Two cytoplasmic domains of mammalian adenylyl cyclase form a Gsα- and forskolin-activated enzyme in vitro

Abstract

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-Δ3, 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 Gsα- and forskolin-activated enzyme activity. The turnover number for Gsα- and forskolin-stimulated enzyme activity of the complex between IC1 and IIC2-Δ3 was 8.2 s-1. The concentration of IIC2-Δ3 to achieve half-maximal activation of IC1 was 0.8 and 1.3 μM when stimulated by forskolin and Gsα, respectively. The concentration of IIC2-Δ3 needed to complex with IC1 was reduced 10-fold (0.08 μM) when the enzyme was activated by both forskolin and Gsα, suggesting that Gsα and forskolin increased the affinity of the two cytoplasmic domains for each other.