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- Title
A network of Gα<sub>i</sub> signaling partners is revealed by proximity labeling proteomics analysis and includes PDZ-RhoGEF.
- Authors
Chandan, Naincy R.; Abraham, Saji; SenGupta, Shuvasree; Parent, Carole A.; Smrcka, Alan V.
- Abstract
G protein–coupled receptors (GPCRs) that couple to the Gαi 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. 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 Gαi1. Multiple targets were identified and validated with a BioID2-tagged, constitutively active Gαi1 mutant, suggesting a network of interactions for activated GαI proteins in intact cells. We showed that active Gαi1, but not Gαi2, 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 Gαi 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 Gαi will aid in the investigation of the functional roles of Gαi-coupled GPCRs in multiple biological processes. Expanding the Gαi interactome: Activated chemoattractant receptors stimulate the G protein αi subunit to induce de-adhesion in migrating neutrophils and fibrosarcoma cells. To identify effectors for active Gαi, Chandan et al. performed proximity labeling and mass spectrometry analysis in cells expressing inactive and constitutively active Gαi. Of the candidates identified by the authors, the RhoGEF PRG was of particular interest because of its previously identified role in neutrophil chemotaxis induced by the activation of Gαi-coupled chemoattractant receptors. In human neutrophils, Gαi was required for PRG-mediated stimulation of RhoA activity upon activation of the chemoattractant receptor FPR1. Thus, the authors' approach could be used to identify binding partners that interact with other G protein subunits in intact cells.
- Subjects
G protein coupled receptors; PROTEOMICS; TISSUE physiology; GUANOSINE triphosphate; CELL physiology; MYOSIN; G proteins; CELL analysis
- Publication
Science Signaling, 2022, Vol 15, Issue 717, p1
- ISSN
1945-0877
- Publication type
Article
- DOI
10.1126/scisignal.abi9869