Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor.
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 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 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 first purified and then photoaffinity labeled with 125I-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 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 desensitized receptors. These data show that catecholamine-induced 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.
D015124:8-Bromo Cyclic Adenosine Monophosphate; D000262:Adenylyl Cyclases / Q000097:blood*; D000818:Animals; D002395:Catecholamines / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D013806:Theophylline / Q000494:pharmacology; D014422:Turkeys
D002395:Catecholamines; D010446:Peptide Fragments; D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; D015124:8-Bromo Cyclic Adenosine Monophosphate; D013806:Theophylline; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D007545:Isoproterenol