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

G protein-coupled receptors (GPCRs) that couple to the G alpha(i) family of G proteins are key regulators of cell and tissue physiology. Our previous work has revealed new roles for G alpha(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 Gan. Multiple targets were identified and validated with a BioID2-tagged, constitutively active G alpha(i)1 mutant, suggesting a network of interactions for activated G alpha(i, )proteins in intact cells. We showed that active G alpha(i1,) but not G alpha(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 G alpha(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 G alpha(i) will aid in the investigation of the functional roles of G alpha(i)-coupled GPCRs in multiple biological processes.