(R2169) Differential GRK Recruitment and the Phosphorylation Barcode as a Mechanism for Biased Agonism at G Protein-Coupled Receptors

Julia Gardner (Duke University)| Dylan Eiger (Duke University)| Christopher Honeycutt (Duke University)| Noelia Boldizsar (Duke University )| Issac Choi (Duke University)| Sudarshan Rajagopal (Duke University)

G Protein-Coupled Receptors (GPCRs) are the largest and most diverse family of cell receptors in the human genome and account for ~30% of all FDA-approved drugs. GPCR signaling is mediated by various effectors, including G proteins, β-arrestins, and GPCR kinases (GRKs). Some ligands preferentially activate G protein- or β-arrestin-dependent signaling pathways, a phenomenon known as biased agonism. One proposed mechanism for GPCR biased signaling—the phosphorylation barcode—hypothesizes that unique phosphorylation patterns of a GPCR induce differential signaling pathways, thereby producing highly specific cellular outputs. 

 To study the phosphorylation barcode hypothesis as a mechanism of biased agonism, we explored the differential recruitment of GRKs 2, 3, 5, and 6 to CXCR3, a chemokine receptor which binds three endogenous biased ligands, CXCL9, CXCL10, and CXCL11. We hypothesized there to be differential recruitment of the GRKs to CXCR3 depending on the endogenous ligand used for stimulation. 

 We first used a split nano-luciferase assay in HEK293T cells to examine the recruitment of GRKs 2, 3, 5, and 6 to the wild type receptor and a variety of phosphorylation deficient mutants following stimulation with the endogenous ligand. An intramolecular biosensor assay known as FlAsh-BRET—fluorescent arsenical hairpin-bioluminescence resonance energy transfer—was also used to monitor β-arrestin2 conformational changes in GRK 2, 3, 5, 6 knockout HEK293 cells following rescue of each individual GRK and stimulation with the endogenous ligands. 

 The split luciferase assay showed evidence of ligand bias across CXCR3's endogenous ligands. Whereas GRKs 2 and 3 were recruited to the receptor, GRKs 5 and 6 were shown to not actively recruit. GRK recruitment at the phosphorylation mutants and the WT receptor revealed evidence of ligand bias in both the magnitude and location (C-terminus vs. receptor core) of GRK recruitment to the receptor for GRKs 2 and 3. The FlAsh-BRET assay to monitor β-arrestin2 conformation changes revealed biased activation of β-arrestin2, which was differentially directed by the individual GRKs, including GRK5 and GRK6. 

 We demonstrate the endogenous ligands of CXCR3 lead to differential recruitment of the GRKs which promote diverse β-arrestin2 conformations. Our work provides supporting evidence that an overall biased cellular response involves the complex interaction between a GPCR ligand, phosphorylated receptor, GRKs, and other effectors. Further exploration of the biased properties of chemokine ligands and the roles of the GRKs and phosphorylation patterns of CXCR3 may underscore the therapeutic promise of developing highly specific biased agonists to treat a variety of disorders involving chemokine receptors and the broader GPCR family.



Differential recruitment of GRKs 2 and 3 to WT CXCR3 upon stimulation with varying concentrations of CXCL9, CXCL10, and CXCL11