Regulation of the AGS4–Gαi Interaction by Chemokine Receptors and the Non‐Receptor Guanine Nucleotide Exchange Factor Ric‐8A

Abstract

Activator of G‐protein signaling‐4 (AGS4), by virtue of its three G‐protein regulatory (GPR) motifs, is a guanine nucleotide dissociation inhibitor (GDI) for GαiGDP subunits free of Gβγ. Our objective is to define the factors that regulate the GPR‐Gαi interaction. We developed a bioluminescence resonance energy transfer (BRET) system to monitor regulation of the AGS4‐Gαi interaction in live cells. Initially, our BRET system was used to assess the relative BRET signals generated between AGS4 and Gαi subunits Gαi1, Gαi2 and Gαi3. Due to its restricted expression in hematopoietic cell lineages, AGS4 is well positioned to modulate G‐protein signaling via G‐protein coupled chemokine receptors (GPCRs). Indeed, AGS4–Gαi complexes were regulated by CXCR4 activation. Interestingly, we also observed Gαi‐dependent interaction between AGS4 and CXCR4 suggesting that AGS4‐Gαi may interface directly with chemokine receptors, which has broad implications with respect to signal processing via chemokine GPCRs. In addition, we determined that the AGS4–Gαi complex is also regulated by the non‐receptor guanine nucleotide exchange factor (GEF) Ric‐8A, but not by the GEFs AGS1 or GIV/Girdin. These data indicate that the AGS4‐Gαi complex is subject to dynamic regulation by both chemokine receptors and Ric‐8A, and reveal novel insights into the unique role of accessory proteins in G‐protein signal processing.