PHOSPHORYLATION OF THE TYPE 1A ANGIOTENSIN-II RECEPTOR BY G-PROTEIN-COUPLED RECEPTOR KINASES AND PROTEIN-KINASE-C

The type 1A angiotensin II receptor (AT(1A)-R), which mediates cardiov ascular effects of angiotensin II, has been shown to undergo rapid ago nist-induced desensitization. We investigated the potential role of se cond messenger-activated kinases and G protein-coupled receptor kinase s (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 corre lated 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 red uced by 40-50% by either overexpression of a dominant negative K220R m utant GRK2 or treatment of the cells with the protein kinase C (PKC) i nhibitor staurosporine, in a virtually additive fashion. Cellular over expression of GRK2(K220R) not only inhibited agonist-induced AT(1A)-R phosphorylation, but also prevented receptor desensitization, as asses sed by angiotensin II-stimulated GTPase activity in membranes prepared from agonist-treated and control cells. In contrast, PKC inhibition b y staurosporine did not affect homologous desensitization of the AT(1A )-R. Overexpression of GRKs 2, 3, or 5 significantly augmented the ago nist-induced AT(1A)-R phosphorylation 1.5- to 1.7-fold (p < 0.001). Th ese findings suggest a role for receptor phosphorylation by one or sev eral GRKs in the rapid agonist-induced desensitization of the AT(1A)-R .