A biased ligand for OXE-R uncouples Gα and Gβγ signaling within a heterotrimer

Abstract

Differential targeting of heterotrimeric G protein versus β-arrestin signaling are emerging concepts in G protein-coupled receptor (GPCR) research and drug discovery, and biased engagement by GPCR ligands of either β-arrestin or G protein pathways has been disclosed. Herein we report on a new mechanism of ligand bias to titrate the signaling specificity of a cell-surface GPCR. Using a combination of biomolecular and virtual screening, we identified the small-molecule modulator Gue1654, which inhibits Gβγ but not Gα signaling triggered upon activation of Gα(i)-βγ by the chemoattractant receptor OXE-R in both recombinant and human primary cells. Gue1654 does not interfere nonspecifically with signaling directly at or downstream of Gβγ. This hitherto unappreciated mechanism of ligand bias at a GPCR highlights both a new paradigm for functional selectivity and a potentially new strategy to develop pathway-specific therapeutics.

A Biased Non-Gαi OXE-R Antagonist Demonstrates That Gαi Protein Subunit Is Not Directly Involved in Neutrophil, Eosinophil, and Monocyte Activation by 5-Oxo-ETE

“…7B). This finding reflects our previous observations that Gue1654 was able to abolish the 5-oxo-ETE-induced Ca 2+ mobilization in eosinophils and neutrophils (14).…”

“…Gue1654 is a small-molecule antagonist of OXE-R that uniquely uncouples Ga-and Gbg-mediated downstream signaling events in human neutrophils and eosinophils, and in recombinant cells overexpressing OXE-R, as we have recently described (14). Gue1654 antagonizes OXE-R in a mode that preserves Ga i -dependent inhibition of adenylyl cyclase but completely impairs the Gbg heterodimer-triggered Ca 2+ mobilization, inositol monophosphate release, and gating of G protein-regulated inwardly rectifying K + channels (14).…”

“…BRET was applied for the detection of bARR2 recruitment in HEK293-bARR2-GFP 2 cells that were transiently coexpressing OXE-R-RLuc, as previously described (14). 5-Oxo-ETE or vehicle was added to 1.8 3 10 5 cells/well in white 96-well plate at 300 rpm for 10 min.…”

“…Cells were cultivated in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 500 mg/ml geneticin (G418). For bioluminescence resonance energy transfer (BRET) assays, cells were transiently transfected with pcDNA3.1 + -OXE-R-RLuc using the gene pulser Xcell electroporator at 250 V, 500 mF, and ' V and were grown for 48 h (14).…”

“…We have recently reported on Gue1654, a newly identified biased OXE-R antagonist that selectively disrupts Gbg, but does not affect Ga i signaling triggered by 5-oxo-ETE (14). In the current study, we elucidated the general importance of Ga i protein activation in the function of human eosinophils, neutrophils, and monocytes by exploiting this to date unique non-Ga i -biased OXE-R antagonist.…”