Constitutively active adenylyl cyclase mutant requires neither G proteins nor cytosolic regulators.
Receptor-mediated G protein-linked adenylyl cyclase systems are universal signal transducers. We exploited the essential role of this cascade in Dictyostelium development to screen for random mutations in the catalytic component, ACA. This enzyme is activated by G protein betagamma-subunits acting in concert with a novel cytosolic regulator, CRAC. By suppression of the CRAC-null phenotype, we isolated constitutively active versions of the enzyme that require neither exogenous stimuli nor internal regulators. One mutant displayed a 15-fold increase in its Vmax. It harbors a single amino acid substitution (L394S) affecting a conserved residue located in the first cytoplasmic loop near the N-terminal hydrophobic domain of ACA. The screening procedure can be adapted for isolation of constitutive mutations in mammalian adenylyl cyclases.
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Department of Biological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
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D000262:Adenylyl Cyclases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000378:metabolism; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D010641:Phenotype; D017354:Point Mutation; D015800:Protozoan Proteins; D015398:Signal Transduction
D005656:Fungal Proteins; D015800:Protozoan Proteins; C089420:dagA protein, Dictyostelium; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases