REGULATION OF RECEPTOR COUPLED ADENYLYLCYCLASE

受体偶联腺苷酸环化酶的调节

• 批准号: 2579539
• 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/2579539
• 关键词: adenylate cyclase; beta adrenergic agent; beta adrenergic receptor; binding proteins; cyclic AMP; forskolin; human genetic material tag; human tissue; messenger RNA; nuclear runoff assay; phosphorylation; protein biosynthesis; receptor coupling; receptor expression; tissue /cell culture; transcription factor

The goal of this project is to identify molecular mechanisms involved in the regulation of the Beta-adrenergic receptor (betaAR)-coupled adenylylcyclase. There are three betaAR subtypes, beta1AR, beta2AR and beta3AR, that may be regulated differently by agonists and other modulators. One regulatory mechanism is receptor down-regulation whereby cells exposed to agonist exhibit a loss of betaAR binding activity with time. We have explored the down-regulation of betaAR subtypes in two different cell lines. Rat C6 glioma cells express both beta1AR and beta2AR. When exposed to agonist or forskolin, we observed a coordinate down-regulation of both subtypes. When we quantified the levels of betaAR mRNA, a different pattern emerged. In both agonist- and forskolin-treated cells, beta1AR mRNA levels exhibited a biphasic change, initially increasing over 1.5-fold by 1 hr, and then decreasing to 50% of control by 3 hr. The change in beta2 AR mRNA levels was monophasic, decreasing with time to <50% of control by 2 hr. Using nuclear run-on analysis, we showed that both the up- and down-regulation of betaAR mRNA were due to changes in gene transcription rate. In this regard, the half-life of either betaAR mRNA was not substantially altered in the treated cells. When protein synthesis was first blocked, both agonist and forskolin treatment resulted in a 4-fold up-regulation of beta1AR mRNA levels by 6 hr and no down-regulation of beta2AR mRNA. As the genes for both rat betaAR subtypes have cAMP responsive elements (CREs), we propose that the up-regulation of beta1AR gene expression is mediated by cAMP- dependent phosphorylation and activation of a CRE-binding protein. By contrast, down-regulation of betaAR gene transcription may be mediated by a inducible cAMP early repressor (ICER), a member of the CRE modulatory protein (CREM) family of transcription factors. Human SK-N-MC neurotumor cells express both beta1AR and beta3AR. When the cells were exposed to agonist, there was a down-regulation of beta1AR but not beta3AR binding sites. Interestingly, forskolin treatment did not mediate a down-regulation of beta1AR, and neither treatment caused a down- regulation of beta1AR mRNA levels. As the human beta1AR gene has CREs, these results were unexpected, but support the concept that in addition to differences in regulation among betaAR subtypes, there are cell- specific differences. Preliminary studies indicated that ICER was induced by agonist in C6 cells but not SK-N-MC cells.

该项目的目标是确定涉及到的分子机制 β-肾上腺素能受体偶联的调节 腺苷环化酶。有三种BetaAR亚型，Beta1AR、Beta2AR和 β3AR，它可能受激动剂和其他 调制器。一种调节机制是受体下调，从而 暴露于激动剂的细胞表现出与β-AR结合活性的丧失 时间到了。我们已经在两个方面探索了BetaAR亚型的下调 不同的细胞系。大鼠C6胶质瘤细胞表达β1受体和 Beta2AR。当接触激动剂或Forsklin时，我们观察到一个坐标 下调这两个子类型的监管。当我们量化这些物质的水平 BetaAR mRNA，出现了一种不同的模式。在激动剂-和 Forsklin处理细胞后，β1AR基因表达水平呈现双相变化， 最初在1小时内增加1.5倍以上，然后减少到50% 控制的时间缩短3小时。β2受体基因表达水平呈单相性变化。 随着时间的推移，在2小时内达到50%的控制率。使用核补给 分析表明，β-AR基因的表达上调和下调 是由于基因转录速率的变化。在这方面， 任何一种BetaAR mRNA的半衰期在 处理过的细胞。当蛋白质合成第一次被阻止时，激动剂和 Forsklin治疗后β-1AR基因表达上调4倍 6小时后，不下调β2AR基因的表达。因为这些基因 我们认为，这两种大鼠βAR亚型都有cAMP反应元件(CRE) β1AR基因表达上调是由cAMP介导的。 依赖于Cre结合蛋白的磷酸化和激活。通过 相反，β-AR基因转录的下调可能是由 由Cre成员可诱导的cAMP早期抑制物(ICER) 调节蛋白(CREM)家族的转录因子。人SK-N-MC 神经肿瘤细胞同时表达β-1AR和β-3AR。当细胞处于 暴露于激动剂后，β1AR表达下调，但不是 β3AR结合位点。有趣的是，福斯科林治疗并没有起到调节作用 β1AR的下调，两种治疗都没有引起下调- β1AR基因表达水平的调控。由于人类的Beta1AR基因有CRES， 这些结果是意想不到的，但支持了另外一个概念 对于不同β-AR亚型之间的调控差异，存在细胞- 具体区别。初步研究表明，ICER是 激动剂诱导的C6细胞，而不是SK-N-MC细胞。