Abstract

P114RhoGEF/ARHGEF18 is a Rho-activating guanine nucleotide exchange factor (RhoGEF) important in the activation of RhoA GTPases. This signaling pathway induces the formation of actin stress fibers, which are crucial to the structure of contractile actomyosin bundles found in non-muscle cells, and also drives cell proliferation via SRF (serum response factor)-mediated gene transcription. Heterotrimeric G proteins of the G12/13 subfamily, consisting of Gα12 and Gα13, stimulate specialized RhoGEFs through interaction with their RGS-homology (RH) domain. Despite lacking a RH domain, p114RhoGEF is bound to Gα12 through a unique structural region that evolved more recently than other components of p114RhoGEF. This specialized region, when examined evolutionarily, appears to be absent in jawless fishes such as lampreys, yet is present in other vertebrates including cartilaginous fishes such as sharks. This bioinformatic analysis suggests the Gα12-interacting region evolved as a relatively recent domain within p114RhoGEF. To understand the significance of this region in the function of p114RhoGEF, three mutations dissecting this region were engineered. The primary mutant construct removed the entire 106-residue Gα12 binding region, and two other mutations converted crucial glutamic acid residues identified in a previous study (Martin et al., 2016) to positively charged arginine residues. Luciferase assays were used to measure SRF-mediated transcriptional activation. Luminometric readings suggest that, in comparison to normal p114RhoGEF, the mutant missing this essential binding region gradually tapers to weaker signaling results at lower DNA concentrations. Future work should include examining the SRF signaling results of the point mutants, thus examining their structural significance in the Gα12 binding region as a whole. These results demonstrate a role of this binding region in modulating or stabilizing the signaling function of p114RhoGEF. This finding improves our understanding of the fine details of a system of interactions that drives Gα12-mediated Rho signaling.

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