G Protein betagamma subunits stimulate p114RhoGEF, a guanine nucleotide exchange factor for RhoA and Rac1: regulation of cell shape and reactive oxygen species production.
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.
D000199:Actins / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism* / Q000494:pharmacology; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism* / Q000494:pharmacology; D017930:Genes, Reporter; D020662:Guanine Nucleotide Exchange Factors / Q000172:deficiency / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D019255:NADPH Oxidases / Q000378:metabolism; D041681:NIH 3T3 Cells; D009928:Organ Specificity; D010750:Phosphoproteins / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D017382:Reactive Oxygen Species / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D027602:Serum Response Element / Q000235:genetics; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
C577230:ARHGEF18 protein, human; D000199:Actins; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020662:Guanine Nucleotide Exchange Factors; D008246:Lysophospholipids; D010750:Phosphoproteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D064067:Rho Guanine Nucleotide Exchange Factors; C079789:neutrophil cytosol factor 67K; D019255:NADPH Oxidases; D013917:Thrombin; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein