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Molecular Characterization of Purinergic Receptor and its Signal Transduction in the Vascular System

Research Project

Project/Area Number 04670045
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field General physiology
Research InstitutionUniversity of Tokyo

Principal Investigator

TAKUWA Yoh  University of Tokyo, Dept.Cardiovascular Biology, Associate Professor, 医学部(医), 客員助教授 (60171592)

Project Period (FY) 1992 – 1993
Project Status Completed (Fiscal Year 1993)
Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1993: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1992: ¥1,200,000 (Direct Cost: ¥1,200,000)
KeywordsBlood Vessel / Purines / Vasoactive Substances / Signal transductions / 平滑筋 / 内皮 / 受容体 / G蛋白 / プリン作動性 / P_1受容体 / P_2受容体
Research Abstract

It is well known that various purines exert diverse biological activities through P_1 and P_2 purinergic receptors in a variety of tissues. We attempted the molecular characterization of a purinergic receptor expressed in the vascular tissue and the signal transduction. Firstly, we cloned a full length rat P_1(A_1 subtype)receptor cDNA from rat liver lambdagt 10 cDNA library by hybridization screening using PCR-amplified dog A_1 receptor cDNA as a probe. Then, we screened rat aortic smooth muscle cDNA library by using rat A_1 receptor cDNA as a probe. However, we failed to detect a clone which represents P_2 receptors. We, then adapted the strategy of the expression cloning, and screened rat aortic smooth muscle cDNA library constructed in the expression vector pCDM8 by measuring the inositol phosphate production in Ltk^-cells transfected with subgroups of the pCDM8 cDNA library. We could not detect any library subgroup which confered a positive response. Finally, we tried molecular cloning of P_2 receptors by PCR using degenerate oligonucleotide primers corresponding to the 3rd and the 6th transmembrane domains which are well conserved among different G protein-coupled receptors. We isolated more than 40 clones which represented G protein-coupled receptors, among which A_1 receptor, ET_A endothelin receptor and alpha_1 and beta_1 adrenergic receptors were included. Two of them were considered to represent novel G protein-coupled receptors. We isolated full length clones of these two receptors. The transfection of either of the two cDNA clones into mammalian cells did not confer responsiveness to the P_2 ligand. We are now trying to find ligands for these two novel receptors.

Report

(3 results)
  • 1993 Annual Research Report   Final Research Report Summary
  • 1992 Annual Research Report
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] Hiroshi Okazaki: "Molecular cloning of a novel putative G protein-coupled receptor expressed in the cardiovascular system" Biochemical and Biophysical Research Communications. 190. 1104-1109 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Nobukazu Ishizaka: "Molecular cloning of a novel putative G protein-coupled receptor from rat aortic smooth muscle Downregutation of the mRNA level by the cyclic AMP messenger pathway" Biochem Biophys Acta. (印刷中). (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hiroshi Okazaki, Nobukazu Ishizaka, Takeshi Sakurai, Kiyoshi Kurokawa, Katsutoshi Goto, Mamoru Kumada, Yoh Takuwa: "Molecular cloning of a novel putative G protein-coupled receptor expressed in the cardiovascular system." Biochem.Biophys.Res.Commun.190. 1104-1109 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Nobukazu Ishizaka, Hiroshi Okazaki, Kiyoshi Kurokawa, Mamoru Kumada, Yoh Takuwa: "Molecular cloning of a novel putative G protein-coupled receptor from rat aortic smooth muscle, Downregulation of the mRNA level by the cyclic AMP messenger pathway." Biochem.Biophys.Acta. (In Press). (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1993 Final Research Report Summary
  • [Publications] Hiroshi Okazaki: "Molecular cloning of a novel putative G protein-coupled receptor expressed in the cardiovascular system" Biochemical and Biophysical Research Communications. 190. 1104-1109 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Noriko Takuwa: "Ca^<2+>-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts" Journal of Biological Chemistry. 268. 138-145 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Yoh Takuwa: "Alterations in protein kinase C activity and membrane lipid metabolism in cerebral vasospasm after subarachnoid hemorrhage" Journal of Cerebral Blood Flow and Metabolism. 13. 409-415 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Yoh Takuwa: "Endothelin in vascular and endocrine systems:biological activities and its mechanisms of action" Endocrine Journal. 40. 489-506 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Wei Zhou: "Protein kinase C-mediated bidirectional regulation of DNA synthesis,PB protein phosphorylation,and cyclin-dependent kinases in human vascular endothelial cells" Journal of Biological Chemistry. 268. 23041-23048 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Jun-ichi Abe: "Suppression of neointinal smooth muscle cell accumulation in vivo by antisense cdc2 and cdk^2 oligonucleotides in rat carotid artery" Biochemical and Biophysical Research Communication. 198. 16-24 (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] 多久和 陽: "カルシウムのシグナル伝達機構" 中外医学社 編集 小島至, 201 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Takao Asano: "Cellular membrane A key to disease Processes" CRC Press Editors:Tsuyoshi Ohnishi,Tomoko Ohnishi, 412 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] Noriko Takuwa: "Ca^<2+>/calmodulin is involved in growth factor-induced retinoblastoma gene product phosphorylation in human vascular endothelial cells" FEBS Letters. 306. 173-175 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Takeshi Sakurai: "Level of ET_B receptor mRNA is down-regulated by endothelins." Biochem. Biophys. Res. Commun.186. 342-347 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Yoshitoshi Kasuya: "A pertussis toxin-sensitive mechanism of endsthelin action in porcine coronary artery smooth muscle." British Journal of Pharmacology.107. 456-462 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] Yoh Takuwa: "Alterations in protein hinase C activity and membrane lipid metabolism in cerebral vasospasm after subarachnrid hemornhage." Journal of cerebral Blood Flow and Metabulism. (1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] Hiroshi Okazaki: "Molecular cloning of a novel putatine G protein-coupled receptor expressed in the cardisvascular system" Biochem. Biophys. Res. Commun.(1993)

    • Related Report
      1992 Annual Research Report
  • [Publications] Takao Asano: "The cellular Membranes" S.T.Ohnishi and T.Ohnishi, 412 (1993)

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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