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.
