GTPγS Regulation of a 12-Transmembrane Guanylyl Cyclase Is Retained after Mutation to an Adenylyl Cyclase

Abstract

DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mammalian adenylyl cyclases containing 12 transmembrane-spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essential for guanylyl cyclase activation by extracellular cAMP. In lysates, guanylyl cyclase activity is strongly stimulated by guanosine 5′-3-O-(thio) triphosphate (GTPγS), which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Expression of the obtained DdGCAkqd in adenylyl cyclase-defective cells restored the phenotype of the mutant. GTPγS stimulated the adenylyl cyclase activity of DdGCAkgd with properties similar to those of the wild-type enzyme (decrease of Km and increase of Vmax), demonstrating that GTPγS stimulation is independent of substrate specificity. Furthermore, GTPγS activation of DdGCAkqd is retained in several null mutants of Gα and Gβ proteins, indicating that GTPγS activation is not mediated by a heterotrimeric G-protein but possibly by a monomeric G-protein.