Biased agonists of the chemokine receptor CXCR3 differentially drive formation of Gαi and β‐arrestin complexes

Abstract

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 InformationNIH