G beta gamma marks the inner side of the plasma membrane where chemotactic GPCRs activate Rac to lead the assembly of actin filaments that push the cell to move forward. Upon dissociation from heterotrimeric Gi, G beta gamma recruits and activates P-Rex1, a Rac guanine nucleotide exchange factor (RacGEF). This cytosolic chemotactic effector is kept inactive by intramolecular interactions. The mechanism by which G beta gamma stimulates P-Rex1 has been debated. We hypothesized that G beta gamma activates P-Rex1 by a two-step mechanism based on independent interaction interfaces to recruit and unroll this RacGEF. Using pulldown assays, we found that G beta gamma binds P-Rex1-DH/PH as well as PDZ-PDZ domains. These domains and the DEP-DEP tandem interact among them and dissociate upon binding with G beta gamma, arguing for a stimulatory allosteric effect. In addition, P-Rex1 catalytic activity is inhibited by its C-terminal domain. To discern P-Rex1 recruitment from activation, we studied Q-Rhox, a synthetic RhoGEF having the PDZ-RhoGEF catalytic DH/PH module, insensitive to G beta gamma, swapped into P-Rex1. G beta gamma recruited Q-Rhox to the plasma membrane, indicating that G beta gamma/PDZ-PDZ interaction interface plays a role on P-Rex1 recruitment. In conclusion, we reconcile previous findings and propose a mechanistic model of P-Rex1 activation; accordingly, G beta gamma recruits P-Rex1 via the G beta gamma/PDZ-PDZ interface followed by a second contact involving the G beta gamma/DH/PH interface to unleash P-Rex1 RacGEF activity at the plasma membrane. (C) 2020 Elsevier Inc. All rights reserved.