Modification of the calcium and calmodulin sensitivity of the type I adenylyl cyclase by mutagenesis of its calmodulin binding domain.

The type I adenylyl cyclase is directly stimulated by Ca2+ and calmodulin in vitro, and the enzyme is also stimulated by increases in intracellular Ca2+ in vivo. Ca2+ stimulation of the enzyme in vivo may be due to direct interactions of the enzyme with Ca2+ and calmodulin or to an indirect mechanism involving stimulation of the enzyme by Ca(2+)-activated protein kinases. In this study, we have made several point mutations within the calmodulin binding domain to determine if the Ca2+ sensitivity of the enzyme can be modified by mutagenesis. The catalytic activities of the mutant enzymes were comparable to wild type type I adenylyl cyclase. Substitution of Cys-507 with Ser-507 did not have significant effects on the calmodulin or Ca2+ sensitivity of the enzyme. However, replacement of Lys-504 with Asp caused a 4-fold decrease in sensitivity to Ca2+. Ca2+ and calmodulin stimulation were abolished by substitution of Phe-503 with Arg-503. Stimulation of type I adenylyl cyclase activity in vivo by intracellular Ca2+ was also greatly diminished with the Arg-503 mutant indicating that Ca2+ stimulation of the enzyme in vivo is due primarily to direct interactions with calmodulin and Ca2+. These data demonstrate that the Ca2+ sensitivity of this enzyme can be modulated by point mutagenesis within the putative calmodulin binding domain and indicate that the enzyme can be directly regulated by Ca2+ and calmodulin in vivo.