(536.12) Phosphorylation barcode ensembles encoded by biased CXCR3 agonists direct non-redundant chemokine signaling

Poster Board Number: B76

Dylan Eiger (Duke University), Jeffrey Smith (Harvard Medical School), Tujin Shi (Pacific Northwest National Laboratory), Tomasz Stepniewski (Pompeu Fabra University - Hospital del Mar Medical Research Institute), Christopher Honeycutt (Duke University), Noelia Boldizsar (Duke University), Julia Gardner (Duke University), Chia-Feng Tsai (Pacific Northwest National Laboratory), Carrie Nicora (Pacific Northwest National Laboratory), Ahmed Moghieb (Pacific Northwest National Laboratory), Kouki Kawakami (Tohoku University), Issac Choi (Duke University), Richard Smith (Pacific Northwest National Laboratory), Asuka Inoue (Tohoku University), Jana Selent (Pompeu Fabra University - Hospital del Mar Medical Research Institute), Jon Jacobs (Pacific Northwest National Laboratory), Sudarshan Rajagopal (Duke University)

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).

NIH T32GM007171 (D.E., J.S.), The Duke Medical Scientist Training Program (D.E., J.S.), AHA 20PRE35120592 (D.E.), NIH 1R01GM122798-01A1 (S.R.)



Working model of GPCR phosphorylation barcodes directing non-redundant chemokine signaling