Rho guanine-nucleotide exchange factors (RhoGEFs) accelerate small GTPase function to drive cell motility, proliferation, and growth. The phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent Rac exchanger (P-Rex) subfamily of RhoGEFs contains P-Rex1 and P-Rex2 which, when misregulated, contribute to diseases. Despite being attractive therapeutic targets, the mechanisms governing P-Rex regulation remain poorly understood, limiting the development of effective isoform-specific inhibitors. P-Rex proteins share conserved upstream signaling pathways, domain architecture, and high sequence similarity, suggesting they may adopt similar structures and regulatory mechanisms; however, they exhibit distinct substrate preferences and regulatory interactions. P-Rex protein activity is modulated by accessory domains which allow them to remain in a cytosolic, autoinhibited state until activated by PIP3 and Gβγ. Although P-Rex1 autoinhibition is structurally and biochemically well-characterized, there is little such information regarding P-Rex2. Previously, our lab determined a moderate resolution cryo-EM structure of autoinhibited P-Rex1 which identified key sites that maintain autoinhibition. Here, we determined the cryo-EM structure of autoinhibited P-Rex2 which revealed that the N-terminal module is similarly arranged to P-Rex1, but oriented differently to the accessory domains, suggesting a potentially divergent regulatory mechanism. Guided by P-Rex1 regulation, we mutated residues in P-Rex2 and contrasted which are required to maintain autoinhibition within the N terminus. Hydrogen-deuterium exchange mass spectrometry showed that P-Rex2 is not stabilized by the PIP3 head group, IP4, like P-Rex1, suggesting that long-range interactions between distal accessory domains and the catalytic core may be less important in P-Rex2 autoinhibition. Together, these experiments uncover the fundamental differences in autoinhibition between P-Rex1 and P-Rex2 and provide the first evidence that P-Rex isoforms exhibit distinct conformations to mediate autoinhibition. Determining the molecular differences between P-Rex proteins will significantly advance the field by enabling rational design of isoform-specific inhibitors with therapeutic potential.
Anderson et al. (Sun,) studied this question.