Gα s Directly Drives PDZ‐RhoGEF Signaling to Cdc42

Abstract

G proteins stimulate RhoGEFs that control cell morphology through actin-cytoskeleton remodeling. Gα12 and Gα13 interact with RH-RhoGEFs (RGS-homology domain RhoGEFs), driving RhoA activation. However, whether additional Gα proteins directly regulate RH-RhoGEFs was not known. To respond this question, we first evaluated the morphological effects of constitutively active DH/PH constructs from p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, consistent with their known specificity. Intriguingly, PRG-DH/PH also induced filopodia-like cell protrusions and directly activated Cdc42. Constitutively active Gαs (GαsQ227L) increased the ability of PRG-DH/PH or endogenous PRG to interact with and activate Cdc42. Using a chemogenetic approach, with DREADD receptors, we demonstrated that Gs-coupled receptors, but not those coupled to Gi or Gq, drive PRG activity towards Cdc42. Given that Gαs interacted with PRG DH/PH region, we looked for potential inhibitory effects of the minimal fragment from PRG (linker) that binds GαsQ227L. This construct, the PRG-linker, interfered with GαsQ227L:PRG interaction and PRG:Cdc42 affinity. Besides, it attenuated Gαs signaling by Gs-DREADDs and Gs-coupled prostaglandin receptors (assessed by phosphorylation of CREB and PKA substrates). Altogether, our results demonstrate that Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.