Catecholamine-Induced Desensitization of Turkey Erythrocyte Adenylate Cyclase is Associated with Phosphorylation of the β -adrenergic Receptor

Preincubation of turkey erythrocytes with catecholamines desensitizes the β -adrenergic receptor-adenylate cyclase complex in the plasma membranes of these cells. Photoaffinity labeling of the β -adrenergic receptors with125I-labeled p-azidobenzylcarazolol (125I-pABC) and subsequent analysis by NaDodSO4/polyacrylamide gel electrophoresis demonstrates an altered mobility of receptor peptides from desensitized cells compared to controls [Stadel, J. M., Nambi, P., Lavin, T. N., Heald, S. L., Caron, M. G. & Lefkowitz, R. J. (1982) J. Biol. Chem. 257, 9242-9245]. The time course of alteration in β -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 first purified and then photoaffinity labeled with125I-pABC. The cyclic nucleotide analog 8-bromoadenosine 3′,5′-cyclic monophosphate partially mimics catecholamines in promoting desensitization of the adenylate cyclase and modification of the receptor. Phosphorylation of the β -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 in32P incorporation into the receptor, which also demonstrates the characteristic alterations in mobility. These effects are blocked by the β -adrenergic antagonist propranolol. Purified turkey erythrocyte β -adrenergic receptors could be phosphorylated by incubation with [γ -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 desensitized receptors. These data show that catecholamine-induced desensitization of adenylate cyclase in turkey erythrocytes correlates with a stable modification of the β -adrenergic receptor and is associated with agonist-promoted phosphorylation of β -receptor peptides.