Interaction of G(sα) with the cytosolic domains of mammalian adenylyl cyclase

Forskolin- and G(sα)-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ~25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native M(r) ~ 120,000). The C(1a) domain from type V adenylyl cyclase (VC₁) and the C₂ domain from type II adenylyl cyclase (IIC₂) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 μmol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC₁, IIC₂, and guanosine 5'-O-(3-thio- triphosphate) (GTPγS)-G(sα) is responsible for the observed enzymatic activity and can be isolated. In addition, GTPγS-G(sα) interacts with homodimers of IIC₂ to form a heterodimeric complex (one molecule each of G(sα) and IIC₂) but not detectably with homodimers of VC₁. Nevertheless, G(sα) can be cross-linked to VC₁ in the activated heterotrimeric complex of VC₁, IIC₂, and G(sα), indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C₁ and C₂.