A Network G Protein Gai Signaling Partners is Revealed by Proximity Labeling Proteomics and Includes PDZ-RhoGEF

G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our previous work has revealed new roles for Gαi in regulating the migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors. We used an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interacted with the GTP-bound form of Gαi1. Multiple targets were identified and validated for selective biotinylation by a constitutively active BioID2-tagged Gai1 mutant, suggesting a network of interactions for activated Gαi proteins in intact cells. We showed that active Gαi1 stimulated one candidate protein, PDZ‐RhoGEF (PRG) and that despite over 85% sequence identity, the ability of Gαi1, Gαi2, and Gαi3 isoforms to activate PRG greatly differed. We also demonstrated that active Gαi likely regulates polarized myosin light chain kinase phosphorylation through activation of PRG in primary human neutrophils, suggesting functional relevance of this interaction. Identification and characterization of new targets regulated by Gαi both individually and in networks provide insights that will aid in the investigation of the functional roles of Gi-coupled GPCRs in multiple biological processes.