PHOSPHORYLATION AND DESENSITIZATION OF THE HUMAN BETA(1)-ADRENERGIC RECEPTOR - INVOLVEMENT OF G-PROTEIN-COUPLED RECEPTOR KINASES AND CAMP-DEPENDENT PROTEIN-KINASE

Persistent stimulation of the beta(1)-adrenergic receptor (beta(1)AR) engenders, within minutes, diminished responsiveness of the beta(1)AR/ adenylyl cyclase signal transduction system. This desensitization rema ins incompletely defined mechanistically, however. We therefore tested the hypothesis that agonist-induced desensitization of the beta(1)AR (like that of the related beta(2)AR) involves phosphorylation of the r eceptor itself, by cAMP-dependent protein kinase (PKA) and the beta ad renergic receptor kinase (beta ARK1) or other G protein-coupled recept or kinases (GRKs). Both Chinese hamster fibroblast and 293 cells demon strate receptor-specific desensitization of the beta(1)AR within 3-5 m in. Both cell types also express beta(1)AR and the associated inhibito ry proteins beta-arrestin-1 and beta-arrestin-2, as assessed by immuno blotting. Agonist-induced beta(1)AR desensitization in 293 cells corre lates with a 2 +/- 0.3-fold increase in phosphorylation of the beta(1) AR, determined by immunoprecipitation of the beta(1)AR from cells meta bolically labeled with P-32(i). This agonist induced beta(1)AR phospho rylation derives approximately equally from PKA and GRK activity, as j udged by intact cell studies with kinase inhibitors or dominant negati ve beta ARK1 (K220R) mutant overexpression. Desensitization, likewise, is reduced by only similar to 50% when PKA is inhibited in the intact cells. Overexpression of rho dopsin kinase, beta ARK1, beta ARK2, or GRK5 significantly increases agonist-induced beta(1)AR phosphorylation and concomitantly decreases agonist-stimulated cellular cAMP producti on (p < 0.05). Furthermore, purified beta ARK1, beta ARK2, and GRK5 al l demonstrate agonist-dependent phosphorylation of the beta(1)AR. Cons istent with a GRK mechanism, receptor-specific desensitization of the beta(1)AR was enhanced by overexpression of beta-arrestin-1 and -2 in transfected 293 cells. We conclude that rapid agonist-induced desensit ization of the beta(1)AR involves phosphorylation of the receptor by b oth PKA and at least beta ARK1 in intact cells. Like the beta(2)AR, th e beta(1)AR appears to bind either beta-arrestin-1 or beta-arrestin-2 and to react with rhodopsin kinase, beta ARK1, beta ARK2, and GRK5.