprising the steps (A) to (C) of step (C); (hereinafter referred to as “the present detection method 2”), and the step (a) of the present detection method 1.
  • the detection method 1 activates the signal transduction activation of the endogenous (endogenous) G12 family induced by the binding of the test substance and the GPCR.
  • the detection method 1 It is possible to specifically detect signaling activation of the endogenous Gq family induced by binding of GPCR to GPCR (FIG. 2B).
  • the present detection method 2 can specifically detect the signal transduction activation of the present G ⁇ protein induced by the binding of the test substance and the GPCR (FIG. 2C).
  • the present detection methods 1 and 2 are performed in vitro.
  • the kit of the present invention includes a binding between a test substance (ligand) and a GPCR comprising a mammalian cell knocked out of the Gq family gene or the G12 family gene, and the labeled substance-binding membrane-bound TGF ⁇ gene or protein.
  • this detection kit 1 A kit for detecting signal transduction activation of G protein (hereinafter referred to as “this detection kit 1”), a mammalian cell in which this Gq family gene and this G12 family gene are knocked out, and the above-mentioned label Induced by binding of test substance and GPCR comprising substance-bound membrane-bound TGF ⁇ gene or protein and the present G ⁇ gene or protein comprising the polynucleotide of any one of (p-1) to (p-3) above
  • the present detection kit 2 are signals of G protein induced by the binding between the test substance and the GPCR. It is limited to the use for detecting transmission activation.
  • the present detection kit 1 or the present detection kit 2 preferably further comprises one or more (two or more) GPCR genes of the present invention or a protein encoding the gene, where “multiple (two or more)” , Preferably 5 or more, more preferably 15 or more, still more preferably 50 or more, still more preferably 100 or more.
  • the form of the GPCR gene of the present invention is preferably a gene library in which the GPCR gene of the present invention is inserted into a vector such as a plasmid, cosmid, or BAC (bacterial-artificial-chromosome).
  • the detection kit 1 and the detection kit 2 include components generally used in this type of detection kit, for example, a reagent for detecting the labeled substance of the present invention, a culture solution, and an instruction manual. Documents are usually included.
  • the mammalian cells of the present invention express normal mammalian cells with normal cell cycle checkpoints, mammalian cancer cells with abnormal cell cycle checkpoints, and SV40-derived T antigen in the normal cells.
  • Pluripotent stem cells embryonic stem cells [ES cells], EG cells, iPS cells, etc.
  • mesenchymal stem cells mesenchymal stem cells
  • neural stem cells e.g., a cord blood cells
  • bone marrow stem cells e.g., fibroblasts, etc.
  • Mammalian stem cells such as germ stem cells are included.
  • mammals examples include rodents such as mice, rats, hamsters, and guinea pigs, rabbits such as rabbits, ungulates such as pigs, cows, goats, horses, and sheep, cats such as dogs and cats, humans, Examples of primates such as monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc., among others, humans can be preferably exemplified.
  • Examples of mammalian cells in which the Gq family gene and / or the G12 family gene are knocked out include nucleotides inserted into the Gq family gene and the G12 family gene present on the chromosome of the mammalian cell, or the Gq family gene. Or a mammalian cell in which the present Gq family gene or the present G12 family gene is disrupted by deleting the nucleotide of the present G12 family gene, and may be present on the chromosome of the mammalian cell.
  • a method of destroying the gene by deleting or inserting nucleotides using homologous recombination may be used.
  • Zinc finger nuclease In terms of effectiveness and time-to-effect Zinc finger nuclease (Document “Urnov, FD et al (2010) Nature Review Genetics. 11, 636-646”), a protein improved from the zinc finger nuclease (Japanese Patent Laid-Open No. 2013-94148), guide RNA ( Using sgRNA (single-guide RNA) and Cas9 endonuclease (literature “Cong et al (2013) Science 339, 819-823”) etc., the double-stranded DNA of this Gq family gene and this G12 family gene region is cleaved.
  • a method for destroying a gene using the fact that nucleotide deletion or insertion occurs during homologous recombination repair and in particular, a gene targeting method using sgRNA and Cas9 endonuclease is used. It is more preferable.
  • NCBI http: //www.ncbi.nlm.nih. Gov / guide / linked to the database, and based on the following Gene ID, the base sequence information of this Gq family gene and this G12 family gene derived from humans, or ortholog genes (chimpanzee, mouse, Rat, cow, etc.).
  • GNAQ gene Gene ID 2776
  • GNA11 gene Gene ID 2767
  • GNA12 gene Gene ID 2768
  • GNA13 gene Gene ID 10672
  • the GPCR gene of the present invention is not particularly limited as long as it is a gene encoding a GPCR having a characteristic that a single polypeptide penetrates the cell membrane seven times.
  • the GPCR specifically includes an adrenergic receptor ( ⁇ 1A, ⁇ 1B, ⁇ 2A, ⁇ 2B, ⁇ 1, ⁇ 2, etc.), cysteinyl leukotriene receptor 1 (CysLTR1), GPCR91, GPCR34, EGF-like module-containing mucin-like hormone receptor-like sequence 2 (EMR2), platelet activating factor receptor Body (PAFR), GPCR105, 7th transmembrane superfamily member 1 (TM7SF1), GPCR37, CD97 antigen (CD97), melanocortin 1 receptor (MC1), FIRE, formyl peptide receptor 1 (fMLP1), GPCR65, Purine receptor P2Y8 (P2Y8), -Pamine receptor D2 (D2), endothelial differentiation lysophosphati
  • the membrane-bound TGF ⁇ gene of the present invention has a cleavage domain by TACE (tumor necrosis factor ⁇ -converting enzyme) at the carboxyl (C) terminus of TGF ⁇ , and a membrane-binding domain at the C-terminus of the cleavage domain. It is not particularly limited as long as it is a gene encoding a membrane-bound TGF ⁇ protein, and the membrane-bound TGF ⁇ protein can be used between TGF ⁇ and a TACE cleavage domain, or between a TACE cleavage domain and a membrane-bound domain. A linker (for example, within the range of 1 to 10 amino acid residues) may be inserted.
  • the membrane-bound TGF ⁇ gene of the present invention is preferably selected from the polynucleotides (r-1) to (r-3) described above.
  • the labeling substance-bound membrane-bound TGF ⁇ gene of the present invention is not particularly limited as long as the labeling substance is bound to the amino (N) terminus of the membrane-bound TGF ⁇ of the present invention.
  • the bond between the TGF ⁇ N-terminal and the labeling substance protein fusion is preferable when the labeling substance is a protein, and when the labeling substance is a non-protein (a compound such as a fluorescent dye), a covalent bond, ionic bond, hydrogen Non-covalent bonds such as bonds and disulfide bonds can be mentioned.
