Phosphorylation of chick heart muscarinic cholinergic receptors by the .beta.-adrenergic receptor kinase

Abstract

Previous studies have demonstrated that muscarinic cholinergic receptors (mAChR) become markedly phosphorylated when intact cardiac cells are stimulated with a muscarinic agonist. This process appears to be related to the process of receptor desensitization. However, the mechanism of agonist-induced phosphorylation of mAChR is not known. In situ phosphorylation studies suggested that agonist-induced phosphorylation of mAChR may involve the participation of a receptor-specific kinase and/or require agonist occupancy. These observations regarding phosphorylation and desensitization of mAChR are similar to observations made for .beta.-adrenergic reeptors. Recent studies have indicated that homologous desensitization of .beta.-adrenergic receptors may be due to the phosphorylation of these receptors by a novel protein kinase that only recognizes the agonist-occupied form of the receptors. As muscarinic receptors are structurally homologous to .beta.-adrenergic receptors, we have initiated studies to identify the protein kinase responsible for the phosphorylation of muscarinic receptors by determining whether the chick heart muscarinic receptor would serve as a substrate for the .beta.-adrenergic receptor kinase (.beta.-AR kinase). We report that the purified and reconstituted chick heart muscarinic receptor serves as an excellent substrate in vitro for the .beta.-AR kinase. Phosphorylation of mAChR receptors by the .beta.-AR kinase was only observed in the presence of a muscarinic receptor agonist and was prevented in the presence of antagonist. Both the extent of phosphorylation (3-4 mol of P/mol of receptor) and the phosphoamino acid composition of the mAChR after incubation in vitro with .beta.-AR kinase were similar to the characteristics of agonist-induced phosphorylation of mAChR in situ. As many of the properties of the in vitro phosphorylation of mAChR with .beta.-AR kinase and the agonist-induced phosphorylation of mAChR in situ are similar, the results suggest that .beta.-AR kinase or a similar enzyme may phosphorylate the mAChR in intact cells. The present results also extend our knowledge of the properties of .beta.-AR kinase by directly demonstrating that this novel enzyme effectively phosphorylates a nonadrenergic receptor. The results support the hypothesis that this protein kinase may be of general importance in the regulation of receptors coupled to the modulation of adenylyl cyclase.