2000 Fiscal Year Final Research Report Summary
ANALYSIS OF REGURATORY MECHANISM OF RGS ON DESENSITIZATION AND TORERANCE.
| Project/Area Number |
11670082
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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 Field |
General pharmacology
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| Research Institution | CHIBA UNIVERSITY |
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Principal Investigator |
MORIO Kayoko Chiba University Graduate School of Medicine Research Assistant, 大学院・医学研究科, 助手 (80110352)
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| Co-Investigator(Kenkyū-buntansha) |
KADOTA Tomoko Chiba University School of Medicine Assistant Professor, 医学部, 助教授 (00089864)
KIMURA Sadao Chiba University Graduate School of Medicine Proffessor, 大学院・医学研究科, 教授 (40134225)
NISHIYAMA Mariko Chiba University Graduate School of Medicine Research Assistant, 大学院・医学研究科, 助手 (00092081)
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| Project Period (FY) |
1999 – 2000
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| Keywords | REGULATOR OF G-PROTEIN SIGNALING (RGS) / G-PROTEIN COUPLED RECEPTOR / ENDOTHELIN RECEPTOR / ANGIOTENSIN RECEPTOR / INTRACELLULAR CA2+TRANSIENT / DESENSITIZATION (TORERANCE) / REVERSE TORERANCE |
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| Research Abstract |
In this study, we determine the effect of RGS (Regulator of G-protein signaling) proteins on desensitization of G protein-coupled receptor signaling. To analyze the effect of RGSs, we prepared several plasmid encoding RGS5, RGS4, GRK and the N-terminal amino acid-deleted RGSs cDNA.In addition, 6-his-tagged RGS proteins were purified from E-coli systems.
1. Wild RGS4 and 5, and their dNRGSs bound to Gi3α, Goα, Gqα subunits and accelerated the catalytic rate of GTP hydrolysis of Gi3α subunit (GAP activity).
2. N-terminus of GRK also bound to Gqα subunit, but did not have any GAP activity against Gi3α.
3. Subcellular distribution of RGSs in HEK293 cells transfected with cDNA encoding RGSs was analyzed using the specific antibodies against RGSs. RGS5 was located in the membrane and cytosolic fractions, while dNRGS5 was localized almost exclusively in the cytosolic fraction. RGS4 and dNRGS4 located both in the membrane and cytosolic fractions.
4. RGS5, RGS4, dNRGS4 and dNRGS5 expressed in HEK cells suppressed angiotensin (AngII) and endothelin (ET)-1-induced intracellular Ca2+ transients in concentration dependent manner. N-terminus of GRK also suppressed intracellular Ca2+ transients induced by Ang-II and ET-1.
These results indicated that RGSs serve as GAP for Gi3α subunits and suppress the AngII and ET-1 induced intracellular Ca2+ transients, suggesting that RGSs negatively regulate the G-protein coupled-receptor signaling.
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