  • examples of the labeling substance of the present invention include peroxidase (for example, HRP [horseradish peroxidase]), alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, glucose-6- Phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, enzymes such as green fluorescent protein (Green Fluorescence Protein; GFP), cyan fluorescent protein (Cyan Fluorescence Protein) CFP), Blue Fluorescence Protein (BFP), Yellow Fluorescence Protein (YFP), Red Fluorescence Protein (RF) ), Luciferase (luciferase) can be cited fluorescent proteins such as, it can be preferably exemplified alkaline phosphatase (
  • the labeling substance of the present invention is a non-protein (compound such as a fluorescent dye)
  • examples of the labeling substance of the present invention include fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride, tetramethyl Fluorescent substances such as rhodamine isothiocyanate, radioisotopes such as 3 H, 14 C, 125 I or 131 I, biotin, avidin and the like can be mentioned.
  • the polynucleotide encoding a chimeric protein in the G ⁇ family gene is an N-terminal domain of a protein encoded by a polynucleotide selected from the above [Gq family gene group] (hereinafter sometimes referred to as “Gq family protein”). And a protein encoded by a polynucleotide selected from the above [G12 family gene group], [Gs family gene group], and [Gi family gene group] (hereinafter sometimes referred to as “G ⁇ protein other than Gq family”)
  • G ⁇ protein other than Gq family A polynucleotide encoding a protein that is capable of coupling both TACE and a G ⁇ protein-coupled receptor other than the Gq family, or the above [G12 family gene group].
  • G12 family protein Selected from the N-terminal domain of a protein encoded by renucleotide (hereinafter sometimes referred to as “G12 family protein”) and the above [Gq family gene group], [Gs family gene group], and [Gi family gene group]
  • G ⁇ protein other than G12 family A chimeric protein with a C-terminal domain of a protein encoded by a polynucleotide (hereinafter sometimes referred to as “G ⁇ protein other than G12 family”), and both TACE and a G ⁇ protein-coupled receptor other than G12 family
  • the polynucleotide is not particularly limited as long as it is a polynucleotide encoding a protein capable of coupling (binding).
  • the chimeric protein includes a fusion protein of an N-terminal domain of a Gq family protein and a C-terminal domain of a G ⁇ protein other than the Gq family, an N-terminal domain of a G12 family protein, and a C-terminal domain of a G ⁇ protein other than the G12 family.
  • a protein in which the amino acid residue in the C-terminal domain of the Gq family protein is substituted with an amino acid residue in a G ⁇ protein other than the Gq family corresponding to the amino acid residue is also included.
  • the first to fifth amino acid residues are preferred, and the third and fourth amino acid residues from the C-terminal are important for the function of G ⁇ (Liu, J., et al. Identification of a receptor / G-protein contact site critical for signaling specificity and G-protein activation. Proc Natl Acad Sci U S A 92, 11642-11646 (1995) '', literature ⁇ Conklin, BR et al. Carboxyl-terminal mutations of Gq alpha and Gs alpha that alter the fidelity of receptor activation. Mol Pharmacol 50, 885-890 (1996).), the third amino acid residue and the fourth amino acid residue from the C-terminal can be preferably exemplified.
  • the amino-terminal domain of the protein encoded by any one of the polynucleotides (GNAQ-1) to (GNAQ-3) of the above [Gq family gene group] is preferable.
  • the “protein N-terminal domain” in the chimeric protein of the present invention is preferably a polypeptide domain in which at least 5 to 1 amino acid residues are deleted from the C terminus of the protein, and 6 to 1 amino acid residues from the C terminus of the protein. More preferred are polypeptide domains that are at least deleted.
  • polypeptide domain in which at least 6 to 1 amino acid residues are deleted from the C terminus of the protein examples include, for example, a polypeptide domain in which 50 to 1 amino acid residues are deleted from the C terminus of the protein, A polypeptide domain that lacks 1 amino acid residue, a polypeptide domain that lacks 20-1 amino acid residues from the C-terminus of the protein, a polypeptide domain that lacks 15-1 amino acid residues from the C-terminus of the protein, A polypeptide domain that deletes 10 to 1 amino acid residues from the C-terminus of the protein, a polypeptide domain that deletes 7 to 1 amino acid residues from the C-terminus of the protein, and a 6 to 1 amino acid residue from the C-terminus of the protein
  • the polypeptide domain to be deleted, etc. can be mentioned. 7 to polypeptide domains lacking one amino acid residue can be preferably exemplified.
  • the “protein C-terminal domain” in the chimeric protein of the present invention is preferably a polypeptide domain containing 5 to 1 amino acid residues from the C terminus of the protein, and contains 6 to 1 amino acid residues from the C terminus of the protein. More preferred are polypeptide domains. Examples of the polypeptide domain containing 6 to 1 amino acid residues from the C terminus of the protein include a polypeptide domain comprising 50 to 1 amino acid residues from the C terminus of the protein, and from 25 to 1 amino acid residues from the C terminus of the protein.
  • preferred examples of the chimeric protein of the present invention include those selected from the proteins (q-1) to (q-3) described above.
  • nucleotide sequence represented by SEQ ID NO: 50 (cDNA sequence of human GNAQ gene) or the nucleotide sequence represented by SEQ ID NO: 51 (human GNA11 CDNA sequence of the gene), nucleotide sequence represented by SEQ ID NO: 52 (cDNA sequence of human GNA14 gene), nucleotide sequence represented by SEQ ID NO: 53 (cDNA sequence of human GNA15 gene), nucleotide represented by SEQ ID NO: 54
  • a sequence (cDNA sequence of human GNA12 gene), a nucleotide sequence represented by SEQ ID NO: 55 (cDNA sequence of human GNA13 gene), a nucleotide sequence represented by SEQ ID NO: 118 (cDNA sequence of human GNAS gene), SEQ ID NO: 119 Indicated by Nucleotide sequence (cDNA sequence of human GNAL gene), nucleotide sequence represented by SEQ ID NO: 120
  • GNAQ gene (Gene ID2776) GNA11 gene (Gene ID2767) GNA14 gene (Gene ID9630) GNA15 gene (Gene ID2769) GNA12 gene (Gene ID2768) GNA13 gene (Gene ID10672) GNAS gene (Gene ID2778) GNAL gene (Gene ID2774) GNAI1 gene (Gene ID 2770) GNAI2 gene (Gene ID 2771) GNAI3 gene (Gene ID 2773) GNAO1 gene (Gene ID 2775) GNAZ gene (Gene ID 2781) GNAT1 gene (Gene ID 2779) GNAT2 gene (Gene ID 2780) GNAT3 gene (Gene ID 346562)
  • protein refers to epitope tags such as FLAG, HA, Myc, GST, maltose binding protein, biotinylated peptide, oligohistidine, etc. for confirmation of the presence or absence of protein expression, isolation and purification.
  • An affinity tag may be further added.
  • a “protein” is prepared using any protein synthesis system known to those skilled in the art that expresses mRNA of a gene encoding a protein and is capable of translating the mRNA into a protein. Can be separated and purified.
