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7回膜貫通型受容体の蛋白間相互作用の解析とその生理的意義

Research Project

Project/Area Number 12771429
Research Category

Grant-in-Aid for Encouragement of Young Scientists (A)

Allocation TypeSingle-year Grants
Research Field Biological pharmacy
Research InstitutionNational Research Institute for Child Health and Development

Principal Investigator

平澤 明  国立小児病院, (小児医療研究センター)・小児薬理研究部, 研究員 (70242633)

Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Keywordsアドレナリン受容体 / GFP / 蛍光標識リカンド / internalization / 細胞内局在 / 細胞内pH / α1Aアドレナリン受容体 / α1Bアドレナリン受容体 / yeast two-hybrid法 / Green Fluorescent Protein / gClqBP / 受容体ヘテロ二量体
Research Abstract

受容体の細胞内局在、およびその制御機構の解明のため、α1アドレナリン受容体をモデルとし、定量的実験が可能なフローサイトメトリーと、画像として可視化が可能なレーザー共焦点顕微鏡を組み合わせ解析を行ってきている。
現在までに、受容体の蛍光標識にはエピトープタグ、GFP(Green Fluorescent protein)を融合した受容体コンストラクトを作製し、細胞内局在、輸送機構の直接的な可視化および、Real-timeでの観察を可能にし、新規の相互作用分子の同定を行った。
本年度は、α1アドレナリン受容体の細胞内局在機構の調節について詳細な解析を行い(Hirasawa et al. Life Sciences 2001)、さらに、蛍光標識したリガンドとしてBODIPY-prazosinと、抗受容体抗体を組み合わせて用い、受容体との相互作用をフローサイトメトリーで詳細に解析し、細胞内局在とリガンドの相互作用の関係を明らかにした(Sugawara et al. Life Sciences 2001)。また、同受容体のシグナル伝達経路に関しても生化学的解析を行った(Yamauchi et al. BBRC 2001)。
新たな手法としてのpH感受性を持つGFP変異型プローブを融合した受容体コンストラクトを用いることで、受容体近傍の微小環境を可視化し、受容体刺激に伴う、pH変化と受容体分子の細胞内への移行現象を同時測定することにも成功し、発表した(Awaji et al. BBRC 2001)。この手法を更に発展させ、受容体に対する相互作用分子による局在変化現象を解析中である。

Report

(2 results)
  • 2001 Annual Research Report
  • 2000 Annual Research Report
  • Research Products

    (11 results)

All Other

All Publications (11 results)

  • [Publications] Yamauchi J, Itoh H, Shinoura H, Tsujimoto G. et al.: "Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in α1β-adrenergic receptor/G αq-induced inhibition of cell proliferation"Biochem. Biophys. Res. Commun.. 281. 1019-1023 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Hirasawa A, Awaji T, Xu Z, Shinoura H, Tsujimoto G.: "Regulation of subcellular localization of α1-adrenoceptor subtypes"Life Sciences. 68. 2259-2267 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Yamauchi J, Hirasawa A, Miyamoto Y, Itoh H, Tsujimoto G: "β2-Adrenergic receptor/cyclic adenosine monophosphate (cAMP)leads to JNK activation through Rho family small GT pases"Biochem. Biophys. Res. Commun.. 284. 1199-1203 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Tsujimoto G, Katsuma S, Shiojima S, Hirasawa A, Tanoue A.: "Functional genomic research of α1 -adrenoceptors"J. Cardiovascular Pharmacology. 38. S1-S5 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Katsuma S, Nishi K, Hirasawa A, Murakami Y, Tsujimoto G, et al.: "Molecular monitoring of bleomycin-induced pulmonary fibrosis by cDNA microarray-bassed gene expression profiling"Biochem. Biophys. Res. Commun.. 288. 747-751 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Yamauchi J, Tsumaya K, Hirasawa A, Kaziro Y, Tsujimoto G, et al.: "Gaq-dependent activation of mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase cascade"Biochem. Biophys. Res. Commun.. 288. 1067-1094 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Katsuma K, Shiojima S, Hirasawa A. Tsujimoto G, et al.: "Genomic analysis of a mouse model of immunoglobulin A nephropathy reveals an enhanced PDGF-EDG5 cascade"The Pharmacogenomics Journal. 1. 211-217 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Awaji T, Hirasawa A, Shirakawa H, Tsujimoto G, Miyazaki S.: "Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains"Biochem. Biophys. Res. Commun.. 289. 457-462 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Shinoura H,Take H,Hirasawa A et al.: "Key amino acids of vasopressin Vla receptor responsible for the species difference in the affinity of OPC-21268."FEBS Letters. 466. 255-258 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Homma N,Hirasawa A et al.: "Both α1A-and α1B-adrenergic receptor subtypes couple to the transient outward current(ITO)in rat ventricular myocytes."Br.J.Pharamacol.. 129(6). 1113-1120 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Yamauchi J,Itoh H et al.: "Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in α1B-adrenergic receptor/G a q-induced inhibition of cell proliferation."Biochem.Biophys.Res.Commun. (in press).

    • Related Report
      2000 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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