Identification of a Giα binding site on type V adenylyl cyclase

The stimulatory G protein alpha subunit G(s alpha) binds within a cleft in adenylyl cyclase formed by the alpha 1-alpha 2 and alpha 3-beta 4 loops of the C-2 domain. The pseudosymmetry of the C-1 and C-2 domains of adenylyl cyclase suggests that the homologous inhibitory alpha subunit G(i alpha) could bind to the analogous cleft within C-1. We demonstrate that myristoylated guanosine 5'-3-O-(thio)triphosphate-G(i alpha 1) forms a stable complex with the C-1 (but not the C-2) 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(i alpha 1). These mutations suggest binding of G(i alpha) within the cleft formed by the alpha 2 and alpha 3 helices of C-1, analogous to the G(s alpha) binding site in C-2. Adenylyl cyclase activity reconstituted by mixture of the C-1 and C-2 domains of type V adenylyl cyclase was also inhibited by G(i alpha). The C-1b domain of the type V enzyme contributed to affinity for G(i alpha), but the source of C-2 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 alpha subunits.