  • a method for introducing a gene into a mammalian cell is operably linked downstream of the promoter sequence of the mammalian expression vector.
  • the target gene may be inserted and introduced into the cell by an appropriate method according to the type of mammalian expression vector.
  • the mammalian expression vector may be a lentiviral vector, a retroviral vector, an adenoviral vector.
  • a method for introducing a mammalian expression vector into which a target gene has been inserted into a cell a plasmid containing the target gene is introduced into an appropriate packaging cell (such as HEK293T cell).
  • a method of infecting the vector into fibroblasts For example, when using a lentiviral vector as a vector, for a specific method, the method described in the document “Science, 318, 1917-1920 (2007)” can be referred to. The method described in ⁇ WO2007 / 69666 '', ⁇ Cell, 126, 663-676 (2006) '', ⁇ Cell, 131, 861-872 (2007) '' and the like can be referred to, and when using an adenovirus vector, Reference can be made to the method described in the document “Science, 322, 945-949” (2008).
  • the mammalian expression vector is a non-viral vector plasmid vector (pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.)
  • the expression of the mammal into which the gene of interest is inserted examples include lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE (diethylaminoethyl) dextran method, microinjection method, gene gun method and the like.
  • Examples of the promoter sequence of the mammalian expression vector include EF-1 ⁇ promoter, CMV (cytomegalovirus) promoter, SR ⁇ promoter, SV40 promoter, LTR promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus). Examples include LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, CAG promoter and the like.
  • the mammalian expression vector includes an enhancer, terminator sequence ( ⁇ globin terminator sequence, SV40 terminator sequence, BGH terminator sequence, etc.), poly A addition signal, selection marker gene (neomycin resistance gene, puromycin resistance gene, blasticidin S). Resistance genes, etc.), SV40 origin of replication and the like.
  • step (a) of the present detection method 1 and the step (A) of the present detection method 2 as a method for introducing a protein into a mammalian cell, for example, a method using a protein introduction reagent, a protein introduction domain (PTD)-or a cell membrane
  • a method using a protein introduction reagent, a protein introduction domain (PTD)-or a cell membrane examples thereof include a method using a permeation peptide (CPP) -fusion protein, a microinjection method, and the like.
  • CPP permeation peptide
  • Protein introduction reagents include Cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systmes), Pro-JectTM Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX), lipid-based Profect -1 (Targeting Systems), Penetrain Peptide (Q Biogene) and Chariot Kit (Active Motif) based on a membrane-permeable peptide, GenomONE (Ishihara Sangyo) using HVJ envelope (inactivated Sendai virus) Etc.) are commercially available.
  • the test substance binds to the GPCR and the endogenous G12 family or It may be cultured under conditions suitable for culturing mammalian cells for a time sufficient to induce activation of the endogenous Gq family signaling pathway, and in the step (B) of the present detection method 2, As a method for culturing the mammalian cells introduced in step (A) in the presence of the test substance, the test substance binds to the GPCR and the activation of the signal transduction pathway of the G ⁇ protein is induced.
  • the culture conditions may be sufficient for a sufficient period of time for culturing mammalian cells.
  • the culture conditions such as time, temperature, type of culture medium, etc. are appropriately determined in consideration of the characteristics of mammalian cells, test substances and GPCRs. To choose It can be.
  • the culture time is usually within the range of 5 minutes to 24 hours, preferably within the range of 10 minutes to 12 hours, more preferably within the range of 20 minutes to 6 hours, even more preferably within the range of 30 minutes to 3 hours, particularly Preferably, it is within the range of 40 minutes to 2 hours.
  • the culture temperature is usually in the range of 20 to 38 ° C., preferably in the range of 35 to 37 ° C.
  • a basic culture solution for animal cell culture usually containing serum (FBS, CS, etc.) in the range of 0.1 to 30 (v / v)%. F-12, F-10, M-199, etc.).
  • step (b) of the present detection method by culturing the mammalian cells introduced in step (a) in the presence of the test substance, the test substance binds to the GPCR, and the endogenous G12 family or endogenous When the activation of the signal transduction pathway of the sex Gq family is induced, or in the step (B) of the present detection method 2, the mammalian cells introduced in the step (A) are cultured in the presence of the test substance.
  • the endogenous TACE cleaves the labeling substance-bound membrane-bound TGF ⁇ , whereby the labeling substance and TGF ⁇ Is produced and released into the culture supernatant.
  • a culture supernatant containing a conjugate of the released labeling substance and TGF ⁇ is collected.
  • the method for detecting the labeling substance may be detected by any method depending on the type of the detection substance.
  • AP is AP
  • a method of detecting by measuring absorbance (OD 405 ) at a wavelength of 405 nm after adding p-nitrophenyl phosphate (P-NPP), which is a substrate of AP, and reacting is given.
  • the detection substance is luciferase
  • ONPG o-nitrophenol- ⁇ -D-galactopyranoside
  • PRG chlorophenol red ⁇ -D-galactopyranoside
  • X-Gal 5-Bromo- 4-Chloro-3-Indolyl- ⁇ -D-Galactoside
  • the Gq family gene knockout mammalian cell when used in the detection method 1 step (a), when the labeling substance is detected in the detection method 1 step (c), the test substance and the GPCR are detected. It can be determined that the activation of endogenous G12 family signaling or the activation of the endogenous G12 family signaling pathway has been induced by the binding of ), When no labeling substance was detected, the activation of the endogenous G12 family signaling pathway was not detected due to the binding of the test substance and the GPCR, or the activation of the signaling pathway of the endogenous G12 family. It can be determined that it was not induced.
  • step (a) of the present detection method 1 when a labeling substance is detected in the step (c) of the present detection method 1, a test substance, a GPCR, It can be determined that the activation of endogenous Gq family signal transduction or the activation of the endogenous Gq family signal transduction pathway is induced by the binding, and step (c) of the present detection method 1 (c) ), When no labeling substance was detected, the activation of the endogenous Gq family signaling pathway was not detected due to the binding between the test substance and the GPCR, or the signaling pathway of the endogenous Gq family was not activated. It can be determined that it was not induced.
  • step (C) of the present detection method 2 When a labeling substance is detected in step (C) of the present detection method 2, signal transduction activation of the G ⁇ family protein is detected by binding of the test substance and GPCR, or the signal of the G ⁇ family protein It can be determined that the activation of the transmission pathway has been induced, and if no labeling substance is detected in step (C) of the detection method 2, the G ⁇ family is detected by the binding of the test substance and the GPCR. It can be determined that the signal transduction activation of the protein was not detected or the activation of the signal transduction pathway of the G ⁇ family protein was not induced.
  • the “Gs-coupled receptor” is a Gs family (protein encoded by GNAS or GNAL gene) such as prostaglandin E receptor (EP2), adrenaline ⁇ 1 or ⁇ 2, histamine H2 receptor (H2R). It means a receptor that is conjugated (bound) to.
