Activated RhoA Positively Regulates Exchange Activity of PDZ-RhoGEF

Abstract

RhoA plays a key role in regulation of the actin cytoskeleton, cell migration and cell shape. Rho GTPases cycle between an inactive GDP-bound state and an active GTP-bound state. This cycle is mediated by guanine nucleotide exchange factors (GEFs) that increase the rate of dissociation of GDP by stabilizing the nucleotide-free state of the GTPases via their DH/PH domains; this facilitates binding of GTP and activation of the protein. The RGS subfamily of RhoGEFs (RGS-RhoGEFs) act as direct mediators of RhoA activation in response to stimulation of the heterotrimeric G12 and G13 proteins by hormone receptors. RhoGEFs usually bind most tightly to the nucleotide free form of RhoA, which represents the intermediate state for exchange of guanine nucleotides. Recently, our lab discovered that PDZ-RhoGEF (PRG), a member of the RGS-RhoGEFs, bound tightly to both nucleotide-free and activated RhoA (RhoA•GTP). Using deletion analysis and pulldown assays, I was able to show that this interaction occurs between the PH domain of PRG and activated RhoA. James Chen was able to define the molecular determinants of this interaction by solving the crystal structure of the PRG-DH•PHRhoA(GTPγS) complex. This structure revealed that the interface is comprised of the switch regions in RhoA and a conserved hydrophobic patch in the PH domain of PRG. Interestingly, activated RhoA does not regulate the exchange activity of PRG in solution. Here, I use reconstitution of the signaling pathway with phospholipid vesicles and recombinant proteins, to show that this interaction serves as a mechanism for spatially regulating PRG exchange activity, a feed-forward mechanism. We hypothesize that this feed-forward mechanism is also applicable in vivo and potentially may serve as a mechanism utilized by a larger group of RhoGEFs known as the Lbc subfamily of RhoGEFs.