Dynamics of Gαi1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of Gi-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 G alpha(i1) subunits with adenylate cyclases. We established a FRET-based assay between G-protein subunits and AC5 (type 5 adenylate cyclase) and monitored receptor-stimulated interactions between G alpha(i1) and AC5 in single intact cells with high temporal resolution. We observed that FRET between G alpha(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 G alpha(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 G alpha(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.