Kassis S, Zaremba T, Patel J, Fishman P H
J Biol Chem. 1985 Jul 25;260(15):8911-7.
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.
将大鼠胶质瘤C6细胞暴露于异丙肾上腺素或12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）会导致异丙肾上腺素刺激的腺苷酸环化酶活性脱敏。两种处理后，β受体对异丙肾上腺素的亲和力均降低。因此，TPA或异丙肾上腺素引起的脱敏似乎涉及β受体与腺苷酸环化酶的刺激性调节成分（Ns）的“解偶联”。通过重组S49 cyc - 腺苷酸环化酶活性测定的Ns活性，在脱敏后未发现变化。通过使C6细胞膜中的Ns和腺苷酸环化酶的催化成分失活并将它们与缺乏β受体的膜融合来测量β受体的活性。与未处理细胞的受体相比，异丙肾上腺素处理的C6细胞的受体在与外源腺苷酸环化酶“偶联”方面活性较低，而TPA处理细胞的受体则完全有活性。对后一意外结果进行了进一步探究。将C6细胞裂解物在线性蔗糖密度梯度上离心，并对梯度级分进行β受体结合活性测定。大多数受体在“重”质膜峰中回收，但一些受体也出现在“轻”膜峰中。用异丙肾上腺素或TPA处理细胞后，轻峰中受体的比例增加。用伴刀豆球蛋白A预先处理细胞可防止异丙肾上腺素或TPA引起的轻受体增加。此外，伴刀豆球蛋白A处理可防止TPA引起的腺苷酸环化酶脱敏，但不能防止异丙肾上腺素引起的脱敏。最后，通过聚乙二醇诱导用TPA处理但未用异丙肾上腺素处理的细胞的膜融合，可逆转腺苷酸环化酶的脱敏。我们得出结论，β激动剂和佛波酯通过不同机制使腺苷酸环化酶脱敏。激动剂导致β受体的功能活性降低，随后受体分离到不含Ns的轻膜组分中。佛波酯不会改变受体的活性，但会导致其分离。
