Dynamics of Gα_i1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of G_i-mediated inhibition of cAMP production

Many physiological and pathophysiological processes are regulated by cAMP. Different therapies directly or indirectly influence the cellular concentration of this second messenger. A wide variety of receptors either activates or inhibits adenylate cyclases in order to induce proper physiological responses. A key event in this signalling system is the direct and dynamic interaction of Gα_i1 subunits with adenylate cyclases.We established a FRET-based assay between G-protein subunits and AC5 (type 5 adenylate cyclase) andmonitored receptor-stimulated interactions between Gα_i1 and AC5 in single intact cells with high temporal resolution. We observed that FRET between Gα_i1 and AC5 developed at much lower concentration of agonist compared with the overall G_i-protein activity resulting in a left-shift of the concentration-response curve by approximately one order of magnitude. Furthermore, G_i1-protein-mediated attenuation of AC5-dependent increases in cAMP occurred at comparable low concentrations of agonist. On analysing the dynamics we found the dissociation of the Gα_i1 subunits and AC5 to occur significantly slower than the G-protein deactivation and to be insensitive to RGS4 (regulator of G-protein signalling type 4) expression. This led us to the conclusion that AC5, by binding active Gα_i1, interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.