GTP gamma S regulation of a 12-transmembrane guanylyl cyclase is retained after mutation to an adenylyl cyclase

DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mamma lian adenylyl cyclases containing 12 transmembrane-spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essen tial for guanylyl cyclase activation by extracellular cAMP. In lysates, gua nylyl cyclase activity is strongly stimulated by guanosine 5'-3-O-(thio) tr iphosphate (GTP gammaS), which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Exp ression of the obtained DdGCA(kqd) in adenylyl cyclase-defective cells rest ored the phenotype of the mutant. GTP gammaS stimulated the adenylyl cyclas e activity of DdGCAkqd with properties similar to those of the wild-type en zyme (decrease of K-m and increase of V-max), demonstrating that GTP gammaS stimulation is independent of substrate specificity. Furthermore, GTP gamm aS activation of DdGCAkqd is retained in several null mutants of Ga and G b eta proteins, indicating that GTP gammaS activation is not mediated by a he terotrimerie G-protein but possibly by a monomeric G-protein.