  • Gi-coupled receptor refers to a Gi family such as histamine H3 receptor (H3R), adrenergic ⁇ 2A or ⁇ 2B receptor, serotonin (5-HT) receptor (GNAI1, GNAI2, GNAI3, It means a receptor that is coupled (coupled) to a protein encoded by GNAO1, GNAZ, GNAT1, GNAT2, or GNAT3 gene.
  • detecting signal transduction activation means detecting the presence or absence of signal transduction activation or detecting the degree of signal transduction activation.
  • identity of at least 80% means that the identity is 80% or more, preferably 85% or more, more preferably 88% or more, still more preferably 90% or more, More preferably, it means 93% or more, particularly preferably 95% or more, and most preferably 98% or more.
  • nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added is, for example, in the range of 1 to 30, preferably in the range of 1 to 20, and more preferably Within the range of 1-15, more preferably within the range of 1-10, more preferably within the range of 1-5, even more preferably within the range of 1-3, more preferably within the range of 1-2. It means a nucleotide sequence in which a number of nucleotides are deleted, substituted or added.
  • a polynucleotide consisting of a nucleotide sequence in which one or several nucleotides are deleted, substituted and / or added (mutant polynucleotide) may be any one known to those skilled in the art such as chemical synthesis, genetic engineering techniques, mutagenesis, etc. It can be produced by a method.
  • HEK293 cells (293A Cell, manufactured by Life Technologies) were collected at 1 ⁇ 10 6 cells, and RNA was extracted using Mammalian Total RNA Miniprep Kit (manufactured by Sigma-Aldrich). CDNA synthesis was performed from the extracted RNA using a High Capacity cDNA Reverse Transcription Kit (manufactured by Applied Biosystems).
  • the G ⁇ gene encoding the ⁇ subunit constituting the trimeric ( ⁇ ) G protein is composed of four types of families (Gs, Gi, Gq, and G12), and among these, the Gs family gene is The gene is composed of two kinds of genes (GNAS and GNAL genes), and the Gi family gene is composed of eight kinds of genes (GNAI1, GNAI2, GNAI3, GNAO1, GNAZ, GNAT1, GNAT2, and GNAT3 genes).
  • the Gq family gene is composed of four types of genes (GNAQ, GNA11, GNA14, and GNA15 genes), and the G12 family gene is composed of two types of genes (GNA12 and GNA13 genes). (FIG. 3).
  • mRNA expression levels of 6 types of G ⁇ genes (4 types of Gq family genes [GNAQ, GNA11, GNA14, and GNA15 genes] and 2 types of G12 family genes [GNA12 and GNA13 genes]) are used for internal control.
  • a primer set (contracted synthesis by Fasmac) and SYBR Premix Ex Taq Kit (manufactured by Takara Bio Inc.) were used, and a 7300 real-time PCR system ( PCR was performed by Applied Biosystems), and amplification and quantification of cDNA fragments of the above six types of G ⁇ gene and ACTB gene were performed.
  • the quantification was performed based on a calibration curve prepared using plasmid vector DNA with known concentrations into which the above six types of G ⁇ gene and ACTB gene were incorporated.
  • PCR reaction conditions were as follows: initial denaturation 95 ° C. (30 seconds) followed by 40 cycles of 95 ° C. (5 seconds) and 60 ° C. (31 seconds), and a melting curve of the reaction product was obtained at the dissociation stage. Analyzed. It should be noted that, by analyzing melting curves, peak temperatures of melting curves of the above 6 types of G ⁇ gene and ACTB gene derived from HEK293 cells, and DNA amplification products of the above 6 types of G ⁇ gene and ACTB gene derived from plasmid DNA. It was confirmed that the peak temperature of the melting curve of the same coincided.
  • Table 1 below shows primer sets for amplifying the cDNA fragments of the above six types of G ⁇ gene and ACTB gene.
  • sgRNA having a 20 base-long complementary sequence (sgRNA binding sequence) of a G ⁇ gene (target gene) existing in the genome and Cas9 nuclease are expressed in cultured cells, and a complex of sgRNA and Cas9 nuclease is targeted. It binds to the sgRNA binding sequence present in the gene, and double-strand breaks of the target gene occur at such sites, and base deletion and insertion occur in the process of joining the double-strand break ends to each other, resulting in loss of target gene function
  • NCBI National Center for Biotechnology Information
  • NM_002072 and GNA11 [Accession No. NM_002067] genes)
  • GNA11 As a binding sequence of sgRNA targeting two types of G12 family genes [GNA12 [Accession No. NM_007353] and GNA13 [Accession No. NM_006572]] genes), DNAs comprising nucleotide sequences represented by SEQ ID NOs: 15 to 18, respectively. Selected.
  • oligonucleotide set consisting of base sequences shown by SEQ ID NOs: 19 and 20, an oligonucleotide consisting of base sequences shown by SEQ ID NOs: 21 and 22, and bases shown by SEQ ID NOs: 23 and 24
  • the oligonucleotide consisting of the sequence and the oligonucleotide consisting of the base sequences shown in SEQ ID NOs: 25 and 26 were mixed, respectively, and a gradient from 95 ° C. (5 minutes) / 95 ° C. to 30 ° C.
  • the pX330 vector in which both Cas9 nuclease and sgRNA targeting the four types of G ⁇ genes are expressed is hereinafter referred to as “Cas9 nuclease / sgRNA expression vector”.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS 10% fetal calf serum
  • HEK293 cells were transfected with 100 ng of pGreenLantern-1 (GIBCO BRL) using 1.25 ⁇ L of LipofectAMINE 2000 (Life Technologies). Cells 24 hours after transfection were detached using 0.05% (v / v) trypsin / 0.53 mM EDTA-containing phosphate buffer (PBS), and GFP positive using a cell sorter SH800Z (Sony).
  • the cells into which the Cas9 nuclease / sgRNA expression vector was introduced were selected by sorting the cells. Such cells were resuspended in a culture solution so as to be 20 cells / mL or 80 cells / mL, and 100 ⁇ L was seeded in each well of a 96-well plate for cell culture (Grenier Bio-One). After culturing for about 2 weeks in the presence of 5% CO 2 , the cells in the wells where cell colonies appeared were detached using trypsin / EDTA, and half of the cells were seeded in a 6-well plate for cell culture (Grenier Bio-One). The remaining half of the cells were used for genotype analysis.
  • the genotype analysis was performed according to the method described in the following [G ⁇ gene genotype analysis], and mutations were introduced into four types of G ⁇ genes (GNAQ, GNA11, GNA12 and GNA13 genes) existing in the genome. After confirming the above, cell clones were cultured until they reached a semi-confluent state in a 6-well plate, detached from the culture vessel using trypsin / EDTA, and half of the cells were seeded in a 100 mm dish for cell culture (Grenier Bio-One).
