| Project/Area Number |
07557151
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Biological pharmacy
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| Research Institution | The University of Tokyo (Granduate School of Pharm.Sci.) |
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Principal Investigator |
NAGAO Taku The University of Tokyo, Graduate School of Pharmaceutical Sciences, Laboratory of Pharmacology and Toxicology, Professor, 大学院・薬学系研究科, 教授 (30217971)
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| Co-Investigator(Kenkyū-buntansha) |
NOMATA Yasuyuki Eiken Chemical CO., Ltd., Research & Development Headquarters, Principle Investi, 生物化学研究所, 主任研究員
KUROSE Hitoshi The University of Tokyo, Graduate School of Pharmaceutical Sciences, Laboratory, 大学院・薬学系研究科, 助手 (10183039)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1996: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | intracellular immunization / monoclonal antibody / single chain Fv molecule / G protein-coupled receptor kinase / subtype specificily / adonovirus / desensitization / G protein-coupled receptors / 抗体分子の遺伝子 / 受容体キナーゼ / 細胞特異性 / 細胞内発現 / 調節機構 / モノクロール抗体 / 抗体遺伝子 |
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| Research Abstract |
To analyze the functional differences among six G protein-coupled receptor kinases (GRKs) in the cells, we tried to establish a novel method, i.e.intracellular immunization. The intracellular immunization is a functional inactivation method by expressing monoclonal antibody (mAb). At first, mAb that reacts with GRK2 (betaARK1) was made by immunizing gluthatione-S-transferase (GST) fusion protein with carboxyl terminus of betaARK1. The resulting mAb specifically recognized betaARKl but not GRK3,5 and 6 with Western blot. The mAb was purified from culture supenatant of hybridoma by ammonium sulfate precipitation and Protein G column. We found that the purified mAb inhibited the basal and agonist-stimulated phosphorylating activities of betaARK1. The mAb also inhibited thc betagammabinding of heterotrimeric G protein to carboxyl terminus of betaARK1. As the GST-carboxyl terminus fusion protein could stimulate the phosphorylating activity of betaARKl, we concluded that antibody bound to the part to be essential for the activity and inhibited the activation of betaARKl. We have also injected mAb into myocytes and found the involvement of betaARKl in the process of beta _1 -adrenergic receptor-mediated modulation of L-type Ca ^<2+> channel. To construct single chain Fv molecule (scFv) to express mAb gene in the cell, the variable regions of heavy and light chains of mAb was amplified from mRNA of hybridoma by PCR.The scFv was expressed in E.coli. and the expressed scFv could recognize the betaARKl. We also ligated scFv into a cosmid vector to make a recombinant adenovirus. We believe that adenovirus-mediated expression of scFv can help to elucidate the roles of betaARKl in desensitization of various G protein-coupled receptors.
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