Receptor-regulated Interaction of Activator of G-protein Signaling-4 and Gαi

Activator of G-protein signaling-4 (AGS4), via its three G-protein regulatory motifs, is well positioned to modulate G-protein signal processing by virtue of its ability to bind G alpha(i)-GDP subunits free of G beta gamma. Apart from initial observations on the biochemical activity of the G-protein regulatory motifs of AGS4, very little is known about the nature of the AGS4-G-protein interaction, how this interaction is regulated, or where the interaction takes place. As an initial approach to these questions, we evaluated the interaction of AGS4 with G alpha(i1) in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and G alpha(i1) reciprocally tagged with either Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) demonstrated saturable, specific BRET signals. BRET signals observed between AGS4-RLuc and G alpha(i1)-YFP were reduced by G-protein-coupled receptor activation, and this agonist-induced reduction in BRET was blocked by pertussis toxin. In addition, specific BRET signals were observed for AGS4-RLuc and alpha(2)-adrenergic receptor-Venus, which were G alpha(i)-dependent and reduced by agonist, indicating that AGS4-G alpha(i) complexes are receptor-proximal. These data suggest that AGS4-G alpha(i) complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation.