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 chemoattractant receptor and guanosine 5'-3-O-(thio)triphosphate stimulation. Both classes of mutants harbored substitutions within the cytoplasmic C1a domain. Mutations that inactivated the enzyme were often at highly conserved 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 and regulation of this important class of enzymes.
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Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
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D000262:Adenylyl Cyclases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion
D017931:DNA Primers; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases