br Materials and methods br Results br Discussion Recently
Materials and methods
Discussion Recently, we characterized the interaction of the PH domains of various Lbc family RhoGEFs with the activated small GTPase, RhoA, and demonstrated that this could serve as a positive feedback mechanism for robust activation of RhoA (Medina et al., 2013). In the current study, we found that the PH domain of one of these, p190RhoGEF, could also bind to activated Rac1, suggesting a mechanism for crosstalk between the regulation evoked by these two GTPases. To better understand the binding between p190-PH and its two GTPase partners, we successfully generated crystal structures of the isolated PH domain of p190RhoGEF in complexes with either activated RhoA or Rac1. The structure of p190-PH bound to RhoA•GTPγS largely mimics the interaction of activated RhoA with the PH domain of PRG (Chen et al., 2010b) and verifies the conservation of this binding motif predicted for the Lbc RhoGEF family (Aittaleb et al., 2009, Medina et al., 2013). Our second structure reveals the unique interaction of p190-PH with activated Rac1. It shows a similar binding pattern between the switch regions on the GTPase and the same patch of mostly hydrophobic surface area located on the C-terminal beta-sheets on the PH domain that interact with activated RhoA. This common binding interface was verified by mutagenesis of key amino dmh express synthesis residues on the PH domain of p190RhoGEF, which abolished binding of the PH domain to both activated RhoA and Rac1 as well as the ability of these GTPases to stimulate the activity of p190RhoGEF on membrane delimited RhoA. Localization of RhoGEFs to the plasma membrane is a proposed mechanism for regulation of these proteins (Rossman et al., 2005, Viaud et al., 2012). We have shown that such a mechanism can utilize the endogenous activity of Lbc RhoGEFs to activate membrane delimited RhoA (Medina et al., 2013) and mediate hormone regulation of the GTPase via the heterotrimeric G12/13 proteins (Carter et al., 2014). This mechanism should be viable for the similar interaction of the PH domain of p190RhoGEF with activated RhoA. However, effective regulation becomes less obvious considering the weaker association of Rac1•GTPγS with p190-PH, as evinced from both biophysical and biochemical techniques. We utilized an in vitro signaling system (Medina et al., 2013) to verify that pre-activated RhoA bound to the vesicles would increase the exchange activity of p190RhoGEF on membrane associated RhoA. The ability of activated Rac1 to function similarly demonstrates that this low affinity interaction is also sufficient for membrane localization. Nevertheless, the smaller improvement in the initial exchange rate seen with Rac1 versus RhoA presumably reflects this difference in binding affinities and a less robust response. The high homology and structural similarity of Cdc42 to Rac1 suggested that it may also show an interaction with p190-PH. While binding between p190-PH and activated Cdc42 was never observed in dot blot or pull-down assays, its ability to interact with p190-PH was observed in the more sensitive vesicle activity assay (Fig. S10). Competition binding data provided an estimated affinity for interaction of activated Rac for p190-PH in the range of 20 μM, fully 10-fold less than binding between p190-PH and RhoA•GTPγS. Is this relevant in the cellular environment? Two observations support the potential of this low affinity interaction for effective action in cells. First, the PH domain of p115RhoGEF was also not observed to bind to activated RhoA in a pull-down assay, but could be shown to inhibit regulation by activated Rho in cells by overexpression (Medina et al., 2013). Second, point mutations at the Center-core site that disrupted binding of activated RhoA to the PH domain severely attenuated the ability of p115RhoGEF to activate RhoA by overexpression. Recent experiments have estimated the affinity of p115-PH:RhoA•GTPγS to be in the range of 50 μM (see Fig. 1 in Chen et al. (Submitted for publication)), lower than the observed affinity of p190-PH:Rac1•GTPγS reported here. Finally, the low affinities reported were measured in solution. Once the RhoGEF is localized to the 2-dimensional space of a membrane surface along with its substrate, effective concentrations become much greater, both for substrate turnover and potential interaction of other membrane components with the RhoGEF. In support of the latter hypothesis, a wide range of binding affinities has also been reported between the PH domains of some members in the Dbl family of RhoGEFs and phospholipids, especially phosphoinositides (Viaud et al., 2012). In this scenario, while clear roles have been shown for specific RhoGEFs, the relatively low affinities of many domains towards phospholipids suggest that these interactions alone are insufficient for membrane localization (Rossman et al., 2005). Both reviews propose that low affinity interactions may facilitate other mechanisms for binding of these proteins to membranes. Such a mechanism may also apply to potential regulation of p190RhoGEF by Rac1.