Identification of G Protein αi Signaling Partners by Proximity Labeling Reveals a Network of Interactions that Includes PDZ-RhoGEF

Abstract

AbstractG protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our recent work has discovered novel roles for Gαi in migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors, but the molecular target(s) of Gαi in these processes remain to be identified. We adopted an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interact with the GTP-bound form of Gαi1. Multiple targets were identified and validated for selective biotinylation by active BioID2-Gαi1(Q204L), suggesting a previously unappreciated network of interactions for activated Gαi proteins in intact cells. Extensive characterization of one candidate protein, PDZ-RhoGEF (PRG), revealed that active-Gαi1 strongly activates PRG. Strikingly, large differences in the ability of Gαi1, Gαi2, and Gαi3 isoforms to activate PRG were observed despite over 85% sequence identity. We also demonstrate the functional relevance of the interaction between active Gαi and PRG ex vivo in primary human neutrophils. Identification and characterization of new targets regulated by Gαi both individually and in networks provide insights that will aid not only in investigation of diverse functional roles of Gi-coupled GPCRs in biology but also in the development of novel therapeutic approaches.SummaryProximity-based labeling approach was used to identify signaling networks and signaling mechanisms downstream of Gi-coupled receptors.