| Project/Area Number |
12835009
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Institution | Yamaguchi University |
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Principal Investigator |
KIMURA Yoshihiro Yamaguchi University, School of Medicine, Assistant Professor, 医学部, 講師 (90301308)
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| Co-Investigator(Kenkyū-buntansha) |
KO Ji-ae Yamaguchi University, School of Medicine, Research Associate, 医学部, 助手 (70314797)
YAMADA Yasue Yamaguchi University, School of Medicine, Research Associate, 医学部, 助手 (00166737)
INUI Makoto Yamaguchi University, School of Medicine, Professor, 医学部, 教授 (70223237)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2000: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | cardiac myocytes / vascular smooth muscle / sarco (endo) plasmic reticulum / calcium ATPase / phospholamban |
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| Research Abstract |
Phospholamban (PLN) reversibly inhibits the Ca^<2+> -ATPase of cardiac sarcoplasmic reticulum (SERCA2) through a direct protein-protein interaction, playing a pivotal role in the regulation of intracellular Ca^<2+> in heart muscle cells. PLN is also thought to be involved in the regulation of vascular tonus. The interaction between PLN and SERCA2 occurs at multiple sites within the cytoplasmic and membrane domains. In this study, we have reconstituted the cytoplasmic protein-protein interaction using bacterially expressed fusion proteins of the cytoplasmic domain of PLN and the long cytoplasmic loop of SERCA2. We have developed a method to evaluate the binding of the fusion proteins employing a 96-well ELISA plate. The PLN fusion protein bound specifically to the SERCA2 fusion protein. The affinity of the binding (K_D) was 0.70 μM. The association was inhibited by cAMP-dependent phosphorylation of the PLN fusion protein and by usage of anti-PLN monoclonal antibody. It was also diminished by substitution at the phosphorylation site of PLN of Ser^<16> to Asp. These results suggest that PLN can bind SERCA2a in the absence of the membrane domains and that the modifications of the cytoplasmic domain of PLN that activate SERCA2a parallel the disruption of the association between the two fusion proteins. Our assay system can be applied to the screening of novel inotropic agents which remove the inhibition of SERCA2a by PLN, improving the relaxation as well as the contractility of the failing heart and reducing vascular resistance.
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