Interaction of AGS4 and Gialpha1 in living cells

Abstract

Activator of G Protein Signaling‐4 (AGS4) is one of several receptor independent activators of G‐protein signaling identified in a yeast‐based functional screen of mammanlian cDNAs. AGS4 encodes a protein of 160 amino acids that contains 3 G‐protein regulatory (GPR) motifs each of which can serve as a docking site for Giα‐GDP free of Gβg. AGS4 and related GPR proteins provide unexpected regulatory mechanisms for G‐protein signaling systems. AGS4 is of particular interest in this regard as it is primarily expressed in the immune system. In addition to defining the functional role of AGS4 in immune cells, two of the key questions for AGS4 are what controls the interaction of these proteins with G‐protein and where does this interaction occur within the cell. As an initial approach to this question, we evaluated the interaction of AGS4 with Giα1 in living cells (HEK‐293) using bioluminescence resonance energy transfer (BRET) to measure interaction between proteins tagged with renilla luciferase (RLuc) and the YFP‐variant Venus. Cells transfected with AGS4‐YFP and Giα1‐RLuc exhibited a net BRET signal that was saturable and specific. The BRET signal was blocked by Gβγ expression and it was not observed with the AGS4‐Q/A mutant in which each of the GPR motifs are rendered incapable of binding Giα. In subsequent experiments, we examined the influence of various signaling systems on the AGS4‐Giα1 interaction. These data indicate a robust interaction of AGS4 and Giα1 in the living cell.