Regulators of G-protein signaling, RGS2 and RGS4, inhibit protease-activated receptor 4-mediated signaling by forming a complex with the receptor and Gα in live cells

BACKGROUND: Protease-activated receptor 4 (PAR4) is a seven transmembrane G-protein coupled receptor (GPCR) activated by endogenous proteases, such as thrombin. PAR4 is involved in various pathophysiologies including cancer, inflammation, pain, and thrombosis. Although regulators of G-protein signaling (RGS) are known to modulate GPCR/Gα-mediated pathways, their specific effects on PAR4 are not fully understood at present. We previously reported that RGS proteins attenuate PAR1- and PAR2-mediated signaling through interactions with these receptors in conjunction with distinct Gα subunits. METHODS: We employed a bioluminescence resonance energy transfer technique and confocal microscopy to examine potential interactions among PAR4, RGS, and Gα subunits. The inhibitory effects of RGS proteins on PAR4-mediated downstream signaling and cancer progression were additionally investigated by using several assays including ERK phosphorylation, calcium mobilization, RhoA activity, cancer cell proliferation, and related gene expression. RESULTS: In live cells, RGS2 interacts with PAR4 in the presence of Gα(q) while RGS4 binding to PAR4 occurs in the presence of Gα(q) and Gα(12/13). Co-expression of PAR4 and Gα(q) induced a shift in the subcellular localization of RGS2 and RGS4 from the cytoplasm to plasma membrane. Combined PAR4 and Gα(12/13) expression additionally promoted translocation of RGS4 from the cytoplasm to the membrane. Both RGS2 and RGS4 abolished PAR4-activated ERK phosphorylation, calcium mobilization and RhoA activity, as well as PAR4-mediated colon cancer cell proliferation and related gene expression. CONCLUSIONS: RGS2 and RGS4 forms ternary complex with PAR4 in Gα-dependent manner and inhibits its downstream signaling. Our findings support a novel physiological function of RGS2 and RGS4 as inhibitors of PAR4-mediated signaling through selective PAR4/RGS/Gα coupling.