Functional consequences of A1 adenosine-receptor phosphorylation by the beta-adrenergic receptor kinase

Treatment of smooth-muscle cells with R-phenylisopropyladenosine (R-PIA) leads to a loss of A1 adenosine receptor (A1AR)-mediated inhibition of adenylate cyclase, a decrease in receptor number and an increase in receptor phosphorylation. In this study, the role of the beta-adrenergic receptor kinase (beta ARK) in the phosphorylation and inactivation of the A1AR was examined. A1ARs were purified from bovine brain and reconstituted into phospholipid vesicles, with or without a 10-fold excess of Gi/Go (a 50:50 mixture). The reconstituted receptor preparations were phosphorylated with beta ARK in the absence (control) or presence (treated) of R-PIA. R-PIA stimulated A1AR phosphorylation by 2-3-fold over control. Phosphorylation of the A1AR was blocked by XAC, and A1AR antagonist, underscoring its agonist dependence. The stoichiometry of phosphorylation obtained was approx. 1.3 mol of phosphate per mol of A1AR. Phosphorylation of the A1AR by beta ARK was enhanced by an additional 42% when G beta gamma (30 nM) was included in the phosphorylation mixture. In order to test the role of phosphorylation on receptor function, the purified A1AR was reconstituted with a mixture of Gi/Go, phosphorylated with beta ARK and used to determine high-affinity [125I]APNEA (A1AR agonist) binding. Agonist binding was reduced by about 50% in the treated preparations compared to control. In contrast, antagonist ([3H]XAC) binding was increased by about 50%. These data are consistent with an uncoupling of the A1AR from G proteins following receptor phosphorylation. In control preparations, R-PIA stimulated GTPase activity from 0.08 to 0.164 pmol Pi released/pmol Gi/Go per min. Phosphorylation of receptor by beta ARK reduced R-PIA-stimulated GTPase activity by 35%. In addition, phosphorylation of the A1AR by beta ARK decreased R-PIA-stimulated GTP gamma S binding by 62%. These data provide evidence that A1AR phosphorylation by beta ARK results in a diminished receptor-G-protein interaction.