  • the remaining half amount was seeded in a 12-well plate, and the proteins encoded by the four types of G ⁇ genes mutated by the TGF ⁇ cleavage assay (G ⁇ q [protein encoded by GNAQ gene], G ⁇ 11 [protein encoded by GNA11 gene], After evaluating the function of G ⁇ 12 [protein encoded by GNA12 gene] and G ⁇ 13 [protein encoded by GNA13 gene]) and confirming the functional deficiency of G ⁇ protein (G ⁇ gene knockout), the cell clone was analyzed in a 100 mm dish.
  • the cells were cultured until they reached confluence, detached from the culture vessel using trypsin / EDTA, and stored frozen in CELLBANKER 1 plus (Nippon Zenyaku Kogyo Co., Ltd.). Further, a Cas9 nuclease / sgRNA expression vector expressing sgRNA targeting the GNAQ gene and a Cas9 nuclease / sgRNA expression vector expressing sgRNA targeting the GNA11 gene are mixed and transfected into parent cells (HEK293 cells). As a result, a double deficient ( ⁇ GNAQ / 11) cell line of G ⁇ q and G ⁇ 11 was isolated.
  • a Cas9 nuclease / sgRNA expression vector expressing sgRNA targeting the GNA12 gene and a Cas9 nuclease / sgRNA expression vector expressing sgRNA targeting the GNA13 gene are mixed and transfected into parent cells.
  • a double deficient ( ⁇ GNA12 / 13) cell line of ⁇ 12 and G ⁇ 13 was isolated.
  • a Cas9 nuclease / sgRNA expression vector expressing sgRNA targeting the GNA12 gene and a Cas9 nuclease / sgRNA expression vector expressing sgRNA targeting the GNA13 gene are mixed and transfected into the ⁇ GNAQ / 11 strain. Isolated a four-deficient ( ⁇ GNAQ / 11/12/13) cell line of G ⁇ q, G ⁇ 11, G ⁇ 12 and G ⁇ 13.
  • G ⁇ protein-deficient cell line was treated with 50 mM aqueous sodium hydroxide solution for 30 minutes, and neutralized by adding Tris-HCl (pH 7.4).
  • PCR was performed using ExTaq (manufactured by Takara Bio Inc.) and a primer set (commissioned synthesis by Fasmac) to amplify DNA fragments of the four types of mutated G ⁇ genes (GNAQ, GNA11, GNA12 and GNA13 genes).
  • the obtained PCR reaction product was digested with restriction enzymes XbaI (GNAQ gene), TaqI (GNA11 and GNA13 genes), HindIII (GNA12 gene) (restriction enzymes were purchased from Takara Bio Inc.), and then 3% agarol gel (Nippon Gene) And the restriction enzyme recognition sites were confirmed to be present in the four types of G ⁇ genes.
  • Table 2 below shows primer sets for amplifying DNA fragments of the above four types of G ⁇ genes.
  • the DNA fragments of the above four types of G ⁇ genes amplified by PCR were incorporated into T-Vector pMD20 vector (Takara Bio) by TA cloning method, introduced into competent E. coli SCSI (manufactured by Agilent Technologies) and transformed. .
  • the transformed Escherichia coli is subjected to drug selection on an LB medium plate containing 100 ⁇ g / mL ampicillin, and using the selected Escherichia coli colony as a template, DNA fragments of the above four types of G ⁇ genes inserted into the T-Vector pMD20 vector are amplified.
  • PCR using a primer set (primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 36 and 37) was performed. It was confirmed by agarol gel electrophoresis that the PCR product was obtained, and the PCR product was used as a template by a dye terminator method using a sequencing primer (primer consisting of the base sequence represented by SEQ ID NO: 36) (consigned to Fasmac) The genotypes (base sequences) of the four types of G ⁇ genes were determined.
  • G ⁇ protein expression vector 1 To clone 6 types of G ⁇ genes (4 Gq family genes [GNAQ, GNA11, GNA14, and GNA15 genes], and 2 G12 family genes [GNA12 and GNA13 genes]), FirstChoice Human Total RNA Survey Panel (Applied Biosystems) as a template, cDNA synthesis using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and using the synthesized cDNA as a template, primer set (consigned synthesis by Fasmac) and polymerase PrimeSTAR HS (PCR was performed using Takara Bio.
  • the primer sets for amplifying the cDNAs of the above six types of G ⁇ genes are shown in Table 3 below.
  • PCR amplification products of the above 6 types of G ⁇ genes obtained by PCR were run on a 1% agarol gel, purified using Wizard ⁇ ⁇ SV Gel and PCR Clean-Up System (Promega), and treated with restriction enzymes KpnI and XhoI. (GNAQ, GNA11, GNA14, GNA15, and GNA12 genes) or restriction enzymes KpnI and EcoRV (GNA13) were treated (restriction enzymes were purchased from Takara Bio Inc.).
  • PCAGGS-MCS vector for mammalian cell expression (literature “Sakagami, H. et al.
  • the transformed E. coli is drug-selected on a 100 ⁇ g / mL ampicillin-containing LB medium plate, the selected E. coli colonies are isolated and grown in a 100 ⁇ g / mL ampicillin-containing LB medium, and then the vector DNA is NucleoBond Xtra Midi Plus Purification was performed using Kit (manufactured by Macherey-Nagle).
  • the purified vector DNA contained the full length cDNAs of the above six types of G ⁇ genes (consigned to Fasmac).
  • the cDNAs of the above six types of G ⁇ genes are DNAs having the nucleotide sequences represented by SEQ ID NOs: 50 to 53 and 54 and 55, respectively.
  • G ⁇ protein expression vector 2 A vector expressing the G ⁇ chimeric protein was produced according to the following procedure. First, the pCAGGS-MCS vector was treated with restriction enzymes EcoRI and XhoI (Takara Bio), electrophoresed on a 1% agarol gel, and purified using Wizard SV Gel and PCR Clean-Up System (Promega).
  • G ⁇ genes 2 Gs family genes [GNAS and GNAL genes], 4 Gi family genes [GNAI1, GNAI3, GNAO1, and GNAZ genes], 3 Gq family genes [GNAQ, GNA14, and GNA15 genes], And carboxyl (C) of proteins encoded by two types of G12 family genes [GNA12 and GNA13 genes] (G ⁇ s and G ⁇ olf, G ⁇ i1, G ⁇ i3, G ⁇ o, and G ⁇ z, G ⁇ q, G ⁇ 14, and G ⁇ 16, and G ⁇ 12 and G ⁇ 13, respectively)
  • a DNA fragment containing DNA encoding a peptide fragment consisting of 7 to 1 amino acid residues from the end (hereinafter referred to as “the C-terminal peptide fragment of 11 types of G ⁇ protein”) was prepared, and 10 pmol of the DNA fragment and the above restriction fermentation
  • the treated and pCAGGS-MCS vector 20ng mixed, subjected to ligase treatment with Mighty Mix (Takara Bio) were introduced and transformed
  • an oligonucleotide consisting of the base sequence shown in SEQ ID NOs: 62 and 63 in the case of the GNAO1 gene, an oligonucleotide consisting of the base sequence shown in SEQ ID NOs: 64 and 65; GNAZ gene
  • an oligonucleotide consisting of the base sequence shown by SEQ ID NO: 68 and 69 in the case of GNAQ gene an oligonucleotide consisting of the base sequence shown by SEQ ID NO: 70 and 71
  • the transformed E. coli is drug-selected on a 100 ⁇ g / mL ampicillin-containing LB medium plate, the selected E. coli colonies are isolated and grown in a 100 ⁇ g / mL ampicillin-containing LB culture solution, and then the vector DNA contained in the E. coli is transformed. It was isolated and purified using GenElute Plasmid Miniprep Kit (manufactured by Sigma-Aldrich).
