Phosphatidylinositol (3,4,5) trisphosphate (PIP 3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine-nucleotide exchange factor that was originally discovered in neutrophils and is regulated by G protein bg subunits and the lipid PIP 3 in response to chemoattractants. P-Rex1 has also become increasingly recognized for its role in promoting metastasis of breast cancer, prostate cancer, and melanoma. Recent structural, biochemical, and biologic work has shown that binding of PIP 3 to the pleckstrin homology (PH) domain of P-Rex1 is required for its activation in cells. Here, differential scanning fluorimetry was used in a medium-throughput screen to identify six small molecules that interact with the P-Rex1 PH domain and block binding of and activation by PIP 3. Three of these compounds inhibit N-formylmethionylleucyl-phenylalanine induced spreading of human neutrophils as well as activation of the GTPase Rac2, both of which are downstream effects of P-Rex1 activity. Furthermore, one of these compounds reduces neutrophil velocity and inhibits neutrophil recruitment in response to inflammation in a zebrafish model. These results suggest that the PH domain of P-Rex1 is a tractable drug target and that these compounds might be useful for inhibiting P-Rex1 in other experimental contexts. SIGNIFICANCE STATEMENT A set of small molecules identified in a thermal shift screen directed against the phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 (P-Rex1) pleckstrin homology domain has effects consistent with P-Rex1 inhibition in neutrophils.

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.

G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our recent work has discovered novel roles for Gαi in migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors, but the molecular target(s) of Gαi in these processes remain to be identified. We adopted an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interact with the GTP-bound form of Gαi1. Multiple targets were identified and validated for selective biotinylation by active BioID2-Gαi1(Q204L), suggesting a previously unappreciated network of interactions for activated Gαi proteins in intact cells. Extensive characterization of one candidate protein, PDZ-RhoGEF (PRG), revealed that active-Gαi1 strongly activates 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 ex vivo in primary human neutrophils. Identification and characterization of new targets regulated by Gαi both individually and in networks provide insights that will aid not only in investigation of diverse functional roles of Gi-coupled GPCRs in biology but also in the development of novel therapeutic approaches.

Cell‐migration and adhesion are fundamental biological processes regulated by chemokine G‐protein‐coupled receptors (GPCRs). GPCRs bind to heterotrimeric G proteins, which are composed of Gα and Gβγ subunits. Chemokine receptors couple to the PTX‐sensitive Gi/o family of Gα (Gαi) proteins. The role of Gβγ subunits released from Gi heterotrimers in chemokine signaling has been well characterized and is thought to be the major transducer of these processes. In contrast, Gαi has been concluded to have no signaling role other than to regulate the release of Gβγ. However, this conclusion is complicated by the fact that tools to inactivate Gαi signaling also inactivates obligatory Gβγ signaling. Previous reports from our laboratory have identified that Gαi‐GTP directly regulates cell adhesion but the direct effector of Gαi has not been identified. To identify the novel effectors of Gαi, we adopted a proximity‐based labeling technique. BioID2 (promiscuous biotin ligase enzyme) generates reactive Biotin‐AMP from biotin. Bio‐AMP then diffuses away from the enzyme’s active site and labels proteins covalently with biotin that are in ∽10 nm radius. HT1080 fibrosarcoma cells were transfected with Gαi1‐WT‐BioID2 (inactive Gαi), Gαi1‐QL‐BioID2 (constitutively active Gαi) or BioID2‐CaaX (membrane‐targeted control samples). Cell extracts were prepared, and biotinylated proteins were selectively isolated by biotin‐streptavidin affinity capture. Tandem mass spectrometry was then performed using quantitative tandem mass tag (TMT) labeling. Known binding partners for Gαi subunits including, GPCRs, Gβ and Gγ, adenylate cyclase, Ric8A, and others were identified as enriched in the Gαi‐WT‐BioID2 or Gαi‐QL‐BioID2 samples. One candidate Gαi effector, significantly enriched in Gαi1‐QL‐BioID2 as compared to Gαi1‐WT‐BioID2 samples, was PDZ‐RhoGEF (ARHGEF11), a Rho guanine nucleotide exchange factor. PDZ‐RhoGEF is a known major regulator of cell adhesion in neutrophils downstream of Gi‐coupled chemoattractant receptors. To determine if PDZ‐RhoGEF is a true effector of Gαi; we co‐transfected PDZ‐RhoGEF with Gαi subunits and an SRE luciferase reporter construct responsive to Rho activation in A293 cells. Gαi1‐QL but not Gαi1‐WT strongly activated SRE luciferase only when co‐expressed with PDZ‐RhoGEF. The activation of RhoA by Gαi1‐QL was confirmed with a Rhotekin‐GST pulldown assay. Activation is specific to the Gαi subfamily of G proteins and requires the RGS domain within PDZ‐RhoGEF. Since PDZ‐RhoGEF is known to be involved in cell migration and adhesion, this protein is likely a key downstream target of Gαi that regulates cell migration. Overall, identification of the relatively ubiquitously expressed PDZ‐RhoGEF as a new Gαi‐GTP target has implications for regulation of downstream signaling by Gi‐coupled GPCRs in multiple biological systems.