Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor.

Abstract

Preincubation of turkey erythrocytes with catecholamines desensitizes the .beta.-adrenergic receptor-adenylate cyclase complex in the plasma membranes of these cells. Photoaffinity labeling of the .beta.-adrenergic receptors with 125I-labeled p-azidobenzylcarazolol (125I-pABC) and subsequent analysis by Na-DodSO4[sodium dodecyl sulfate](polyacrylamide gel electrophoresis demonstrates an altered mobility of receptor peptides from desensitized cells compared to controls. The time course of alteration in .beta.-adrenergic receptor mobility correlates with that for desensitization of isoproterenol-stimulated adenylate cyclase activity. The altered mobility of the receptor peptides from desensitized cells is also observed if the receptors are 1st purified and then photoaffinity labeled with 125I-pABC. The cyclic nucleotide analog 8-bromo-cAMP partiallly mimics catecholamines in promoting desensitization of the adenylate cyclase and modification of the receptor. Phosphorylation of the .beta.-adrenergic receptor in intact turkey erythrocytes was assessed by preincubating the cells with [32P]orthophosphate, desensitizing them with catecholamine, purifying the receptors, and then subjecting them to NaDodSO4/polyacrylamide gel electrophoresis. Desensitization is associated with a 2- to 3-fold increase in 32P incorporation into the receptor, which also demonstrates the characteristic alterations in mobility. These effects are blocked by the .beta.-adrenergic antagonist propranolol. Purified turkey erythrocyte .beta.-adrenergic receptors could be phosphorylated by incubation with [.gamma.-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase. The mobility of the phosphorylated receptor peptides on NaDodSO4/polyacrylamide gel electrophoresis appears to correspond to that of the desensitization of adenylate cyclase in turkey erythrocytes correlates with a stable modification of the .beta.-adrenergic receptor and is associated with agonist-promoted phosphorylation of .beta.-receptor peptides.