  • the purified vector DNA contained DNA fragments encoding the C-terminal peptide fragments of the above 11 types of G ⁇ proteins. That is, a DNA fragment containing a DNA encoding a C-terminal peptide fragment of the above 11 types of G ⁇ protein is a restriction enzyme PvuII recognition sequence and a DNA encoding a C-terminal peptide fragment of the above 11 types of G ⁇ protein (including a stop codon).
  • the DNAs encoding the C-terminal peptide fragments of the above 11 types of G ⁇ proteins were DNAs having the base sequences represented by SEQ ID NOs: 78 to 88, respectively.
  • a pCAGGS-MCS vector containing DNA fragments encoding the C-terminal peptide fragments of the above 11 types of G ⁇ proteins was treated with restriction enzymes KpnI and PvuII (manufactured by Takara Bio Inc.), migrated with 1% agarol gel, and then restricted.
  • the fragment cleaved with the enzyme was purified using Wizard SV-Gel and PCR Clean-Up System (Promega).
  • pCAGGS-MCS containing cDNAs of six types of G ⁇ genes prepared by the method described in the above item [Preparation of G ⁇ protein expression vector 1] is used as a template.
  • Primer set for amplifying DNA encoding a peptide fragment lacking the C-terminal peptide fragment of the protein encoded by these 6 types of G ⁇ genes (hereinafter referred to as “6 types of N-terminal peptide fragments of G ⁇ protein”) PCR was performed using polymerase PrimeSTAR HS (Takara Bio Inc.).
  • Table 4 below shows primer sets for amplifying DNA encoding the N-terminal peptide fragments of the above six types of G ⁇ proteins.
  • DNA amplification products encoding the N-terminal peptide fragments of the above 6 types of G ⁇ proteins obtained by PCR were run on a 1% agarol gel, and then purified using WizardWSV Gel and PCR Clean-Up System (manufactured by Promega). Then, restriction enzyme KpnI (manufactured by Takara Bio Inc.) was treated. 100 ng of the DNA amplification product treated with the restriction enzyme and 50 ng of pCAGGS-MCS vector containing DNA fragments encoding the C-terminal peptide fragments of the 11 types of G ⁇ proteins treated with the restriction enzymes were mixed, and Mighty Mix (manufactured by Takara Bio Inc.) was mixed.
  • E. coli SCSI manufactured by Agilent Technologies
  • the transformed E. coli is drug-selected on a 100 ⁇ g / mL ampicillin-containing LB medium plate, the selected E. coli colonies are isolated and grown in a 100 ⁇ g / mL ampicillin-containing LB medium, and then the vector DNA is NucleoBond Xtra Midi Plus Purification was performed using Kit (manufactured by Macherey-Nagle).
  • the purified vector DNA contains DNA encoding a chimeric protein (G ⁇ chimeric protein) of the N types of the G-proteins and the C-terminal peptides of the 11 types of G ⁇ proteins, Confirmed by the dye terminator method (consigned to Fasmac).
  • the G ⁇ chimeric protein is specifically a fusion protein (G ⁇ q / s) of an N-terminal peptide fragment of G ⁇ q and a C-terminal peptide fragment of G ⁇ s, an N-terminal peptide fragment of G ⁇ q, and a C-terminal peptide of G ⁇ olf.
  • G ⁇ protein expression vector 3 Production of a G ⁇ q (siRNA resistant G ⁇ q) expression vector in which a silent silent mutation is introduced to siRNA targeting the mRNA of the wild-type GNAQ gene, and a G ⁇ chimeric protein expression vector based on siRNA resistant G ⁇ q. The following procedure was followed. First, using pCAGGS-MCS containing GNAQ cDNA prepared by the method described in the above item [G ⁇ protein expression vector preparation 1] as a template, two types of primer sets (from the nucleotide sequences represented by SEQ ID NOs: 38 and 111).
  • PCR was performed using a primer set consisting of the nucleotide sequence shown in SEQ ID NOS: 39 and 110) and polymerase PrimeSTAR HS (manufactured by Takara Bio Inc.). Two types of PCR products obtained using two types of primer sets were run on a 1% agarol gel, purified using Wizard SV Gel and PCR Clean-Up System (Promega), mixed, and then PCR was performed using two kinds of PCR products as a template and a primer set (primer set consisting of the nucleotide sequences shown in SEQ ID NOs: 38 and 39), and the PCR product was run on a 1% agarol gel, then Wizard SV Gel and PCR The product was purified using Clean-Up System (Promega) and treated with restriction enzymes KpnI and XhoI (Takara Bio).
  • the purified vector DNA contained DNA encoding siRNA-resistant G ⁇ q.
  • the DNA encoding siRNA resistance G ⁇ q is a DNA consisting of the base sequence represented by SEQ ID NO: 112.
  • TGF ⁇ cleavage assay Alkaline phosphatase (AP) fusion TGF ⁇ (AP-TGF ⁇ ) and 10 G protein coupled receptors (GPCR) (3 prostaglandin E receptors [EP1, EP2, and EP3], histamine H3 receptor [H3R] , Adrenergic ⁇ 1A receptor [ ⁇ 1A], thromboxane receptor [TP], type A endothelin receptor [ETA], type I angiotensin II receptor [AT1], prostaglandin F receptor [FP], and cannabinoid receptor 1 [CB1])
  • GPCR G protein coupled receptors
  • Vectors were obtained or created.
  • the cells are suspended in the present culture solution to 2 ⁇ 10 5 cells / mL, and 1 mL is seeded in each well of a 12-well plate for cell culture (manufactured by Greiner Bio-One), in the presence of 5% CO 2 .
  • 250 ng of AP-TGF ⁇ expression vector and 100 ng of the above 10 types of GPCR expression vector, or 250 ng of AP-TGF ⁇ expression vector, 100 ng of the above 10 types of GPCR expression vector, and G ⁇ protein expression are added to each well.
  • 50 ng of the vector was transfected into the cells using 1.25 ⁇ L of LipofectAMINE 2000 (Life Technologies).
  • AP-TGF ⁇ is a protein in which placenta AP (protein encoded by ALPP gene) is fused to the N-terminal side of membrane-bound pro-TGF ⁇ (protein encoded by TGFA gene).
