Heterotrimeric G protein Gαs subunit attenuates PLEKHG2, a Rho family-specific guanine nucleotide exchange factor, by direct interaction

Abstract

PLEKHG2 is a Gβγ-dependent guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42, and has been shown to mediate signalling pathways such as actin cytoskeleton reorganization and serum response element (SRE)-dependent gene transcription. Here we show that the constitutively active mutant of the Gαs subunit significantly attenuated PLEKHG2-induced SRE-mediated gene transcription. Strikingly, we observed that the constitutive activation of endogenous Gαs by treatment with CTx caused a similar inhibitory effect on PLEKHG2-induced activation of SRE. However, both the enforced expression of the catalytic subunit β of protein kinase A and the treatment with dibutyl-cyclic AMP failed to mimic the inhibitory effect of Gαs on PLEKHG2. Furthermore, the dominant negative mutant of protein kinase A had no effect on PLEKHG2-mediated SRE activation. Performing immunoprecipitation and an in vitro pulldown assay, we found that PLEKHG2 directly interacted with the active form of the Gαs subunit in cells. The interaction between PLEKHG2 and Gαs required the N-terminal region of PLEKHG2, which includes the DH domain, a functional domain of GEF, suggesting that Gαs directly masks the DH domain of PLEKHG2. In a previous study, we reported that Gβγ accelerates PLEKHG2-mediated SRE-dependent gene transcription. Interestingly, Gαs also inhibited the hyperactivation of SRE induced by the co-expression of Gβγ and PLEKHG2; however, Gαs and Gβγ bind to different regions of PLEKHG2. This is the first report to show that PLEKHG2 is a novel effector of Gαs, and is negatively regulated by the Gαs subunit through direct interaction.

Keywords: Gene transcription; Gαs/olf; Interaction; RhoGEF.