REGULATION OF RECEPTOR COUPLED ADENYLYLCYCLASE

这个项目的目标是确定相关的分子机制。 在受体偶联的腺苷环化酶(AC)的调节中。1) 佛波酯在不同条件下对蛋白激酶C的激活作用 已知不同类型的细胞会导致脱敏或 受体偶联AC的增强作用。尽管潜在的基础是 因为这些相反的影响是未知的，所以有人认为它们 可能由不同形式的PKC介导。我们现在报道，揭露 人神经肿瘤SK~N~MC细胞对佛波醇酯的诱导作用 增强AC活性及其β_1-脱敏作用 肾上腺素能受体(β1AR)。使用几种生化方法， 我们确定这种增强作用不涉及G蛋白， GS和GI调节AC，但最有可能的是催化剂本身。 有趣的是，SK~N~MC也表达D1多巴胺受体 未经佛波酯处理脱敏的。基于西部 免疫印迹显示SK~N~MC细胞仅表达一种佛波酯敏感的PKC， PKC-α。当细胞暴露在佛波酯中时，PKC~α 迅速地从胞浆转移到细胞膜。我们建议， AC的类型可以决定是否发生PKC的增强作用。 这可能对以下机制有重要影响： 不同的细胞信号系统相互交叉调节。2)我们有 能够证实和扩展我们之前的证据，人类 β_1AR和β_2AR受激动剂的调节不同。稳定地 构建了转基因仓鼠细胞系，表达了 不同级别的亚型。当暴露在激动剂中时，细胞 表达高水平或低水平的Beta2AR表现出快速的， 激动剂刺激AC的典型脱敏模式。两者都有 最大刺激(Vmax)降低，剂量反应(Kact) 转向更低的敏感度。相比之下，激动剂处理的细胞 表达高水平的Beta1AR不会降低Vmax，并且 表达低水平的细胞只会缓慢、适度地减少。两个单元格 然而，台词在Kact中表现出了转变。据信， 后者是由蛋白激酶A通过第三个蛋白的磷酸化而介导的 受体的细胞内环。Vmax的降低被认为是 通过β-肾上腺素能受体激酶介导 C~末端的磷酸化。脱敏作用的差异 两种人类BetaAR亚型之间的关系可能与结构有关 它们在C~末端的差异是高度分化的。

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.