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2002 Fiscal Year Final Research Report Summary

Real-time imaging of alpha2 adrenergic receptor subtypes in living cells using green fluorescent protein

Research Project

Project/Area Number 13671605
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Anesthesiology/Resuscitation studies
Research InstitutionKyoto Prefectural University of Medicine

Principal Investigator

MIZOBE Toshiki  Associate Professor, Department of Anesthesiology, Kyoto Prefectural University of Medicine, 医学部, 講師 (50239266)

Co-Investigator(Kenkyū-buntansha) NISHI Mayumi  Associate Professor, Department of Anesthesiology and Neurobiology, Kyoto Prefectural University of Medicine, 医学部, 講師 (40295639)
Project Period (FY) 2001 – 2002
Keywordsadrenergic receptor / green fluorescent protein (GFP)
Research Abstract

Background and Goals: Despite the increasing knowledge of beta2 adrenergic receptor, the study of agonist-induced intracellular trafficking of alpha2 adrenoceptor has been limited to immunocytochemical analysis. Thus, we examine the real time subtype-specific trafficking of alpha2 adrenoceptors in living cells using a respective fusion protein of green fluorescent protein (GFP) and human alpha2 adrenoceptor subtypes.
Material and Methods: Using recombinant DNA techniques, the C terminus of each alpha2A, B, and C adrenoceptor subtype was fused to the N terminus of GFP (alpha2 AR-GFP). The DNA sequence of each alpha2 AR-GFP was confirmed by dideoxy sequencing both strands. For the radiolabeled ligand binding assay, the recombinant constructs cloned into Pcdna3 vector were transfected transiently in COS-1 cells using DEAE dextram method. Satruration binding isotherms were performed by incubating membranes with varying cocentrations of 3 [H]-RX-821002, and nonspecific binding was determined … More by adding 10 Um rauwolscine. Equilibrium dissociation constants were determined from saturation isothems using a nonlinear least-square surve-fitting technique (GraphPDA Software Inc., San Diego, CA). Protein content for the membrane preparations were assayed according to Lowry et al. The recombinant constructs were transfected transiently in HEK293 cells using liposome method for the living cell imaging. We examined dynamic manners of trafficking of alpha2 AR-GFP in response to dexmedetomidne using cooled CCD camera attached to an epifluorescent microscope. The distributon into endosomal compartments were also observed using three-dimensional analysis of time-lapse images
Results and Discussions: : At steady state, alpha2A and 2B AR subtypes are localized in the plasma membrane, though a significant portion of alpha2C AR subtype is localized in an intracellular pool. We observed that alpha2B AR subtype underwent agonist-induced internalization and seemed to follow the same endosomal pathway within 15 min used by beta2 AR. Agonist treatment of alpha2A and 2C AR subtypes results in some receptor internalization Less

  • Research Products

    (13 results)

All Other

All Publications (13 results)

  • [Publications] 溝部俊樹: "βアドレナリン受容体ノックアウトマウスと血圧調整"自律神経. 37(2). 204-209 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 溝部俊樹: "アドレナリン受容体とノックアウトマウス(1)"麻酔. 49(12). 1349-1357 (2000)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] 溝部俊樹: "アドレナリン受容体とノックアウトマウス(2)"麻酔. 50(1). 12-19 (2001)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Mizobe T. et al.: "Searching for structural similarity of beta 2 adrenoceptor to bacterior hodopsin or opioid M-receptor by making chimeric receptors"The management of acute and chronic pain. 65-70 (2000)

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      「研究成果報告書概要(和文)」より
  • [Publications] Yoshida M., Nishi M.et al.: "Subcellular and Subnuelear Distributions of Estrogen Receptor α in Living Cells Using Green R/warescent protein"Acta Histochem Cytochem. 34(6). 413-422 (2001)

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      「研究成果報告書概要(和文)」より
  • [Publications] Matsuda K., Nishi M et al.: "Colocalization and ligand-dependent discrete distribution of the estrogen receptor (ER)α and ERβ"Molecular Endocvinolgy. 16(10). 2215-2230 (2002)

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      「研究成果報告書概要(和文)」より
  • [Publications] MIZOBE T: "β adrenergic receptor knockout mouse and cardiovascular control"The Autonomic Nervous System. 37. 204-209 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] MIZOBE T: "A drenergic receptor and knockout mouse 1) β adrenergic receptor knockout mouse"Japanese Journal of Anesthesiology. 49(12). 1349-1357 (2000)

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      「研究成果報告書概要(欧文)」より
  • [Publications] MIZOBE T. and TANAKA Y: "Searching for structural similarity of beta2 adrenoceptor to bacteriorhodopsin or opioid mu receptor by making chimeric receptors. The Management of Acute and Chronic Pain"The Use of the Tools of the Trade, Monduzzi Editore, Bologna. 65-70 (2000)

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      「研究成果報告書概要(欧文)」より
  • [Publications] MIZOBE T: "A drenergic receptor and knockout mouse 2) α adrenergic receptor knockout mouse"Japanese Journal of Anesthesiology. 50(1). 12-19 (2001)

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      「研究成果報告書概要(欧文)」より
  • [Publications] NISHI M., OGAWA H., ITO T. et al: "Dynamic changes in subcellular localization of mineralocorticoid receptor in living cells. In comparison with glucocorticoid receptor using dual-color labeling with green fluorescent protein spectral variants"Molecular Endocrinology. 15(7). 1077-1092 (2001)

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  • [Publications] YOSHIDA M., NISHI M., KIZAKI Z. et al: "Subcellular and subnuclear distribution of estrogen receptor alpha in living cells using green fluorescent protein and immunohistochemistry"Acta Histochem Cytochem. 34(6). 413-422 (2001)

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      「研究成果報告書概要(欧文)」より
  • [Publications] MATSUDA K., OCHIAI I., NISHI M., et al: "Colocalization and ligand-dependent discrete distributon of the estrogen receptor alpha and beta"Molecular Endocrinology. 16. 2215-2230 (2002)

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Published: 2004-04-14  

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