Two cytoplasmic domains of mammalian adenylyl cyclase form a Gs alpha- and forskolin-activated enzyme in vitro.
Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C1 and C2). The first cytoplasmic domain of type I enzyme (IC1) and the second cytoplasmic domain of type II enzyme (IIC2-delta 3, a construct in which 36 N-terminal amino acids of the C2 region are deleted) were expressed and purified to homogeneity. Alone, each had no adenylyl cyclase activity; however, mixing of the two domains in vitro resulted in Gs alpha- and forskolin-activated enzyme activity. The turnover number for Gs alpha- and forskolin-stimulated enzyme activity of the complex between IC1 and IIC2-delta 3 was 8.2 s-1. The concentration of IIC2-delta 3 to achieve half-maximal activation of IC1 was 0.8 and 1.3 microM when stimulated by forskolin and Gs alpha, respectively. The concentration of IIC2-delta 3 needed to complex with IC1 was reduced 10-fold (0.08 microM) when the enzyme was activated by both forskolin and Gs alpha, suggesting that Gs alpha and forskolin increased the affinity of the two cytoplasmic domains for each other.
Yan
S Z
SZ
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637, USA.
Hahn
D
D
Huang
Z H
ZH
Tang
W J
WJ
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D005576:Colforsin / Q000494:pharmacology*; D003593:Cytoplasm / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data
D005576:Colforsin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases