Phosphorylation barcode ensembles encoded by biased CXCR3 agonists direct non‐redundant chemokine signaling

Abstract

Chemokine receptors, a group of G protein‐coupled receptors (GPCRs), interact with transducers such as G proteins, β‐arrestins, and GPCR kinases (GRKs). In the chemokine system, many chemokine agonists act as “biased agonists” that preferentially activate distinct signaling effectors when binding to the same receptor, resulting in distinct physiological effects. Although one third of FDA‐approved drugs target GPCRs, there has been limited success in targeting the chemokine system. Currently, there is little evidence that differential receptor phosphorylation, or “phosphorylation barcodes,” direct these biased responses at chemokine receptors. To address this knowledge gap, we used mass spectrometry to demonstrate that chemokines of CXCR3 promote different ensembles of phosphorylation barcodes that are associated with differential activation of G proteins, β‐arrestins and GRKs. Chemokine stimulation also resulted in distinct changes throughout the kinome in global phosphoproteomic studies. Mutation of specific CXCR3 phosphosites altered β‐arrestin conformation and impacted β‐arrestin activation in molecular dynamics simulations. T‐cells expressing phosphorylation‐deficient CXCR3 mutants resulted in distinct agonist‐ and receptor‐specific chemotactic and signaling profiles that were not completely explained by engagement of G proteins, β‐arrestins, and GRKs alone. Our results directly link distinct GPCR phosphorylation patterns with non‐redundant chemokine signaling (Figure 1).