Innovative Approach Reveals How Receptors Control G Protein State Equilibria.

G protein-coupled receptors (GPCRs) constitute the largest family of drug targets, acting through heterotrimeric G proteins. Agonist bound GPCRs promote GDP release from G proteins allowing their activation upon GTP binding. Here, we analyze this process with innovative tools that detect the main states of native Gi proteins: the GDP-bound, the open nucleotide-free and the active GTP-bound states. We show that receptor activation increases the amount of open-state Gi proteins, consistent with GDP release from inactive Gi proteins. We also observe that the active receptor can release GTP from the active Gi protein, to return to an inactive open state. Consistently, receptor activation also promotes the formation of the Gαβγ complex from the GTP-bound state in cells, in agreement with the formation of the receptor-G protein ternary complex. These data suggest that the amount of active GTP-bound form produced upon GPCR activation can be controlled by the receptor-induced GTP release. This control of G protein state equilibrium that depends on the receptor subtype may affect coupling efficacy. These data reveal receptor-dependent G-protein state equilibria and identify GTP release as an additional, context-dependent control point that can shape apparent coupling efficacy.