Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins

A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G_q-coupled receptors exert their effects primarily via Ca²⁺. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)–cAMPS (adenosine 3′,5′-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca²⁺ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A₂ activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G_i-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G_i-proteins and AC5. FRET between Gα_i1 and AC5 developed at much lower concentration of agonist compared with the overall G_i-protein activity. We found the dissociation of Gα_i1 subunits and AC5 to occur slower than the G_i-protein deactivation. 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.