Reconstitution of catecholamine-stimulated adenylate cyclase activity using three purified proteins.
beta-Adrenergic receptors, the GTP-binding regulatory protein that stimulates adenylate cyclase (Gs), and adenylate cyclase were each purified and reconstituted into unilamellar vesicles composed of phosphatidylethanolamine and phosphatidylserine (3:2, w/w). The molar ratio of receptor:Gs:adenylate cyclase was estimated to be about 1:10:1. Adenylate cyclase activity in the vesicles was stimulated up to 2.6-fold by beta-adrenergic agonists. Stimulation was dependent on the presence of guanine nucleotide, displayed appropriate beta-adrenergic selectivity and stereoselectivity for agonists, and was blocked appropriately by beta-adrenergic antagonists. Therefore, while additional proteins may modulate adenylate cyclase activity in native membranes, these results show that these three proteins are sufficient for the expression of hormone-stimulated adenylate cyclase.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002395:Catecholamines / Q000494:pharmacology*; D002417:Cattle; D000242:Cyclic AMP / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004837:Epinephrine / Q000494:pharmacology; D005614:Freeze Fracturing; D019204:GTP-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008051:Lipid Bilayers / Q000378:metabolism; D008854:Microscopy, Electron; D011433:Propranolol / Q000494:pharmacology; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D013237:Stereoisomerism; D013726:Terbutaline / Q000494:pharmacology; D014422:Turkeys
D002395:Catecholamines; D008051:Lipid Bilayers; D011943:Receptors, Adrenergic, beta; D011433:Propranolol; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D013726:Terbutaline; D004837:Epinephrine