IDENTIFICATION OF A G(I-ALPHA) BINDING-SITE ON TYPE-V ADENYLYL-CYCLASE

The stimulatory G protein alpha subunit G(s alpha) binds within a clef t 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 domain s of adenylyl cyclase suggests that the homologous inhibitory alpha su bunit G(i alpha) could bind to the analogous cleft within C-1. We demo nstrate that myristoylated guanosine 5'-3-O-(thio)triphosphate-G(i alp ha 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 iden tified residues whose alteration either increased or substantially dec reased the IC50 for inhibition by G(i alpha 1). These mutations sugges t binding of G(i alpha) within the cleft formed by the alpha 2 and alp ha 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). T he C-1b domain of the type V enzyme contributed to affinity for G(i al pha), but the source of C-2 had little effect. Mutations in this solub le system faithfully reflected the phenotypes observed with the membra ne-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase p ermits bidirectional regulation of activity by homologous G protein al pha subunits.