Identification of a G(iα) binding site on type V adenylyl cyclase

The stimulatory G protein α subunit G(sα) binds within a cleft in adenylyl cyclase formed by the α1-α2 and α3-β4 loops of the C₂ domain. The pseudosymmetry of the C₁ and C₂ domains of adenylyl cyclase suggests that the homologous inhibitory α subunit G(iα) could bind to the analogous cleft within C₁. We demonstrate that myristoylated guanosine 5'-3-O- (thio)triphosphate-G(iα1) forms a stable complex with the C₁ (but not the C₂) domain of type V adenylyl cyclase. Mutagenesis of the membrane-bound enzyme identified residues whose alteration either increased or substantially decreased the IC₅₀ for inhibition by G(iα1). These mutations suggest binding of G(iα) within the cleft formed by the α2 and α3 helices of C₁, analogous to the G(sα) binding site in C₂. Adenylyl cyclase activity reconstituted by mixture of the C₁ and C₂ domains of type V adenylyl cyclase was also inhibited by G(iα). The C(1b) domain of the type V enzyme contributed to affinity for G(iα), but the source of C₂ had little effect. Mutations in this soluble system faithfully reflected the phenotypes observed with the membrane-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase permits bidirectional regulation of activity by homologous G protein α subunits.