Niu Jiaxin, Profirovic Jasmina, Pan Haiyun, Vaiskunaite Rita, Voyno-Yasenetskaya Tatyana
Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, Ill 60612, USA.
Circ Res. 2003 Oct 31;93(9):848-56. doi: 10.1161/01.RES.0000097607.14733.0C. Epub 2003 Sep 25.
Rho GTPases integrate the intracellular signaling in a wide range of cellular processes. Activation of these G proteins is tightly controlled by a number of guanine nucleotide exchange factors (GEFs). In this study, we addressed the functional role of the recently identified p114RhoGEF in in vivo experiments. Activation of endogenous G protein-coupled receptors with lysophosphatidic acid resulted in activation of a transcription factor, serum response element (SRE), that was enhanced by p114RhoGEF. This stimulation was inhibited by the functional scavenger of Gbetagamma subunits, transducin. We have determined that Gbetagamma subunits but not Galpha subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that Gbetagamma subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, Gbetagamma subunits stimulated SRE activity induced by full-length and DH/PH domain of p114RhoGEF. Using in vivo pull-down assays and dominant-negative mutants of Rho GTPases, we have determined that p114RhoGEF activated RhoA and Rac1 but not Cdc42 proteins. Functional significance of RhoA activation was established by the ability of p114RhoGEF to induce actin stress fibers and cell rounding. Functional significance of Rac1 activation was established by the ability of p114RhoGEF to induce production of reactive oxygen species (ROS) followed by activation of NADPH oxidase enzyme complex. In summary, our data showed that the novel guanine nucleotide exchange factor p114RhoGEF regulates the activity of RhoA and Rac1, and that Gbetagamma subunits of heterotrimeric G proteins are activators of p114RhoGEF under physiological conditions. The findings help to explain the integrated effects of LPA and other G-protein receptor-coupled agonists on actin stress fiber formation, cell shape change, and ROS production.
Rho GTP酶在广泛的细胞过程中整合细胞内信号传导。这些G蛋白的激活受到多种鸟嘌呤核苷酸交换因子（GEF）的严格控制。在本研究中，我们在体内实验中探讨了最近鉴定出的p114RhoGEF的功能作用。用溶血磷脂酸激活内源性G蛋白偶联受体导致转录因子血清反应元件（SRE）的激活，p114RhoGEF可增强这种激活。这种刺激被Gβγ亚基的功能性清除剂转导素抑制。我们已经确定异源三聚体G蛋白的Gβγ亚基而非Gα亚基刺激了p114RhoGEF依赖性的SRE活性。使用免疫共沉淀分析，我们已经确定Gβγ亚基与p114RhoGEF的全长和DH/PH结构域相互作用。同样，Gβγ亚基刺激了由p114RhoGEF的全长和DH/PH结构域诱导的SRE活性。使用体内下拉分析和Rho GTP酶的显性负性突变体，我们已经确定p114RhoGEF激活了RhoA和Rac1，但未激活Cdc42蛋白。p114RhoGEF诱导肌动蛋白应激纤维和细胞变圆的能力确立了RhoA激活的功能意义。p114RhoGEF诱导活性氧（ROS）产生并随后激活NADPH氧化酶复合物的能力确立了Rac1激活的功能意义。总之，我们的数据表明新型鸟嘌呤核苷酸交换因子p114RhoGEF调节RhoA和Rac1的活性，并且在生理条件下异源三聚体G蛋白的Gβγ亚基是p114RhoGEF的激活剂。这些发现有助于解释溶血磷脂酸和其他G蛋白受体偶联激动剂对肌动蛋白应激纤维形成、细胞形状改变和ROS产生的综合作用。
