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

Abstract

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 PAR4mediated 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.

ZHX3 promotes the progression of urothelial carcinoma of the bladder via repressing of RGS2 and is a novel substrate of TRIM21

“…To date, as the most potent negative regulator of G q α, RGS2 has been reported to act as a tumor suppressor in certain human cancers, such as breast, prostate, ovarian, and bladder cancers 18,32‐34 . A previous study stated that RGS2 can inhibit the activation of RhoA by negatively regulating heterotrimeric G protein signaling 35 . As a classical oncogene, RhoA is generally considered to be tumorigenic 36 .…”

“…18,[32][33][34] A previous study stated that RGS2 can inhibit the activation of RhoA by negatively regulating heterotrimeric G protein signaling. 35 As a classical oncogene, RhoA is generally considered to be tumorigenic. 36 Similarly, high RhoA expression was closely associated with muscle invasion and metastasis in UCB.…”

Protease Activated Receptors and Arthritis

“…Nevertheless, regulators of G protein signalling (RGS) eventually recognize the receptor and form complexes to terminate its signalling. Specifically, RGS2 interacts with G q and RGS4 interacts with G 12/13 , inhibiting the downstream processes of these G proteins [126]. Upon internalization, PAR-4 is sorted between lysosomes for degradation or endosomes for recycling [122].…”

Acute and 28-days subacute toxicity studies of Gαq-RGS2 signaling inhibitor

“…Previous reported study synthesized the triazolone bearing compound Gαq-RGS2 signaling inhibitor and extensively evaluated the pharmacological activity against the urinary incontinence, which showed the beneficial activity in the urinary incontinence [ 4 ]. Furthermore, the Gαq-RGS2 signaling inhibitor has demonstrated Gαq signaling inhibitor activity by acting at the intersection of RGS2 and G-αq proteins, resulting in attenuated the Gαq signaling which reduced calcium fluxes and reduced muscle contraction [ 5 ]. In our previous study, we showed the beneficial effect of Gαq-RGS2 signaling inhibitor in the aminophylline induced cardiac arrhythmia [ 6 ].…”