2 CYTOPLASMIC DOMAINS OF MAMMALIAN ADENYLYL-CYCLASE FORM A G(S-ALPHA)-ACTIVATED AND FORSKOLIN-ACTIVATED ENZYME IN-VITRO

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C -1 and C-2). The first cytoplasmic domain of type I enzyme (IC1) and t he second cytoplasmic domain of type II enzyme (IIC2-Delta 3, a constr uct in which 36 N-terminal amino acids of the C-2 region are deleted) were expressed and purified to homogeneity. Alone, each had no adenyly l cyclase activity; however, mixing of the two domains in vitro result ed in G(s alpha)- and forskolin activated enzyme activity. The turnove r number for G(s alpha)- and forskolin-stimulated enzyme activity of t he complex between IC1 and IIC2-Delta 3 was 8.2 s(-1). The concentrati on of IIC2-Delta 3 to achieve half-maximal activation of IC1 was 0.8 a nd 1.3 mu M when stimulated by forskolin and G(s alpha), respectively. The concentration of IIC2-Delta 3 needed to complex with IC1 was redu ced 10-fold (0.08 mu M) when the enzyme was activated by both forskoli n and G(s alpha), suggesting that G(s alpha) and forskolin increased t he affinity of the two cytoplasmic domains for each other.