REGULATION OF RECEPTOR COUPLED ADENYLYLCYCLASE

该项目的目标是确定参与的分子机制， β-肾上腺素能受体（β AR）偶联的调节 腺苷酸环化酶 有三种β AR亚型，β 1 AR、β 2 AR和β 3 AR。 β 3AR，其可通过激动剂和其他 调制器。 一种调节机制是受体下调， 暴露于激动剂的细胞表现出β AR结合活性的丧失， 时间我们已经在两个实验中探索了β AR亚型的下调， 不同的细胞系。 大鼠C6神经胶质瘤细胞同时表达β 1 AR和β 2 AR。 β 2AR。当暴露于激动剂或毛喉素，我们观察到一个坐标 两种亚型的下调。 当我们量化 在β AR mRNA中，出现了不同的模式。 在激动剂和 毛喉素处理的细胞，β 1 AR mRNA水平呈现双相变化， 最初增加超过1.5倍1小时，然后下降至50% β 2 AR mRNA水平的变化呈双相性， 2小时后随时间降低至<50%对照。 分析，我们发现β AR mRNA的上调和下调 是由于基因转录速率的变化。 在这方面 β-AR mRNA的半衰期没有实质性改变， 处理的细胞。当蛋白质合成首次被阻断时，激动剂和 毛喉素处理导致β 1 AR mRNA上调4倍 6小时的水平，且β 2 AR mRNA无下调。 作为基因， 两种大鼠β AR亚型都有cAMP反应元件（CREs），我们提出， β 1 AR基因表达的上调是由cAMP介导的， 依赖性磷酸化和CRE结合蛋白的活化。 通过 相反，β AR基因转录的下调可能是由 通过诱导型cAMP早期阻遏物（ICER），CRE的一个成员 调节蛋白（CREM）家族的转录因子。 人SK-N-MC 神经瘤细胞同时表达β 1 AR和β 3 AR。 当细胞 激动剂作用下，β 1 AR表达下调，但β 1 AR表达无明显变化。 β 3AR结合位点。有趣的是，毛喉素治疗没有介导 β 1 AR下调，两种治疗均未导致β 1 AR下调， β 1 AR mRNA水平的调节。 由于人β 1 AR基因具有CREs， 这些结果是出乎意料的，但支持的概念，除了 对于β AR亚型之间的调节差异，有细胞- 具体差异。 初步研究表明，ICER 在C6细胞中而不是在SK-N-MC细胞中由激动剂诱导。

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.