Published online before print September 25, 2003, doi: 10.1161/01.RES.0000097607.14733.0C
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© 2003 American Heart Association, Inc.
Cellular Biology |
G Protein ß
Subunits Stimulate p114RhoGEF, a Guanine Nucleotide Exchange Factor for RhoA and Rac1
Regulation of Cell Shape and Reactive Oxygen Species Production
From the Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, Ill.
Correspondence to Tatyana Voyno-Yasenetskaya, University of Illinois, Department of Pharmacology (MC 868), 835 S Wolcott Ave, Chicago, IL 60612. E-mail tvy{at}uic.edu
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 Gß
subunits, transducin. We have determined that Gß subunits but not G
subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that Gß subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, Gß 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 Gß 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.
Key Words: Rho GTPases • guanine nucleotide exchange factor • actin cytoskeleton • serum response element • reactive oxygen species
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