Isolation of inactive and G protein-resistant adenylyl cyclase mutants using random mutagenesis

We used random mutagenesis and phenotypic rescue of adenylyl cyclase-null Dictyostelium cells to isolate loss-of-function mutations in the enzyme. Mutants were (i) catalytically inactive or (ii) resistant to chemoattrac-tant receptor and guanosine 5*-3-O-(thio)triphosphate stimulation. Both classes of mutants harbored substitu-tions within the cytoplasmic C1a domain. Mutations that inactivated the enzyme were often at highly con-served positions. Those that blocked activation were grouped in two distinct regions: one close to the plane of the plasma membrane and another halfway within the C1 loop. Missense mutations or deletions within the transmembrane domains resulted in missorting of the protein. Our screen provides a simple and efficient method to separately define the sites of catalysis an