Biased agonists of the chemokine receptor CXCR3 differentially signal through G alpha(i):beta-arrestin complexes

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