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. 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. In live cells, RGS2 interacts with PAR4 in the presence of Gαq while RGS4 binding to PAR4 occurs in the presence of Gαq and Gα12/13. Co-expression of PAR4 and Gαq induced a shift in the subcellular localization of RGS2 and RGS4 from the cytoplasm to plasma membrane. Combined PAR4 and Gα12/13 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. 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.

中文翻译：

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G 蛋白信号传导调节剂 RGS2 和 RGS4 通过在活细胞中与受体和 Gα 形成复合物来抑制蛋白酶激活受体 4 介导的信号传导。

蛋白酶激活受体 4 (PAR4) 是一种七跨膜 G 蛋白偶联受体 (GPCR)，由内源性蛋白酶（如凝血酶）激活。PAR4 参与各种病理生理学，包括癌症、炎症、疼痛和血栓形成。尽管已知 G 蛋白信号传导 (RGS) 的调节剂可调节 GPCR/Gα 介导的通路，但目前尚未完全了解它们对 PAR4 的具体影响。我们之前报道过，RGS 蛋白通过与这些受体以及不同 Gα 亚基的相互作用减弱了 PAR1 和 PAR2 介导的信号传导。我们采用生物发光共振能量转移技术和共聚焦显微镜来检查 PAR4、RGS 和 Gα 亚基之间的潜在相互作用。通过使用多种测定法，包括 ERK 磷酸化、钙动员、RhoA 活性、癌细胞增殖和相关基因表达，还研究了 RGS 蛋白对 PAR4 介导的下游信号传导和癌症进展的抑制作用。在活细胞中，RGS2 在 Gαq 存在下与 PAR4 相互作用，而 RGS4 在 Gαq 和 Gα12/13 存在下与 PAR4 结合。PAR4 和 Gαq 的共表达诱导 RGS2 和 RGS4 的亚细胞定位从细胞质到质膜的转变。PAR4 和 Gα12/13 的联合表达还促进了 RGS4 从细胞质到膜的易位。RGS2 和 RGS4 都消除了 PAR4 激活的 ERK 磷酸化、钙动员和 RhoA 活性，以及​​ PAR4 介导的结肠癌细胞增殖和相关基因表达。RGS2 和 RGS4 以 Gα 依赖性方式与 PAR4 形成三元复合物并抑制其下游信号传导。我们的研究结果支持 RGS2 和 RGS4 作为 PAR4 介导的信号传导抑制剂通过选择性 PAR4/RGS/Gα 偶联的新生理功能。