696.2 - Location Bias Contributes to Functionally Selective Responses of Biased CXCR3 Agonists to Regulate Inflammation

Julia Gardner (Duke University), Dylan Eiger (Duke University), Noelia Boldizsar (Duke University ), Christopher Honeycutt (Duke University ), Stephen Kirchner (Duke University ), Chloe Hicks (Duke University ), Issac Choi (Duke University ), Kevin Zheng (Duke University ), Anmol Warman (Duke University), Jeffrey Smith (Harvard Medical School), Jennifer Zhang (Duke University ), Sudarshan Rajagopal (Duke University )

G Protein-Coupled Receptors (GPCRs) represent the largest and most diverse family of cell receptors in the human genome and are targeted by ~35% of all FDA-approved drugs. GPCR signaling is mediated by various effectors, including G proteins, β-arrestins, and GPCR kinases (GRKs). Recent studies have uncovered that different ligands can bind to the same GPCR and differentially activate specific signaling pathways, a phenomenon known as biased agonism. Most research has focused on characterizing biased agonism at the plasma membrane, but GPCRs are also known to traffic to and signal from a variety of subcellular compartments. To determine if GPCR subcellular localization contributes to biased signaling, we studied how the chemokine family GPCR CXCR3 signals from endosomes following stimulation by the three endogenous ligands CXCL9, CXCL10, and CXCL11.

We determined that the biased ligands of CXCR3 promote different amounts of receptor mediated endocytosis and also show markedly different G protein and β-arrestin signaling profiles at both the plasma membrane and the endosome. The biased activation of G proteins and β-arrestins promotes differential activation of cytoplasmic and nuclear ERK1/2, as well as cellular transcription in both HEK293 cells and primary CD8+ T-cells. Inhibition of receptor internalization greatly diminishes the biased signaling observed between the endogenous chemokines. Finally, we demonstrate in a murine model of contact hypersensitivity that receptor internalization is a key component of the ability of a synthetic β-arrestin biased CXCR3 agonist to potentiate the inflammatory response. The present study shows the CXCR3 biased ligands induce unique signaling profiles at the plasma membrane and the endosome, and that signaling from endosomes greatly contributes to the overall biased responses seen at CXCR3. These results demonstrate that signaling from subcellar compartments is a critical component of GPCR signaling. Our findings have implications in the development of pharmaceutical drugs targeting chemokine receptors and other GPCRs.

T32GM007171 (DE), Duke Medical Scientist Training Program (DE); AHA 20PRE35120592 (DE), 1R01GM122798 (SR)



Working model demonstrating how location bias contributes to functionally selective cellular signaling and inflammatory responses at CXCR3