Biased agonists of the chemokine receptor CXCR3 differentially signal through Gα _i :β-arrestin complexes

Abstract

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.

GPCR kinases differentially modulate biased signaling downstream of CXCR3 depending on their subcellular localization

“…Previous publications measuring relative amounts of G protein signaling and β-arrestin recruitment demonstrate that CXCL11 is relatively β-arrestin–biased, whereas CXCL10, VUF10661, and VUF11418 are relatively G protein–biased. CXCL9 acts as a β-arrestin–biased partial agonist ( 52 , 53 ).…”

“…CXCR3 is a chemokine receptor with endogenous ligands that exhibit biased signaling in various forms, such as in their differential formation of Gαi:β-arrestin complexes and markedly different abilities to induce G protein-or βarrestin-mediated signaling (52)(53)(54)(55). CXCR3 is primarily found on activated CD8 + T cells and natural killer cells; directs cellular functions like chemotaxis and T cell polarization; and is implicated in inflammatory conditions such as cancer, atherosclerosis, autoimmunity, and allergic contact dermatitis (11).…”

Phosphorylation barcodes direct biased chemokine signaling at CXCR3

“…We next examined β-arrestin recruitment ( Figure 2H ). Consistent with prior work, CXCL11 was significantly more effective in recruiting β-arrestin to CXCR3-WT compared to CXCL9 and CXCL10 ( Figure 2I- K ) (Colvin et al ., 2004; Smith et al ., 2017; Zheng et al ., 2022). All phosphodeficient mutant receptors treated with CXCL11 demonstrated significantly less recruitment of β-arrestin when compared to CXCR3-WT ( Figure 2K ).…”

“…Like most other chemokine receptors, CXCR3 signals through both Gαi family G proteins and β-arrestins (Colvin et al, 2006; Colvin et al, 2004; Smith et al, 2017). CXCL11 is β-arrestin-biased compared to CXCL9 and CXCL10, with each chemokine displaying distinct profiles of G protein signaling, β-arrestin recruitment and receptor internalization (Rajagopal et al ., 2013; Zheng et al, 2022). Synthetic CXCR3 biased agonists have shown distinct physiological effects in a mouse model of allergic contact dermatitis, with a β-arrestin-biased agonist promoting inflammation through increased T cell recruitment (Smith et al, 2018a).…”

Keras/TensorFlow in Drug Design for Immunity Disorders

“…Biased ligands of CXCR3 (biaryl-type VUF10661 and VUF11418) have also been discovered in addition to the biased signaling observed for endogenous agonists of CXCR3 (CXCL11 bias towards β-arrestin). Recently, these three agonists of CXCR3 have been shown to activate the formation of the Gαi:β-arrestin complex in non-canonical GPCR signaling [33]. This emphasizes the importance of drug design for CXCR3 in numerous diseases, such as cancer, inflammatory diseases, and autoimmune disorders.…”