Biased agonists of the chemokine receptor CXCR3 differentially signal through Gαi:ß-arrestin complexes.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and signal through the proximal effectors, G proteins and ß-arrestins, to influence nearly every biological process. The G protein and ß-arrestin signaling pathways have largely been considered separable; however, direct interactions between Gα proteins and ß-arrestins have been described that appear to be part of a distinct GPCR signaling pathway. Within these complexes, Gαi/o, but not other Gα protein subtypes, directly interacts with ß-arrestin, regardless of the canonical Gα protein that is coupled to the GPCR. Here, we report that the endogenous biased chemokine agonists of CXCR3 (CXCL9, CXCL10, and CXCL11), together with two small-molecule biased agonists, differentially formed Gαi:ß-arrestin complexes. Formation of the Gαi:ß-arrestin complexes did not correlate well with either G protein activation or ß-arrestin recruitment. ß-arrestin biosensors demonstrated that ligands that promoted Gαi:ß-arrestin complex formation generated similar ß-arrestin conformations. We also found that Gαi:ß-arrestin complexes did not couple to the mitogen-activated protein kinase ERK, as is observed with other receptors such as the V2 vasopressin receptor, but did couple with the clathrin adaptor protein AP-2, which suggests context-dependent signaling by these complexes. These findings reinforce the notion that Gαi:ß-arrestin complex formation is a distinct GPCR signaling pathway and enhance our understanding of the spectrum of biased agonism.

Biased agonists of the chemokine receptor CXCR3 differentially control chemotaxis and inflammation.

The chemokine receptor CXCR3 plays a central role in inflammation by mediating effector/memory T cell migration in various diseases; however, drugs targeting CXCR3 and other chemokine receptors are largely ineffective in treating inflammation. Chemokines, the endogenous peptide ligands of chemokine receptors, can exhibit so-called biased agonism by selectively activating either G protein- or ß-arrestin-mediated signaling after receptor binding. Biased agonists might be used as more targeted therapeutics to differentially regulate physiological responses, such as immune cell migration. To test whether CXCR3-mediated physiological responses could be segregated by G protein- and ß-arrestin-mediated signaling, we identified and characterized small-molecule biased agonists of the receptor. In a mouse model of T cell-mediated allergic contact hypersensitivity (CHS), topical application of a ß-arrestin-biased, but not a G protein-biased, agonist potentiated inflammation. T cell recruitment was increased by the ß-arrestin-biased agonist, and biopsies of patients with allergic CHS demonstrated coexpression of CXCR3 and ß-arrestin in T cells. In mouse and human T cells, the ß-arrestin-biased agonist was the most efficient at stimulating chemotaxis. Analysis of phosphorylated proteins in human lymphocytes showed that ß-arrestin-biased signaling activated the kinase Akt, which promoted T cell migration. This study demonstrates that biased agonists of CXCR3 produce distinct physiological effects, suggesting discrete roles for different endogenous CXCR3 ligands and providing evidence that biased signaling can affect the clinical utility of drugs targeting CXCR3 and other chemokine receptors.