Biased Signaling at Chemokine Receptors

Jenny Corbisier,Céline Galès,Alexandre Huszagh,Marc Parmentier,Jean-Yves Springael

doi:10.1074/jbc.m114.596098

Abstract

The ability of G protein-coupled receptors (GPCRs) to activate selective signaling pathways according to the conformation stabilized by bound ligands (signaling bias) is a challenging concept in the GPCR field. Signaling bias has been documented for several GPCRs, including chemokine receptors. However, most of these studies examined the global signaling bias between G protein- and arrestin-dependent pathways, leaving unaddressed the potential bias between particular G protein subtypes. Here, we investigated the coupling selectivity of chemokine receptors CCR2, CCR5, and CCR7 in response to various ligands with G protein subtypes by using bioluminescence resonance energy transfer biosensors monitoring directly the activation of G proteins. We also compared data obtained with the G protein biosensors with those obtained with other functional readouts, such as β-arrestin-2 recruitment, cAMP accumulation, and calcium mobilization assays. We showed that the binding of chemokines to CCR2, CCR5, and CCR7 activated the three Gαi subtypes (Gαi1, Gαi2, and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies that generally correlate to their binding affinities. In addition, we showed that the binding of chemokines to CCR5 and CCR2 also activated Gα12, but not Gα13. For each receptor, we showed that the relative potency of various agonist chemokines was not identical in all assays, supporting the notion that signaling bias exists at chemokine receptors.

Highlights

G protein-coupled receptors (GPCRs) can activate selective signaling pathways according to the nature of the bound ligand
Signaling Selectivity at Chemokine Receptor CCR7—In a competition binding assay, we first confirmed that CCL19 and CCL21 compete with radiolabeled CCL19 for CCR7 binding with nearly equivalent pIC50 values (Fig. 1A and Table 1)
We investigated the panel of G proteins activated by CCR7 upon binding of CCL19 or CCL21 by using bioluminescence resonance energy transfer (BRET)-based biosensors directly monitoring the activation of G proteins

Summary

Background

GPCRs can activate selective signaling pathways according to the nature of the bound ligand. It is important to identify ligands and receptors that possess biased signaling properties and to understand how agonist binding induces the selective activation of a signaling pathway within the cell. Signaling selectivity has been first described for the two natural ligands of the chemokine receptor CCR7, CCL19, and CCL21 Both CCL19 and CCL21 were reported to activate G protein-dependent pathways, but only CCL19 was proposed to recruit ␤-arrestin and activate MAPK Erk1/2 [11,12,13]. CCR7 became a prototypical example of biased signaling, with respect to natural agonists Based on these first studies, it was anticipated that other chemokine receptors might display a similar bias by preferentially activating either G protein- or arrestin-dependent pathways. We combined the use of these sensors with other functional assays to revisit the signaling bias previously identified for CCR7 and to analyze the signaling selectivity of various chemokine ligands through activation of the closely related receptors CCR2 and CCR5

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION