Desensitization of the turkey erythrocyte beta-adrenergic receptor in a cell-free system. Evidence that multiple protein kinases can phosphorylate and desensitize the receptor.
Desensitization of the turkey erythrocyte beta-adrenergic receptor in a cell-free system. Evidence that multiple protein kinases can phosphorylate and desensitize the receptor.
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DOI：
10.1016/s0021-9258(18)89532-1
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发表时间：
1985-02
期刊：
影响因子：
--
通讯作者：
P. Nambi;J. Peters;D. Sibley;R. Lefkowitz
中科院分区：
文献类型：
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作者：
P. Nambi;J. Peters;D. Sibley;R. Lefkowitz
我们已经使用了最近开发的无细胞系统（细胞裂解液）来自火鸡红细胞探索cAMP激活和其他蛋白激酶系统脱敏腺苷酸环化酶偶联β-肾上腺素能受体的潜在作用。由激动剂异丙肾上腺素脱敏需要比受体的激动剂的简单占领，因为在腺苷酸环化酶不被激活的条件下，没有脱敏发生。在整个细胞中，除了环核苷酸的细胞裂解物产生的最大异丙肾上腺素诱导的脱敏可获得的只有约50%。此外，纯化的cAMP依赖性蛋白激酶全酶加异丙肾上腺素分离的火鸡红细胞质膜模仿次最大脱敏诱导的裂解物中的cAMP。这种作用被cAMP依赖性蛋白激酶的特异性抑制剂完全阻断。相比之下，最大脱敏诱导的裂解物中异丙肾上腺素的蛋白激酶抑制剂仅衰减约50%。在裂解物制剂中，异丙肾上腺素也显示出以立体特异性方式诱导β-肾上腺素能受体的磷酸化。cAMP依赖性蛋白激酶抑制剂可减弱异丙肾上腺素促进的磷酸化，其程度与脱敏相同（即约50%）。佛波醇二酯也促进细胞裂解物中受体脱敏和磷酸化。脱敏模仿孵育分离的火鸡红细胞膜与部分纯化的蛋白激酶C加佛波醇二酯的制剂。在细胞裂解液中，钙调素也促进受体磷酸化和脱敏，EGTA可阻断这种作用。腺苷酸环化酶的脱敏异丙肾上腺素，佛波醇二酯，钙调素没有被观察到是加性的。这些研究结果表明：（a）多种蛋白激酶系统，包括cAMP依赖性、蛋白激酶C依赖性和Ca 2 +/钙调蛋白依赖性激酶，能够通过磷酸化反应调节β-肾上腺素能受体功能，（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的唯一介质。
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.