Phosphorylation of the beta-adrenergic receptor in intact cells: relationship to heterologous and homologous mechanisms of adenylate cyclase desensitization.
We have recently shown that both heterologous and homologous forms of adenylate cyclase desensitization involve phosphorylation of beta-adrenergic receptors. In order to compare these two reactions, we wished to identify a single cell system in which both processes could be studied. Using the frog erythrocyte, which has been previously shown to exhibit cAMP-independent homologous desensitization, we have found that under appropriate conditions cAMP-dependent heterologous desensitization can be elicited. Incubation of intact cells with the membrane-permeable cAMP analogs dibutyryl cAMP or 8-bromo cAMP promotes about a 50% desensitization of isoproterenol- and prostaglandin E1-stimulated adenylate cyclase activity in a time-, temperature-, and dose-dependent fashion. There is also a 20% desensitization in the abilities of guanine nucleotides (GTP and guanyl-5'-yl-imidodiphosphate) and NaF to stimulate adenylate cyclase maximally. In contrast, there is no effect on forskolin- or MnCl2-stimulated enzyme activities. The desensitization response is specific for cAMP as dibutyryl cGMP, 8-bromo cGMP, or 8-bromo AMP produce little or no desensitization. Incubation of the cells with dibutyryl cAMP does not affect the number of cell surface beta-adrenergic receptors. In contrast, incubation with isoproterenol promotes homologous desensitization and sequestration of the receptors. Incubation of 32P-labeled erythrocytes with either dibutyryl cAMP or isoproterenol promotes a stoichiometric threefold increase in the phosphorylation state of the beta-adrenergic receptor which occurs predominantly on serine residues. However, if the cells are coincubated with both dibutyryl cAMP and isoproterenol then the desensitization of isoproterenol-stimulated enzyme activity and phosphorylation of the beta-adrenergic receptor are greater than those observed with either agent alone. These results indicate that heterologous and homologous desensitization of adenylate cyclase-coupled beta-adrenergic receptors are mediated by different biochemical pathways involving phosphorylation of the receptor protein on distinct sites.
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000222:Adaptation, Physiological; D000262:Adenylyl Cyclases / Q000097:blood*; D000527:Alprostadil / Q000494:pharmacology; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D010766:Phosphorylation; D011894:Rana pipiens; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology
D011943:Receptors, Adrenergic, beta; D015124:8-Bromo Cyclic Adenosine Monophosphate; D006165:Guanylyl Imidodiphosphate; D003994:Bucladesine; D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D000527:Alprostadil; D007545:Isoproterenol; D015056:1-Methyl-3-isobutylxanthine