Nambi P, Peters J R, Sibley D R, Lefkowitz R J
J Biol Chem. 1985 Feb 25;260(4):2165-71.
We have used a recently developed cell-free system (cell lysate) derived from turkey erythrocytes to explore the potential role of cAMP-activated and other protein kinase systems in desensitizing the adenylate cyclase-coupled beta-adrenergic receptor. Desensitization by the agonist isoproterenol required more than simple occupancy of the receptor by the agonist since under conditions where adenylate cyclase was not activated, no desensitization occurred. As in whole cells, addition of cyclic nucleotides to the cell lysate produced only approximately 50% of the maximal isoproterenol-induced desensitization obtainable. Addition of the purified cAMP-dependent protein kinase holoenzyme plus isoproterenol to isolated turkey erythrocyte plasma membranes mimicked the submaximal desensitization induced in lysates by cAMP. This effect was entirely blocked by the specific inhibitor of the cAMP-dependent protein kinase. By contrast, maximal desensitization induced in lysates by isoproterenol was only approximately 50% attenuated by the protein kinase inhibitor. In the lysate preparations, isoproterenol was also shown to induce, in a stereospecific fashion, phosphorylation of the beta-adrenergic receptor. Phosphorylation promoted by isoproterenol was attenuated by cAMP-dependent protein kinase inhibitor to the same extent as desensitization (i.e. approximately 50%). Phorbol diesters also promoted receptor desensitization and phosphorylation in cell lysates. The desensitization was mimicked by incubation of isolated turkey erythrocyte membranes with partially purified preparations of protein kinase C plus phorbol diesters. In the cell lysate, calmodulin also promoted receptor phosphorylation and desensitization which was blocked by EGTA. Desensitization of adenylate cyclase by isoproterenol, phorbol diesters, and calmodulin was not observed to be additive. These findings suggest that: (a) multiple protein kinase systems, including cAMP-dependent, protein kinase C-dependent, and Ca2+/calmodulin-dependent kinases, are capable of regulating beta-adrenergic receptor function via phosphorylation reactions and that (b) cAMP may not be the sole mediator of isoproterenol-induced phosphorylation and desensitization in these cells.
我们使用了一种最近开发的源自火鸡红细胞的无细胞系统（细胞裂解物），以探究环磷酸腺苷（cAMP）激活的蛋白激酶系统及其他蛋白激酶系统在使腺苷酸环化酶偶联的β-肾上腺素能受体脱敏过程中的潜在作用。激动剂异丙肾上腺素引起的脱敏作用需要的不仅仅是激动剂对受体的简单占据，因为在腺苷酸环化酶未被激活的条件下，不会发生脱敏现象。与完整细胞的情况一样，向细胞裂解物中添加环核苷酸仅能产生约50%的异丙肾上腺素诱导的最大脱敏效果。向分离的火鸡红细胞质膜中添加纯化的依赖cAMP的蛋白激酶全酶加异丙肾上腺素，模拟了cAMP在裂解物中诱导的次最大脱敏作用。这种效应被依赖cAMP的蛋白激酶的特异性抑制剂完全阻断。相比之下，异丙肾上腺素在裂解物中诱导的最大脱敏作用仅被蛋白激酶抑制剂减弱了约50%。在裂解物制剂中，异丙肾上腺素还被证明以立体特异性方式诱导β-肾上腺素能受体的磷酸化。异丙肾上腺素促进的磷酸化被依赖cAMP的蛋白激酶抑制剂减弱的程度与脱敏作用相同（即约50%）。佛波酯也能促进细胞裂解物中受体的脱敏和磷酸化。用部分纯化的蛋白激酶C制剂加佛波酯孵育分离的火鸡红细胞膜可模拟这种脱敏作用。在细胞裂解物中，钙调蛋白也能促进受体的磷酸化和脱敏，而这种作用被乙二醇双四乙酸（EGTA）阻断。未观察到异丙肾上腺素、佛波酯和钙调蛋白对腺苷酸环化酶的脱敏作用具有叠加性。这些发现表明：（a）多种蛋白激酶系统，包括依赖cAMP的、依赖蛋白激酶C的以及依赖Ca2+/钙调蛋白的激酶，能够通过磷酸化反应调节β-肾上腺素能受体的功能；（b）cAMP可能不是这些细胞中异丙肾上腺素诱导的磷酸化和脱敏作用的唯一介质。