  • placenta AP protein encoded by ALPP gene
  • pro-TGF ⁇ protein encoded by TGFA gene
  • Test compound at each concentration prostaglandin E2 [Prostaglandin E2], histamine, noradrenaline, U-46619, Endothelin-1), CP-55940, Angiotensin II, or Angiotensin II analog [Sar (1) -Ile (4 ) -Ile (8) AngII; SII] added) by 10 [mu] L, it was further allowed to stand for one hour in the presence of 5% CO 2.
  • a 96-well plate was centrifuged (190 ⁇ g, 2 minutes, room temperature), and 80 ⁇ L of 100 ⁇ L of the culture supernatant was transferred to another 96-well plate using a 12-channel multi-channel electric pipettor eLine (Sartorius).
  • OD 405 was measured again (OD 405 after reaction).
  • the OD 405 after the reaction in the cell plate is reduced by the OD 405 (background) before the reaction in the cell plate to calculate “OD 405 of the cell”, and the OD 405 after the reaction in the culture supernatant plate is The “supernatant OD 405 ” is calculated by subtracting the OD 405 (background) before the reaction in the culture supernatant plate, and the values of the “cell OD 405 ” and the “supernatant OD 405 ” are AP activity (%) was calculated by entering into the equation. The value obtained at this time is referred to as “stimulated AP activity value”.
  • HEK293 cells were suspended in this culture solution so as to be 1 ⁇ 10 5 cells / mL, and 1 mL was seeded in each well of a 12-well plate. After culturing for 24 hours in the presence of 5% CO 2 , 12 pmol (final concentration of siRNA (Stealth siRNA [manufactured by Life Technologies)] targeting 4 kinds of G ⁇ genes (GNAQ and GNA11, and GNA12 and GNA13) is added to each well. 10 nM) was transfected into cells using 1 ⁇ L of LipofectAMINER RNAiMAX (Life Technologies).
  • the DNAs targeted by the siRNA targeting the four types of G ⁇ genes are DNAs consisting of the nucleotide sequences represented by SEQ ID NOs: 113 and 114, and 115 and 116, respectively. is there. After culturing for 24 hours in the presence of 5% CO 2 , the culture solution was removed, and 1 mL of new main culture solution was added to each well.
  • the mRNA expression levels of Gq family gene and G12 family gene in HEK293 cells are determined according to the method described in the above section [G ⁇ gene mRNA expression analysis]. Analyzed. The result is shown in FIG.
  • TGF ⁇ cleavage assay using knockout cell lines of Gq and G12 family genes is an assay system that can specifically detect both the Gq family signaling pathway and the G12 family signaling pathway, HEK293 wild-type strain (Parent) expressing exogenous GPCR (EP2, EP3, ⁇ 1A, EP3, TP, and ETA) and five test compounds (prostaglandin E2, histamine, noradrenaline, U-46619, and endothelin) Using the ligand of -1), a TGF ⁇ cleavage assay was performed according to the method described in the above item [TGF ⁇ cleavage assay]. The result is shown in FIG.
  • TGF ⁇ cleavage assay using a knockout cell ( ⁇ GNAQ / 11, ⁇ GNA12 / 13, and ⁇ GNAQ / 11/12/13) strain of Gq or G12 family gene, a TGF ⁇ cleavage assay according to the method described in the above item [TGF ⁇ cleavage assay]. It was examined whether or not Gq family signaling activation and G12 family signaling activation could be detected separately. The result is shown in FIG.
  • HEK293 AP activation was detected as in the case of using the wild type strain, whereas TP and ETA receptor-expressing Gq and G12 family gene knockout cells ( ⁇ GNAQ / 11/12/13) strains, When stimulated with -46619 and endothelin-1 (" ⁇ ⁇ GNAQ / 11/12/13" in FIGS. 5E and F) , Activation level of AP was detected. This result supports the results in FIGS.
  • Gs and Gi family proteins can also be detected by performing a TGF ⁇ cleavage assay using a G ⁇ or G12 family protein and a G ⁇ chimeric protein fused with Gs or Gi. .
  • a TGF ⁇ cleavage assay using a G ⁇ or G12 family protein and a G ⁇ chimeric protein fused with Gs or Gi.
  • an N-terminal peptide fragment of four Gq family proteins (G ⁇ q, G ⁇ 11, G ⁇ 14, and G ⁇ 16) and a C-terminal peptide fragment of one Gs family protein (G ⁇ s) were fused.
  • G ⁇ chimeric proteins (G ⁇ q / s, G ⁇ 11 / s, G ⁇ 14 / s, and G ⁇ 16 / s), two G12 family proteins (G ⁇ 12 and G ⁇ 13), and a C-terminal peptide fragment of G ⁇ s, respectively.
  • HEK293 cells expressing the fused G ⁇ chimeric protein (G ⁇ 12 / s and G ⁇ 13 / s)
  • TGF ⁇ cleavage assay was performed according to the method described in the above item [TGF ⁇ cleavage assay]. The result is shown in FIG.
  • G ⁇ q As a control, six types of G ⁇ proteins (G ⁇ q, G ⁇ 11, G ⁇ 14, and G ⁇ 16, and G ⁇ 12 and G ⁇ 13) were further expressed in HEK293 cells expressing EP2 (Gs-coupled) receptor, and prostaglandin E2 (EP2 receptor) was expressed.
  • EP2 Gs-coupled
  • EP2 receptor prostaglandin E2
  • a ⁇ GNAQ / 11/12/13 strain expressing a G ⁇ chimeric protein having a Gq family protein as a backbone and fused with a Gs, Gi, or G12 family protein is used as described in the above item of [TGF ⁇ cleavage assay].
  • TGF ⁇ cleavage assay When a TGF ⁇ cleavage assay is performed according to the method, it is clarified which of the four types of G ⁇ family proteins (Gs, Gi, Gq, and G12) can be activated by the target ligand. It was examined whether the activation levels of the four types of G ⁇ proteins by the ligands can be evaluated relatively. Note that G ⁇ q that can be detected with the highest sensitivity in the results of FIG. 6 was used as the Gq protein used for the backbone of the G ⁇ chimeric protein. The result is shown in FIG.
  • AP activation is detected. It was done.
  • the detected activation level of AP was quantified using the Emax / EC50 value obtained from the dose response curve, it was possible to induce ligand stimulation (AP activation) at the lowest prostaglandin F2 ⁇ concentration.
  • G ⁇ q / s was expressed, and hereinafter, the prostaglandin F ⁇ concentration capable of inducing ligand stimulation in the order of G ⁇ q, G ⁇ q / 13, G ⁇ q / 12, and G ⁇ q / i1 was low. This result indicates that when prostaglandin F2 ⁇ binds to the FP receptor, G ⁇ s signal transduction is activated at the highest level, and thereafter G ⁇ q, G ⁇ 13, G ⁇ 12, and G ⁇ i1 increase in this order.
