Construction of a soluble adenylyl cyclase activated by Gs alpha and forskolin.
A soluble adenylyl cyclase was constructed by linkage of portions of the cytosolic domains of the mammalian type I and type II enzymes. The soluble enzyme was stimulated by both forskolin and the alpha subunit of the heterotrimeric guanine nucleotide-binding protein (G protein) Gs (Gs alpha). Expression of the construct complemented the catabolic defect in a strain of Escherichia coli that is deficient in adenylyl cyclase activity. The active, approximately 60-kilodalton enzyme accumulated in the cytoplasmic fraction of E. coli to yield activities in excess of 1 nanomole per minute per milligram of protein. The two sets of transmembrane helices of mammalian adenylyl cyclases are thus not necessary for the catalytic or the most characteristic regulatory activities of the enzyme. This system may be useful for both genetic and biochemical analysis of G protein-regulated adenylyl cyclases.
Tang
W J
WJ
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
Gilman
A G
AG
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008969:Molecular Sequence Data; D012995:Solubility; D014170:Transformation, Genetic
D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases