Genetic selection of mammalian adenylyl cyclases insensitive to stimulation by Gsalpha.
We describe the development of a genetic system allowing for the isolation of mutant mammalian adenylyl cyclases defective in their responses to G protein subunits, thus allowing for the identification of structural elements within the cyclase that are responsible for the recognition of these regulators. Expression of mammalian type V adenylyl cyclase in a cyclase-deleted yeast strain can conditionally complement the lethal phenotype of this strain. Type V adenylyl cyclase-expressing yeast grow only when the cyclase is activated by coexpression of Gsalpha or addition of forskolin to the medium; however, growth arrest is observed in the presence of both activators or under basal conditions. Utilizing this genetic system, we have isolated 25 adenylyl cyclase mutants defective in their response to Gsalpha. Sequence analysis and biochemical characterization of these mutants have identified residues in both cytoplasmic domains of the cyclase that are involved in the specific binding of and regulation by Gsalpha.
Zimmermann
G
G
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0636, USA.
Zhou
D
D
Taussig
R
R
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012641:Selection, Genetic; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases