A network of Gα_i signaling partners is revealed by proximity labeling proteomics analysis and includes PDZ-RhoGEF

G protein–coupled receptors (GPCRs) that couple to the Gα_i family of G proteins are key regulators of cell and tissue physiology. Our previous work has revealed new roles for Gα_i in regulating the migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors. Here, we used an intact cell proximity–based labeling coupled to tandem mass tag (TMT)–based quantitative proteomics analysis to identify proteins that selectively interacted with the GTP-bound form of Gα_i1. Multiple targets were identified and validated with a BioID2-tagged, constitutively active Gα_i1 mutant, suggesting a network of interactions for activated Gα_I proteins in intact cells. We showed that active Gα_i1, but not Gα_i2, stimulated one candidate protein, PDZ-RhoGEF (PRG), despite more than 85% sequence identity between the G proteins. We also demonstrated in primary human neutrophils that active Gα_i likely regulated the polarization of phosphorylated myosin light chain, a process critical for migration, through the activation of PRG. The identification and characterization of new targets directly or indirectly regulated by Gα_i will aid in the investigation of the functional roles of Gα_i-coupled GPCRs in multiple biological processes.