Phosphorylation of the type 1A angiotensin II receptor by G protein-coupled receptor kinases and protein kinase C

被引:202
作者
Oppermann, M
Freedman, NJ
Alexander, RW
Lefkowitz, RJ
机构
[1] DUKE UNIV, MED CTR, DEPT MED, HOWARD HUGHES MED INST, DURHAM, NC 27710 USA
[2] DUKE UNIV, MED CTR, DEPT BIOCHEM, DURHAM, NC 27710 USA
[3] EMORY UNIV, SCH MED, DEPT MED, DIV CARDIOL, ATLANTA, GA 30322 USA
关键词
D O I
10.1074/jbc.271.22.13266
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The type 1A angiotensin II receptor (AT(1A)-R), which mediates cardiovascular effects of angiotensin II, has been shown to undergo rapid agonist-induced desensitization. We investigated the potential role of second messenger-activated kinases and G protein-coupled receptor kinases (GRKs) in the regulation of this receptor. In 293 cells transfected with the AT(1A)-R, a 3-min challenge with angiotensin II engendered a 46% decrease in subsequent angiotensin II-stimulated phosphoinositide hydrolysis in intact cells. This agonist-induced desensitization correlated temporally and dose-dependently with the phosphorylation of the receptor to a stoichiometry of 1 mol of phosphate/mol of receptor, as assessed by immunoprecipitation of receptors from cells metabolically labeled with P-32(i). Agonist-induced receptor phosphorylation was reduced by 40-50% by either overexpression of a dominant negative K220R mutant GRK2 or treatment of the cells with the protein kinase C (PKC) inhibitor staurosporine, in a virtually additive fashion. Cellular overexpression of GRK2(K220R) not only inhibited agonist-induced AT(1A)-R phosphorylation, but also prevented receptor desensitization, as assessed by angiotensin II-stimulated GTPase activity in membranes prepared from agonist-treated and control cells. In contrast, PKC inhibition by staurosporine did not affect homologous desensitization of the AT(1A)-R. Overexpression of GRKs 2, 3, or 5 significantly augmented the agonist-induced AT(1A)-R phosphorylation 1.5- to 1.7-fold (p < 0.001). These findings suggest a role for receptor phosphorylation by one or several GRKs in the rapid agonist-induced desensitization of the AT(1A)-R.
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页码:13266 / 13272
页数:7
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