Identification of PDZ‐RhoGEF (PRG) as a Novel Gα i Target

Cell‐migration and adhesion are fundamental biological processes regulated by chemokine G‐protein‐coupled receptors that couple to PTX‐sensitive Gi proteins. The role of Gβγ subunits released from Gi in chemokine signaling has been well characterized. In contrast, Gα i has been thought to have no direct signaling function. Our laboratory recently uncovered a role for Gα i ‐GTP in regulating cell adhesion. However, a direct effector of Gα i responsible was not identified. To discover novel targets of active Gα i , we used BioID2 proximity‐based labeling coupled to TMT quantitative proteomic mass spectrometry. We subsequently compared the abundance ratios of proteins in Gα i1 ‐QL‐BioID2 (constitutively active) to Gα i1 ‐WT‐BioID2 (inactive) samples. BioID2‐CaaX was used as a membrane‐targeted control. Experiments were done in HT1080 fibrosarcoma cells where we previously demonstrated Gα i regulation of adhesion. Known binding partners for Gα i subunits including, GPCRs, Gβ, Gγ, adenylate cyclase, and Ric8A were identified by mass spectrometry further validating the method. A candidate Gα i effector identified was PDZ‐RhoGEF (PRG), a Rho guanine nucleotide exchange factor that regulates cell adhesion in neutrophils. To determine if PRG can be activated by Gα i ; we co‐transfected PRG with Gα i1 subunit and an SRE luciferase Rho reporter in A293 cells. Gα i1 ‐QL but not Gα i1 ‐WT strongly activated PRG dependent SRE luciferase, which was confirmed in a rhotekin assay for active RhoA. PRG activation was specific to the Gα i subfamily and required the PRG‐RGS domain. A proximal interaction was confirmed in aproximity ligation assay in A293 cells transfected with PRG and Gα i1 cDNAs. Purified Cy5‐labeled myr‐His‐Gα i bound specifically to GST‐PRG in a TR‐FRET assay. This binding was independent of the nucleotide state of Gα i1 but was inhibited by the addition of Gβγ subunits. In neutrophils, activation of the formyl peptide receptor (FPR) by fMLP, and subsequent PRG activation leads to Rho‐dependent accumulation of phosphorylated‐myosin light chain (P‐MLC) at the trailing edge and has been proposed to result from FPR‐dependent Gα 13 activation. In our preliminary experiments, stimulation of primary human neutrophils with 10 nM fMLP promoted polarized P‐MLC accumulation that was inhibited by PTX. This supports a model where at physiological concentrations of chemoattractant, activation of PRG/Rho is through Gα i ‐GTP rather than Gα 13 in primary human neutrophils. Overall, we identified relatively ubiquitously expressed PRG as a novel Gα i ‐GTP target. Identification of a novel signaling pathway regulated by Gi‐coupled GPCRs will have a significant impact on our understanding of the biology regulated by these ubiquitous and pharmacologically important receptors.