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–mediated or β-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 β-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 β-arrestin–biased, but not a G protein–biased, agonist potentiated inflammation. T cell recruitment was increased by the β-arrestin–biased agonist, and biopsies of patients with allergic CHS demonstrated coexpression of CXCR3 and β-arrestin in T cells. In mouse and human T cells, the β-arrestin–biased agonist was the most efficient at stimulating chemotaxis. Analysis of phosphorylated proteins in human lymphocytes showed that β-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.

Biased agonism, the ability of a receptor to differentially activate downstream signaling pathways depending on binding of a “biased” agonist compared to a “balanced” agonist, is a well-established paradigm for G protein-coupled receptor (GPCR) signaling. Biased agonists have the promise to act as smarter drugs by specifically targeting pathogenic or therapeutic signaling pathways while avoiding others that could lead to side effects. A number of biased agonists targeting a wide array of GPCRs have been described, primarily based on their signaling in pharmacological assays. However, with the promise of biased agonists as novel therapeutics, comes the peril of not fully characterizing and understanding the activities of these compounds. Indeed, it is likely that some of the compounds that have been described as biased, may not be if quantitative approaches for bias assessment are used. Moreover, cell specific effects can result in “system bias” that cannot be accounted by current approaches for quantifying ligand bias. Other confounding includes kinetic effects which can alter apparent bias and differential propagation of biological signal that results in different levels of amplification of reporters downstream of the same effector. Moreover, the effects of biased agonists frequently cannot be predicted from their pharmacological profiles, and must be tested in the vivo physiological context. Thus, the development of biased agonists as drugs requires a detailed pharmacological characterization, involving both qualitative and quantitative approaches, and a detailed physiological characterization. With this understanding, we stand on the edge of a new era of smarter drugs that target GPCRs.

G protein–coupled receptors (GPCRs) are the largest family of cell surface receptors and signal through the proximal effectors, G proteins and β-arrestins, to influence nearly every biological process. The G protein and β-arrestin signaling pathways have largely been considered separable; however, direct interactions between Gα proteins and β-arrestins have been described that appear to be part of a distinct GPCR signaling pathway. Within these complexes, Gα i/o , but not other Gα protein subtypes, directly interacts with β-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 Gα i :β-arrestin complexes. Formation of the Gα i :β-arrestin complexes did not correlate well with either G protein activation or β-arrestin recruitment. β-arrestin biosensors demonstrated that ligands that promoted Gα i :β-arrestin complex formation generated similar β-arrestin conformations. We also found that Gα i :β-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 Gα i :β-arrestin complex formation is a distinct GPCR signaling pathway and enhance our understanding of the spectrum of biased agonism.

G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, signal through the proximal effectors G proteins and β-arrestins to influence nearly every biological process. Classically, the G protein and β-arrestin signaling pathways have largely been considered separable. Recently, direct interactions between Gα protein and β-arrestin have been described and suggest a distinct GPCR signaling pathway. Within these newly described Gα:β-arrestin complexes, Gαi/o, but not other Gα protein subtypes, have been appreciated to directly interact with β-arrestin, regardless of canonical GPCR Gα protein subtype coupling. However it is unclear how biased agonists differentially regulate this newly described Gαi:βarrestin interaction, if at all. Here we report that endogenous ligands (chemokines) of the GPCR CXCR3, CXCL9, CXCL10, and CXCL11, along with two small molecule biased CXCR3 agonists, differentially promote the formation of Gαi:β-arrestin complexes. The ability of CXCR3 agonists to form Gαi:β-arrestin complexes does not correlate well with either G protein signaling or β-arrestin recruitment. Conformational biosensors demonstrate that ligands that promoted Gαi:β-arrestin complex formation generated similar β-arrestin conformations. We find these Gαi:β-arrestin complexes can associate with CXCR3, but not with ERK. These findings further support that Gαi:β-arrestin complex formation is a distinct GPCR signaling pathway and enhance our understanding of biased agonism.

