Agonist-independent phosphorylation of purified cardiac muscarinic cholinergic receptors by protein kinase C.
The results of several studies have suggested that muscarinic cholinergic receptors (mAChR) may be regulated by multiple pathways involving phosphorylation of the receptors. Previous studies have demonstrated that chick heart mAChR are phosphorylated by the beta-adrenergic receptor kinase (beta-AR kinase) in an agonist-dependent manner, and it has been suggested that this process may be linked to receptor desensitization. In this work, we present evidence that protein kinase C can phosphorylate the purified, reconstituted chick heart mAChR and can modify the interaction of the receptors with GTP binding proteins (G-proteins) that couple the receptors to effectors. Phosphorylation of the mAChR with protein kinase C occurred to an extent of approximately 5 mol of P/mol of receptor. Neither the rate nor the extent of the protein kinase C mediated phosphorylation of mAChR was agonist-dependent. Under the conditions tested, the initial rate of phosphorylation of the mAChR by protein kinase C was significantly more rapid than that obtained with the beta-AR kinase. At equilibrium, phosphorylation of mAChR by protein kinase C and beta-AR kinase was partially additive. The functional effects of protein kinase C mediated phosphorylation of the mAChR were assessed by comparing the abilities of purified G-proteins (Gi and Go) to reconstitute high-affinity agonist binding to phosphorylated and nonphosphorylated receptors. A significantly larger percentage of the receptors phosphorylated with protein kinase C exhibited G-protein-dependent high-affinity agonist binding, suggesting that phosphorylation of the receptors by protein kinase C modulates receptor function in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Richardson
R M
RM
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
Hosey
M M
MM
D000818:Animals; D002642:Chick Embryo; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism; D006352:Heart Ventricles; D008081:Liposomes; D009206:Myocardium / Q000737:chemistry*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015290:Second Messenger Systems; D051552:beta-Adrenergic Receptor Kinases
D008081:Liposomes; D011976:Receptors, Muscarinic; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins