Novel regulatory properties of adenylate cyclase type 6

Several studies have examined the regulation of recombinant adenylate cyclase type 6 (AC6) in vitro. Both PKC phosphorylation and calcium inhibit AC6 in vitro. In cultured cells or in tissues, neither PKC activation nor calcium release has been demonstrated to inhibit AC6. Furthermore, downstream effectors of PKC and calcium, Raf1 and calmodulin, respectively, have been implicated in the heterologous sensitization of AC6, suggesting that both PKC and calcium signaling may differentially regulate AC6 in vitro vs. in intact cells. To specifically investigate the regulation of AC6 by PKC, we constructed HEK293 cells that stably express recombinant AC6. PKC activation in intact cells enhances AC6 activation and the positive effects of PKC on AC6 activity are Raf1-dependent. Activation of Raf1 via receptor tyrosine kinases also enhances AC6 activation and AC6 and Raf1 may interact physically in intact cells. Subsequent studies investigated the ability of Gαqcoupled receptors upstream of PKC to potentiate AC6. Activation of endogenous Gαqcoupled muscarinic receptors in HEK-AC6 cells potentiates drug-stimulated cyclic AMP accumulation, however this potentiation was independent of PKC. Chelation of intracellular calcium or antagonism of calmodulin attenuated the effects of Gαq-coupled muscarinic receptors on AC6 activation. These studies suggest that although calcium directly inhibits AC6, Gαq-mediated release of intracellular calcium potentiates AC6 activation. To investigate the role of these novel signaling mechanisms in heterologous sensitization of AC6, we constructed HEK-AC6–D2L cells. AC6 is robustly sensitized after pretreatment (2 hr) with the D2L agonist, quinpirole. Neither inhibition of PKC nor Raf1 effectively attenuated D2L-mediated sensitization of AC6. However, activation of PKC robustly potentiated D2L-receptor mediated sensitization without altering acute D2L-mediated inhibition of AC6. The potentiation of sensitization of AC6 by PKC was blocked by inhibition of novel PKC isoforms and Raf1. These data demonstrate that although PKC and Raf1 are not required for heterologous sensitization of AC6, they robustly potentiate the magnitude of sensitization. Due to the broad expression pattern of AC6 in human tissue, these studies may aid in understanding the pathology and treatment of neuronal disorders and cardiac disease