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Regulation of the GPR‐Gαi Signaling Complex by a Non‐Receptor Guanine Nucleotide Exchange Factor, Ric‐8A

Group II Activators of G‐protein Signaling (AGS) serve as alternative binding partners for Gαi via one or more G‐protein regulatory motifs (GPR). We recently reported the regulation of the GPR‐Gαi signaling module by a cell surface receptor and asked if the GPR‐Gαi signaling module was also regulated by the non‐receptor guanine nucleotide exchange factor Ric‐8 in this system. Interaction of AGS3‐ or AGS4‐Rluc with Gαi1‐YFP was monitored in HEK cells by BRET. AGS‐Rluc·Gαi1‐YFP BRET was regulated by coexpression of Ric‐8A but not Ric‐8B, and this regulation was dependent upon the stoichiometry of the expressed proteins. AGS‐Rluc·Gαi1‐YFP BRET was observed in both membrane and cytosolic fractions. Membrane AGS‐Rluc·Gαi1‐YFP BRET was regulated by the α2A/D‐AR, whereas both membrane and cytosolic AGS‐Rluc·Gαi1‐YFP BRET were inhibited by Ric‐8A. Ric‐8A expression also increased the amount of Gαi1‐YFP in the cells. The effect of Ric‐8A on AGS‐Rluc·Gαi1‐YFP BRET, but not its effect on Gαi1‐YFP expression, was blocked by pertussis toxin treatment. Receptor‐ and non‐receptor GEF‐mediated regulation of the AGS·Gαi complex may serve to mediate distinct signaling events at different locations within the cell.