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.
Wu
Z
Z
Department of Pharmacology, University of Washington, Seattle 98195.
Wong
S T
ST
Storms
D R
DR
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002384:Catalysis; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011494:Protein Kinases / Q000378:metabolism
D002147:Calmodulin; D009838:Oligodeoxyribonucleotides; D011494:Protein Kinases; D000262:Adenylyl Cyclases; D002118:Calcium