Biased agonists of the chemokine receptor CXCR3 differentially drive formation of G_αi:β-arrestin complexes

Abstract

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.

Noncanonical interactions of G proteins and β‐arrestins: from competitors to companions

“…While the formation of Gai:b-arrestin complexes was consistent across GPCRs, the Gai:b-arrestin complexes did not form the same ERK scaffolding functions downstream of all GPCRs, even within a homogenous cellular background. For example, downstream of CXCR3 Gai:b-arrestin: ERK complexes was not observed [92]. These data suggest that multiple other factors influencing Gai, b-arrestin, biased agonists, cellular context, and/or post-translational modifications (e.g., phosphorylation 'barcode') dictate scaffold formation.…”

Subcellular localization of GPCR kinases differentially modulate biased signaling at CXCR3

“…CXCR3 is a chemokine receptor with three known endogenous ligands that exhibit biased signaling in various forms, such as in their differential formation of G i:β-arrestin complexes and markedly different abilities to induce G protein-or β-arrestin-mediated signaling [43][44][45][46] . We first determined if activation of CXCR3 using biased ligands promotes distinct patterns of GRK recruitment to the plasma membrane in HEK293 cells.…”

Biochemical insights into structure and function of arrestins

“…This demonstrated that the formation of a supercomplex of receptor, G protein, and arr critically depends on the conformation arr adapts upon binding to specific C termini. Most recently, a complementation assay leading to BRET in tripartite arr-receptor-G protein supercomplexes [110] has been proposed.…”