  • AT1 receptor-expressing ⁇ GNAQ / 11/12/13 strain was stimulated with angiotensin II (AT1 receptor ligand) (“ ⁇ No G ⁇ ” in FIG. 7D), AP activation was not detected.
  • AT ⁇ receptor-expressing ⁇ GNAQ / 11/12/13 strain is further allowed to express G ⁇ q and four types of G ⁇ chimeric proteins (G ⁇ q / s, G ⁇ q / i1, G ⁇ q / 12, and G ⁇ q / 13), and angiotensin When stimulated with II (“G ⁇ q” in FIG.
  • TGF ⁇ cleavage assay is performed using Gq and G12 family gene knockout cell lines expressing G ⁇ chimeric protein fused with Gs, Gi, or G12 protein using Gq family protein or G12 family protein as the backbone. And revealing the highest level of signal transduction of four types of G ⁇ family (Gs, Gi, Gq, and G12) proteins activated by the binding of a ligand to its receptor. It was shown that the activation level of signal transduction can be relatively evaluated among the four types of G ⁇ family proteins.
  • Gq family gene knocked down cell (GNAQ / 11 siRNA) strain G12 family gene knocked down cell (GNA12 / 13 siRNA) strain, Gq and G12 family gene knocked down cell (GNAQ / 11/12 / 13 siRNA) strains were prepared, and these cell lines were used to perform a TGF ⁇ cleavage assay according to the method described in the above item [TGF ⁇ cleavage assay]. The result is shown in FIG.
  • G ⁇ q and three types of G ⁇ chimeric proteins were further expressed in the TP receptor-expressing GNAQ / 11/12/13 siRNA strain and stimulated with U-46619.
  • G ⁇ q the activation level of AP increased
  • the control “ ⁇ No G ⁇ ” in FIG. 10A)
  • FIG. 10C the case of using the EP1 receptor-expressing ⁇ GNAQ / 11/12/13 strain
  • G ⁇ q and four types of G ⁇ chimeric proteins are further expressed in the FP receptor-expressing GNAQ / 11/12/13 siRNA strain, and prostagland When stimulated with gin F2 ⁇ (a ligand of FP receptor) (“G ⁇ q”, “G ⁇ q / s”, “G ⁇ q / i”, “G ⁇ q / 12”, and “G ⁇ q / 12” in FIG.
  • the TGF ⁇ cleavage assay using Gq and G12 family gene knockdown cell lines cannot detect G ⁇ signaling activation induced by the binding of the ligand and its receptor with high sensitivity.
  • the TGF ⁇ cleavage assay of the present invention using Gq and G12 family gene knockout cell lines is a method with excellent sensitivity and specificity.
  • the present invention can detect signal transduction activation of a large number of G ⁇ s induced by a ligand and its GPCR simply, highly sensitively, specifically and quantitatively, it specifically activates signal transduction activation of individual G ⁇ s. It is useful for screening a target drug of GPCR (biased agonist).
  • GPCR target drugs biassed agonists
  • GPCR target drugs that can specifically activate signal transduction activation of individual G ⁇ s are specific to endogenous ligands and a plurality of physiological activities induced by the GPCR. Therefore, according to the present invention, it is expected that a target drug (biased agonist) for GPCR with few side effects can be screened more easily.
  • a target drug (antagonist) of GPCR capable of specifically inhibiting the signal transduction activation of individual G ⁇ may specifically inhibit a single one of a plurality of physiological activities induced by the ligand and the GPCR. Therefore, according to the present invention, it is expected that a target drug (antagonist) of GPCR with few side effects can be screened more easily. Moreover, since the signal transduction activation of G12 and G13 can be detected with high sensitivity and specificity by using the ⁇ GNAQ / 11 cell line of the present invention, the target drug for GPCR conjugated with G12 or G13 can be accurately evaluated. In addition to the expansion of GPCR targets that are targets for drug discovery, it is expected that the drug efficacy and side effects through the signal transduction pathways of the GPCR target drugs G12 and G13 can be accurately predicted.

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Abstract

　La présente invention aborde le problème consistant à [1] la fourniture d'un procédé par lequel l'activation de la transduction de signal endogène de la famille Gq ou G12 induite par la liaison d'une substance d'essai (ligand) et d'un récepteur couplé à une protéine G peut être spécifiquement détecté avec une sensibilité élevée dans une cellule de mammifère, ou [2] la fourniture d'un procédé par lequel l'activation de quatre types (Gs, Gi, Gq, et G12) de la transduction de signal de la famille Gα induite par la liaison d'un ligand et d'un GPCR peut être spécifiquement détecté avec une sensibilité élevée dans une cellule de mammifère. Dans la présente invention, l'activation de la transduction de signal de la famille Gq induite par la liaison d'un ligand et le GPCR de ce dernier est spécifiquement détectée par dosage de l'élimination de TGFα à l'aide de lignées de cellules inactivées (∆ GNAQ /11) pour deux types de gènes et gènes (gènes GNAQ et GNA11) de la famille Gq. L'activation de la transduction de signal de la famille G12 induite par la liaison d'un ligand et le GPCR de ce dernier est également détectée spécifiquement par dosage d'élimination de TGFα à l'aide de lignées de cellules inactivées (∆ GNA12 /13) pour deux types de gènes (gènes GNA12 et GNA13) de la famille G12. Une protéine Gα chimère sur la base d'une structure de la famille Gq ou G12 est également amené à être exprimée dans les lignées de cellules inactivées (∆ GNAQ/11/12/13) pour la deux types de gènes mentionnés ci-dessus de la famille Gq et la deux types de gènes mentionnés ci-dessus de la famille G12, et l'activation de quatre types (Gs, Gi, Gq, et G12) de la transduction de signal de la famille Gα induite par la liaison d'un ligand et le GPCR de ce dernier est également détectée spécifiquement par dosage d'élimination de TGFα.
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JPWO2020026979A1 (ja) * 2018-07-31 2021-08-02 国立大学法人 東京大学 膜タンパク質活性測定法
JP7367974B2 (ja) 2018-07-31 2023-10-24 国立大学法人 東京大学 膜タンパク質活性測定法
WO2020050208A1 (fr) 2018-09-05 2020-03-12 ヤマサ醤油株式会社 Procédé et kit de mesure rapide de l'activité d'un auto-anticorps par rapport au récepteur tsh
JPWO2020050208A1 (ja) * 2018-09-05 2021-09-16 ヤマサ醤油株式会社 Ｔｓｈ受容体に対する自己抗体活性の測定方法及びキット
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WO2022065354A1 (fr) 2020-09-23 2022-03-31 国立大学法人京都大学 Antagoniste du récepteur alpha-2 adrénergique
KR20230074209A (ko) 2020-09-23 2023-05-26 고쿠리츠 다이가쿠 호진 교토 다이가쿠 알파 2 아드레날린 수용체 안타고니스트

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