Hains Melinda D, Wing Michele R, Maddileti Savitri, Siderovski David P, Harden T Kendall
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7365, USA.
Mol Pharmacol. 2006 Jun;69(6):2068-75. doi: 10.1124/mol.105.017921. Epub 2006 Mar 22.
Because phospholipase C epsilon (PLC-epsilon) is activated by Galpha(12/13) and Rho family GTPases, we investigated whether these G proteins contribute to the increased inositol lipid hydrolysis observed in COS-7 cells after activation of certain G protein-coupled receptors. Stimulation of inositol lipid hydrolysis by endogenous lysophosphatidic acid (LPA) or thrombin receptors was markedly enhanced by the expression of PLC-epsilon. Expression of the LPA(1) or PAR1 receptor increased inositol phosphate production in response to LPA or SFLLRN, respectively, and these agonist-stimulated responses were markedly enhanced by coexpression of PLC-epsilon. Both LPA(1) and PAR1 receptor-mediated activation of PLC-epsilon was inhibited by coexpression of the regulator of G protein signaling (RGS) domain of p115RhoGEF, a GTPase-activating protein for Galpha(12/13) but not by expression of the RGS domain of GRK2, which inhibits Galpha(q) signaling. In contrast, activation of the G(q)-coupled M1 muscarinic or P2Y(2) purinergic receptor was neither enhanced by coexpression with PLC-epsilon nor inhibited by the RGS domain of p115RhoGEF but was blocked by expression of the RGS domain of GRK2. Expression of the Rho inhibitor C3 botulinum toxin did not affect LPA- or SFLLRN-stimulated inositol lipid hydrolysis in the absence of PLC-epsilon but completely prevented the PLC-epsilon-dependent increase in inositol phosphate accumulation. Likewise, C3 toxin blocked the PLC-epsilon-dependent stimulatory effects of the LPA(1), LPA(2), LPA(3), or PAR1 receptor but had no effect on the agonist-promoted inositol phosphate response of the M1 or P2Y(2) receptor. Moreover, PLC-epsilon-dependent stimulation of inositol phosphate accumulation by activation of the epidermal growth factor receptor, which involves Ras- but not Rho-mediated activation of the phospholipase, was unaffected by C3 toxin. These studies illustrate that specific LPA and thrombin receptors promote inositol lipid signaling via activation of Galpha(12/13) and Rho.
由于磷脂酶Cε（PLC-ε）可被Gα(12/13)和Rho家族GTP酶激活，我们研究了这些G蛋白是否参与某些G蛋白偶联受体激活后在COS-7细胞中观察到的肌醇脂质水解增加。内源性溶血磷脂酸（LPA）或凝血酶受体刺激引起的肌醇脂质水解，在PLC-ε表达后显著增强。LPA(1)或PAR1受体的表达分别使细胞对LPA或SFLLRN产生的肌醇磷酸生成增加，并且这些激动剂刺激的反应在共表达PLC-ε后显著增强。p115RhoGEF（一种Gα(12/13)的GTP酶激活蛋白）的G蛋白信号调节（RGS）结构域的共表达抑制了LPA(1)和PAR1受体介导的PLC-ε激活，但抑制Gα(q)信号的GRK2的RGS结构域的表达则无此作用。相反，与PLC-ε共表达既未增强G(q)偶联的M1毒蕈碱或P2Y(2)嘌呤能受体的激活，p115RhoGEF的RGS结构域也未对其产生抑制作用，但GRK2的RGS结构域的表达可阻断该激活。在不存在PLC-ε时，Rho抑制剂C3肉毒杆菌毒素的表达不影响LPA或SFLLRN刺激的肌醇脂质水解，但完全阻止了PLC-ε依赖的肌醇磷酸积累增加。同样，C3毒素阻断了LPA(1)、LPA(2)、LPA(3)或PAR1受体的PLC-ε依赖的刺激作用，但对M1或P2Y(2)受体的激动剂促进的肌醇磷酸反应无影响。此外，表皮生长因子受体激活引起的PLC-ε依赖的肌醇磷酸积累刺激，涉及Ras而非Rho介导的磷脂酶激活，不受C3毒素影响。这些研究表明，特定的LPA和凝血酶受体通过激活Gα(12/13)和Rho促进肌醇脂质信号传导。
