Identification and Characterization of G Protein Signaling Networks by Proximity Labeling-Coupled Proteomics

Abstract

Cell signaling networks control the ability of the cells to function and maintain equilibrium with intracellular and extracellular milieus. These networks are highly complex and tightly regulated, as they control essential functions in the body. Dysregulation of any of the pathways can lead to various pathophysiological disorders. Cell migration and adhesion are fundamental biological processes regulated by chemokine G-protein-coupled receptors (GPCRs). Chemokine receptors primarily couple to the Gi/o family of G proteins, composed of Gαi and Gβγ subunits. In the inactive form, Gαi is bound to GDP and Gβγ. Receptor activation triggers the exchange of GDP for GTP on Gαi, leading to its dissociation from the receptor and the Gβγ complex.

The role of Gβγ subunits released from Gi heterotrimers in chemokine signaling has been well characterized and is thought to be the major signal transducer. Lack of tools to manipulate Gαi signaling lead to the conclusion that it has no signaling role other than to regulate the release of Gβγ. By developing the selective activator of Gβγ, without Gαi activation; our recent work has discovered novel roles for Gαi in migration of neutrophils and fibrosarcoma cells, downstream of chemoattractant receptors. However, the molecular targets of Gαi in these processes remain to be identified. Driven by the possibility that multiple targets of Gi-coupled receptors remain to be identified, we adopted an intact cell proximity-based labeling approach using BioID2 (promiscuous biotin ligase enzyme)-coupled to mass spectrometry (MS). We screened for proteins that are differentially labeled in Gαi1-WT (inactive) vs Gαi1-QL (constitutively active), expressing cells. We confirmed that BioID2 fused Gαi1 performs unaltered signaling functions and localizes to the PM in HT1080 fibrosarcoma cells. Tandem mass tag (TMT)-MS with BioID2-fused Gαi1, Gαi1-QL and BioID2-CaaX (as a membrane- targeted control) was performed and known interactors of G protein α subunits including GPCRs, Gβ and Gγ, adenylate cyclase (AC), Resistance to inhibitors of cholinesterase 8A (Ric8A), and others were identified. Following the success of the BioID2-Gαi screen, we performed proximity-based labeling coupled MS screen for another G proteins subtype, Gαq. This was done using TurboID, an improved enzyme with faster labeling kinetics. Multiple known interactors, including PLCβ isoforms 1,3 and 4, RhoGEF; Trio, Kalirin, and p63RhoGEF, were selectively enriched in the TurboID-Gαq-QL samples relative to TurboID-Gαq.

In our screen, multiple potential candidate interactor proteins were identified and validated for selective biotinylation by BioID2-Gαi1-QL. This suggests a previously unappreciated network of interactions for activated Gαi proteins in intact cells. Extensive characterization of one candidate protein, PDZ‐RhoGEF (PRG), using in vitro cell-based and biochemical assays revealed that active Gαi1 strongly activates and interacts with 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 in primary human neutrophils. Besides, we identified a number of RasGAP proteins in MS, which we validated by co-immunoprecipitation and are currently characterizing it.

Given the ubiquitous presence of Gi and Gq-coupled receptors and G proteins, identification, and characterization of their binding partners both individually and in networks provides insights that will unravel multiple novel physiological roles of these receptors. In addition, it can identify novel therapeutic targets and contribute to understanding the on and off-target effects of drugs that directly target GPCRs.