Phorbol esters and beta-adrenergic agonists mediate desensitization of adenylate cyclase in rat glioma C6 cells by distinct mechanisms.
Exposure of rat glioma C6 cells to either isoproterenol or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in desensitization of isoproterenol-stimulated adenylate cyclase activity. After either treatment, the affinity of beta-receptors for isoproterenol was reduced. Thus, desensitization by TPA or isoproterenol appeared to involve an "uncoupling" of the beta-receptor from the stimulatory regulatory component (Ns) of adenylate cyclase. The activity of Ns, assayed by reconstitution of S49 cyc- adenylate cyclase activity, was found to be unchanged after desensitization. The activity of beta-receptors was measured by inactivating Ns and the catalytic component of adenylate cyclase in C6 membranes and fusing them with membranes lacking beta-receptors. Receptors from isoproterenol-treated C6 cells were less active in "coupling" to the foreign adenylate cyclase than receptors from untreated cells, whereas receptors from TPA-treated cells were fully active. This unexpected latter result was explored further. Lysates from C6 cells were centrifuged on linear sucrose density gradients and the gradient fractions assayed for beta-receptor binding activity. Most of the receptors were recovered in a "heavy" plasma membrane peak but some receptors also appeared in a "light" membrane peak. After treatment of the cells with isoproterenol or TPA, the proportion of receptors in the light peak increased. Prior treatment of the cells with concanavalin A prevented the increase in light receptors caused by isoproterenol or TPA. In addition, the concanavalin A treatment prevented the desensitization of adenylate cyclase caused by TPA but not that caused by isoproterenol. Finally, desensitization of adenylate cyclase was reversed by polyethylene glycol-induced fusion of membranes from cells treated with TPA but not isoproterenol. We conclude that beta-agonists and phorbol esters desensitize adenylate cyclase by distinct mechanisms. Agonists cause a reduction in the functional activity of the beta-receptors followed by a segregation of the receptors into a light membrane fraction devoid of Ns. Phorbol esters do not alter the activity of the receptors but do cause their segregation.
Kassis
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Zaremba
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Patel
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Fishman
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D000262:Adenylyl Cyclases / Q000032:analysis*; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D004789:Enzyme Activation; D005910:Glioma / Q000201:enzymology*; D007545:Isoproterenol / Q000494:pharmacology; D010704:Phorbols / Q000494:pharmacology*; D010766:Phosphorylation; D011493:Protein Kinase C; D011494:Protein Kinases / Q000032:analysis; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000032:analysis / Q000187:drug effects / Q000378:metabolism; D011955:Receptors, Drug; D011971:Receptors, Immunologic / Q000032:analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000318:Adrenergic beta-Agonists; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D010704:Phorbols; D011943:Receptors, Adrenergic, beta; D011955:Receptors, Drug; D011971:Receptors, Immunologic; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D003208:Concanavalin A; D011494:Protein Kinases; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate