Mechanism of Gαi-mediated inhibition of type V adenylyl cyclase

The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C-1 and C-2). The stimulatory G protein, Galpha(s), binds within a cleft in the C-2 domain of adenylyl cyclase while Galpha(i) binds within the opposite cleft in the C-2 domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Galpha(i). or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another. We show herein that Galpha(i) reduces C-1/C-2 domain interaction and thus formation of the adenylyl cyclase catalytic site. Mutants that increase the affinity of C-1 for C-2 decrease the ability of Gai to inhibit the enzyme. In addition, Galpha(i) can influence binding of molecules to the catalytic site, which resides at the C-1/C-2 interface. Adenylyl cyclase can bind substrate analogs in the presence of Galpha(i) but cannot simultaneously bind Galpha(i) and transition state analogs such as 2'd3'-AMP. Galpha(i) also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs. Thus homologous G protein alpha-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme.