Abstract
Persistent stimulation of the beta 1-adrenergic receptor (beta 1AR) engenders, within minutes, diminished responsiveness of the beta 1 AR/adenylyl cyclase signal transduction system. This desensitization remains incompletely defined mechanistically, however. We therefore tested the hypothesis that agonist-induced desensitization of the beta 1AR (like that of the related beta 2AR) involves phosphorylation of the receptor itself, by cAMP-dependent protein kinase (PKA) and the beta-adrenergic receptor kinase (beta ARK1) or other G protein-coupled receptor kinases (GRKs). Both Chinese hamster fibroblast and 293 cells demonstrate receptor-specific desensitization of the beta 1 AR within 3-5 min. Both cell types also express beta ARK1 and the associated inhibitory proteins beta-arrestin-1 and beta-arrestin-2, as assessed by immunoblotting. Agonist-induced beta 1AR desensitization in 293 cells correlates with a 2 +/- 0.3-fold increase in phosphorylation of the beta 1AR, determined by immunoprecipitation of the beta 1AR from cells metabolically labeled with 32P(i). This agonist-induced beta 1AR phosphorylation derives approximately equally from PKA and GRK activity, as judged by intact cell studies with kinase inhibitors or dominant negative beta ARK1 (K220R) mutant overexpression. Desensitization, likewise, is reduced by only approximately 50% when PKA is inhibited in the intact cells. Overexpression of rhodopsin kinase, beta ARK1, beta ARK2, or GRK5 significantly increases agonist-induced beta 1AR phosphorylation and concomitantly decreases agonist-stimulated cellular cAMP production (p < 0.05). Furthermore, purified beta ARK1, beta ARK2, and GRK5 all demonstrate agonist-dependent phosphorylation of the beta 1AR. Consistent with a GRK mechanism, receptor-specific desensitization of the beta 1AR was enhanced by overexpression of beta-arrestin-1 and -2 in transfected 293 cells. We conclude that rapid agonist-induced desensitization of the beta 1AR involves phosphorylation of the receptor by both PKA and at least beta ARK1 in intact cells. Like the beta 2AR, the beta 1AR appears to bind either beta-arrestin-1 or beta-arrestin-2 and to react with rhodopsin kinase, beta ARK1, beta ARK2, and GRK5.
Highlights
From the Howard Hughes Medical Institute, Departments of"J;.Medicine (Cardiology), **Biochemistry, and IICell Biology, Duke University Medical Center, Durham, North Carolina 27710
Under assay conditions favoring the detection of j3ARK1-initiated desensitization [29], the maximum adenylyl cyclase response in membranes from !SO-pretreated cells was 69 ::': 8% and 72 ::': 10% of control values for the j31AR and f32AR, respectively; EC50 remained essentially unchanged for the j31AR and increased 2.7 ::': 0.7-fold for the f3zAR
In contrast to experiments in {31AR-expressing cells, overexpression of neither {3ARK1 nor J3-arrestin-2 affects !SO-stimulated cAMP production in {33AR-expressing cells (Fig. 8). In this series of experiments, we have demonstrated for the first time that rapid desensitization of the J31AR in intact cells involves agonist-induced phosphorylation of the receptor itself
Summary
Pulmonary/Critical Care Medicine, University of Cincinnati College of Medicine, Cincinnati, OH 45267. Second messenger-independent G protein-coupled receptor kinases (GRKs) [7], by contrast, initiate a two-step process of homologous desensitization by phosphorylating only activated receptors. In the lethal condition of human chronic heart failure, depressed f3-adrenergic responsiveness accompanies a 2-3-fold up-regulation in the expression of the /3-adrenergic receptor kinase (f3ARK1 or GRK2) [11]. Despite this provocative association, the only mechanistic investigation of short term f3 1AR desensitization published far suggests that only a PKA-dependent mechanism, and not a GRK-dependent mechanism, effects f3 1AR desensitization [5]. F3rAdrenergic Receptor Phosphorylation and Desensitization tion and intact cell model systems to assess directly the role of GRKs and {3-arrestin isoforms in both the agonist-induced phosphorylation and desensitization of the {31AR
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