G‐protein‐coupled receptors (GPCRs) are the most common cell surface receptor class and influence nearly every biological process within a cell. Approximately 30% of all FDA‐approved medications target GPCRs. Following receptor activation, G proteins and β‐arrestins are currently appreciated to be the two primary proximal intracellular signaling pathways that regulate GPCR signaling. It has been thought that G protein and β‐arrestin signaling pathways are largely discernable, evidenced in part by ‘biased agonists’ that can preferentially activate one of these pathways relative to the other. However, increased crosstalk between G protein and β‐arrestin pathways is beginning to be appreciated. We recently described the ability of Gα proteins to directly interact with β‐arrestins to form signaling scaffolds, with Gαi/o found to directly interact with β‐arrestin following GPCR activation regardless of canonical GPCR G protein coupling. We previously demonstrated in other work that all GPCRs tested, including the vasopressin type 2 receptor, β2‐adrenergic receptor, neurotensin receptor type 1, and the dopamine D1 and D2 receptors, formed a Gαi:β‐arrestin complex. Now that we appreciate this novel GPCR signaling paradigm, it is unclear if biased agonists that preferentially stimulate either G protein‐ or β‐arrestin‐dependent signaling differentially regulate Gαi:β‐arrestin interactions, if at all. To address this question, we utilized the chemokine receptor CXCR3 that binds three endogenous chemokines with high affinity. We demonstrate that these endogenous biased CXCR3 chemokines, CXCL9, CXCL10, and CXCL11, along with its synthetic biased agonists, VUF10661 and VUF11418, differentially form Gαi:β‐arrestin complexes. The ability of CXCR3 agonists to form Gαi:β‐arrestin complexes does not correlate highly with more well‐established GPCR signaling pathways including G protein recruitment, G protein signaling (e.g., regulation of cAMP), or β‐arrestin recruitment, suggesting that Gαi:β‐arrestin complex formation is distinct from conventional G protein or β‐arrestin signaling events. Additionally, differential Gαi:β‐arrestin complex formation was found to correlate with similar degrees of Gαi:β‐arrestin:CXCR3 “megaplex” formation, suggesting that formation of a long‐lived complex may depend upon close coupling of the two effectors at the receptor initially. This work provides further support for a separable Gαi:β‐arrestin signaling pathway and enhances our understanding of GPCR signaling and biased agonism. Support or Funding Information NIH

Allergic contact dermatitis (ACD) is a disease with few targeted therapies. Chemokines play an important role in ACD through the recruitment of T-cells that express the chemokine receptor (CKR) CXCR3. Chemokines signal through CKRs, a subgroup of the G proteincoupled receptor (GPCR) family, which are targeted in >30% of drugs. However, few drugs target CKRs. Classically, GPCRs were thought to act as simple switches turned on by agonists and off by antagonists. We now appreciate that GPCRs adopt multiple conformations that link to distinct signaling pathways, such as G-proteins and ß-arrestins (ßarrs). These pathways can be selectively activated by a novel class of receptor ligands, termed biased agonists, which signal through some pathways while blocking signaling through others. The purpose of this study was to determine the roles that G-proteins and ßarrs play in ACD by selectively targeting signaling with CXCR3 biased agonists. Mouse and human cell chemotaxis was determined through transwell migration, and the effects of CXCR3 ligands on ACD were assessed in the DNFB allergic contact hypersensitivity (CHS) mouse model. Patient biopsies of patch tested skin were analyzed. Our results show that ßarr signaling through CXCR3 is necessary for full efficacy chemotaxis of both mouse and human T-cells. A topically applied ßarr-biased ligand doubled (ph0.05) the CHS inflammatory response in WT, but not in ßarr2 KO or CXCR3 KO, mice. Flow cytometry of mouse skin demonstrated increased T-cells following ßarr-biased drug treatment. We conclude that CXCR3 ßarr-mediated signaling is critical for effector T-cell recruitment that underlies the inflammatory response in CHS. These findings suggest that biased ligands could be utilized to selectively target CKRs for therapeutic benefit.