Uncovering Modes of P-Rex2 Regulation to Inform Drug Design

Abstract ID 94118 Poster Board 550 Rho guanine-nucleotide exchange factors (RhoGEFs) accelerate the function of small GTPases to drive cell motility, proliferation, and growth. The phosphatidylinositol 3,4,5-triphosphate (PIP₃)-dependent Rac exchanger (P-Rex) subfamily of RhoGEFs contains P-Rex1 and P-Rex2. When misregulated, P-Rex1 plays a role in atherosclerosis pathogenesis, while P-Rex2 is a biomarker of ischemic heart disease, which can lead to heart attacks, the leading cause of death in the United States. P-Rex proteins have low basal activity in the cytosol (autoinhibited state) and are synergistically activated by PIP₃ and the beta gamma subunits of the heterotrimeric G proteins at the membrane (active state). Despite high sequence similarity, these proteins have divergent physiological functions for unclear reasons. For example, P-Rex2 GTPase specificity is more narrow than P-Rex1, and P-Rex2 is uniquely regulated by the canonical tumor suppressor PTEN whereas P-Rex1 is not. Although the P-Rex1 autoinhibited state is structurally and biochemically well-characterized, there is little such information regarding P-Rex2, and how these proteins transition to the fully active state is unknown. Our lab previously determined the cryo-EM structure of autoinhibited P-Rex1 at moderate resolution. Interestingly, our preliminary, low-resolution cryo-EM map of autoinhibited P-Rex2 indicates that P-Rex2 has a distinct domain architecture from P-Rex1 that could explain differences in substrate specificity and regulation. To elucidate these differences, we are investigating full-length P-Rex1 and P-Rex2 substrate specificity against different GTPases. Using nanodiscs as a model membrane, we are studying the activated structures of P-Rex1 and P-Rex2. Here we are using cryo-EM and biochemical assays to directly correlate the activated structures to their activity. Collectively, these experiments will give us insights into the mechanistic details of P-Rex activation and allow us to identify unique regulatory features between P-Rex1 and P-Rex2. Determining the molecular differences between P-Rex proteins will significantly advance the field by allowing us to rationally design highly specific inhibitors toward P-Rex1 or P-Rex2.