REGULATION OF RECEPTOR COUPLED ADENYLYLCYCLASE

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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.