REGULATION OF RECEPTOR COUPLED ADENYLYLCYCLASE

受体偶联腺苷酸环化酶的调节

• 批准号: 3782329
• 负责人: P H FISHMAN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3782329
• 关键词: adenylate cyclase; beta adrenergic agent; beta adrenergic receptor; clone cells; dopamine receptor; human tissue; phorbols; phosphorylation; protein kinase A; protein kinase C; protein structure function; receptor binding; receptor coupling; receptor expression; receptor sensitivity; tissue /cell culture; transfection; western blottings

The goal of this project is to identify molecular mechanisms involved in the regulation of receptor~coupled adenylylcyclase (AC). 1) Activation of protein kinase C (PKC) by phorbol esters in different types of cells is known to result in either a desensitization or a potentiation of the receptor~coupled AC. Although the underlying basis for these opposing effects is unknown, it has been suggested that they may be mediated by different forms of PKC. We now report that exposing human neurotumor SK~N~MC cells to phorbol esters resulted in both a potentiation of AC activity and a desensitization of their beta1- adrenergic receptors (beta1AR). Using several biochemical approaches, we established that the potentiation did not involve the G proteins, Gs and Gi, which regulate AC, but most likely the catalyst itself. Interestingly, SK~N~MC also express D1 dopamine receptors which were not desensitized by phorbol ester treatment. Based on Western blotting, SK~N~MC cells expressed only one phorbol ester~sensitive PKC, PKC-alpha. When the cells were exposed to phorbol ester, PKC~alpha was rapidly translocated from cytosol to cell membrane. We propose that the type of AC may determine whether or not potentiation by PKC occurs. This may have important implications for the mechanisms by which different cell signaling systems cross~regulate each other. 2) We have been able to confirm and extend our previous evidence that human beta1AR and beta2AR are regulated differently by agonists. Stably transfected hamster cell lines were constructed which expressed either subtype at different levels. When exposed to agonist, the cells expressing either high or low levels of beta2AR exhibited a rapid, typical pattern of desensitization of agonist~stimulated AC. Both maximum stimulation (Vmax) was reduced and dose response (Kact) was shifted to lower sensitivity. By contrast, agonist~treated cells expressing high levels of beta1AR displayed no reduction in Vmax, and cells expressing low levels only a slow, modest reduction. Both cell lines, however, exhibited a shift in Kact. It is believed that the latter is mediated by protein kinase A via phosphorylation of the third intracellular loop of the receptors. The reduction in Vmax is believed to be mediated by the beta~adrenergic receptor kinase via phosphorylation of the C~terminus. The difference in desensitization between the two human betaAR subtypes may relate to tructural differences in their C~termini which are highly divergent.

该项目的目标是确定参与的分子机制 受体偶联腺苷酸环化酶（AC）的调节。 第一章 佛波酯对不同组织中蛋白激酶C（PKC）的激活作用 已知细胞类型导致脱敏或 受体偶联AC的增强。虽然基础 由于这些相反的影响是未知的，有人认为， 可能由不同形式的PKC介导。 我们现在报告， 人神经瘤SK~N~MC细胞对佛波酯的反应， AC活性增强和β 1- 肾上腺素能受体（β 1 AR）。使用几种生物化学方法， 我们确定增强作用不涉及G蛋白， Gs和Gi，它们调节AC，但最有可能是催化剂本身。 有趣的是，SK~N~MC也表达D1多巴胺受体， 不被佛波醇酯治疗脱敏。 基于西方 免疫印迹法显示SK~N~MC细胞只表达一种佛波酯敏感性PKC， 蛋白激酶C-α 当细胞暴露于佛波酯时，PKC~ α被激活， 从细胞质迅速转移到细胞膜。我们建议 AC的类型可以决定是否发生PKC增强。 这可能对以下机制具有重要意义： 不同的细胞信号系统相互交叉调节。 2)我们有 能够证实并扩展我们先前的证据，即人类 β 1 AR和β 2 AR受激动剂的调节不同。稳定 构建转染的仓鼠细胞系，其表达 不同层次的子类型。 当暴露于激动剂时，细胞 表达高水平或低水平β 2 AR的人表现出快速， 激动剂刺激AC的典型脱敏模式。 两 最大刺激（Vmax）降低，剂量反应（Kact） 降低敏感度。 相反，激动剂处理的细胞 表达高水平的β 1 AR没有显示Vmax降低，并且 细胞表达水平低，只是缓慢，适度的减少。 两种细胞 然而，线表现出Kact的变化。 据信 后者是由蛋白激酶A通过磷酸化第三 受体的细胞内环。 Vmax的降低被认为是 由β-肾上腺素能受体激酶介导， C末端的磷酸化。 脱敏的区别在于 两种人类β AR亚型之间的差异可能与结构性 C-末端的差异是高度分歧的。