Some G protein-coupled receptor (GPCR) ligands act as “biased agonists” that preferentially activate specific signaling transducers over others. Although GPCRs are primarily found at the plasma membrane, GPCRs can traffic to and signal from many subcellular compartments. Here, we determine that differential subcellular signaling contributes to the biased signaling generated by three endogenous ligands of the GPCR CXC chemokine receptor 3 (CXCR3). The signaling profile of CXCR3 changes as it traffics from the plasma membrane to endosomes in a ligand-specific manner. Endosomal signaling is critical for biased activation of G proteins, β-arrestins, and extracellular-signal-regulated kinase (ERK). In CD8 + T cells, the chemokines promote unique transcriptional responses predicted to regulate inflammatory pathways. In a mouse model of contact hypersensitivity, β-arrestin-biased CXCR3-mediated inflammation is dependent on receptor internalization. Our work demonstrates that differential subcellular signaling is critical to the overall biased response observed at CXCR3, which has important implications for drugs targeting chemokine receptors and other 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.
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