G Protein βγ Subunits Stimulate p114RhoGEF, a Guanine Nucleotide Exchange Factor for RhoA and Rac1: Regulation of Cell Shape and Reactive Oxygen Species Production

Document Type

Article

Publication Title

Circulation Research

Abstract

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 Gβγ subunits, transducin. We have determined that Gβγ subunits but not Gα subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that Gβγ subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, Gβγ 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 Gβγ 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.

First Page

848

Last Page

856

DOI

10.1161/01.RES.0000097607.14733.0C

Publication Date

10-31-2003

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