Location Bias of G Protein‐Coupled Receptor Kinases Promotes Biased Signaling at CXCR3

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G protein‐coupled receptors (GPCRs) are the largest family of cell‐surface receptors and are known to canonically signal through two main effector proteins: G proteins and β‐arrestins. Biased agonism at GPCRs refers to the functional selectivity of a ligand towards certain signaling pathways over others. In addition to biased agonists, there are various other sources of bias that influence GPCR signaling, such as receptor and location bias, which affect the downstream pathways of receptor activation, leading to disproportionate levels of signaling between the G protein and β‐arrestin pathway. One of the mechanisms underlying this signaling bias is the phosphorylation barcode hypothesis which states that ligand induced receptor phosphorylation promotes differential engagement with downstream transducers. Across the entire GPCR superfamily, GPCR kinases (GRKs) are known to be primarily responsible for receptor phosphorylation. Of the seven known isoforms in the GRK family, GRK2, GRK3, GRK5, and GRK6 are expressed ubiquitously, whereas GRK1 and GRK7 are mainly found in the eye while GRK4 is predominantly located in the testes. Although there is supportive evidence demonstrating that differential phosphorylation promotes biased signaling, there is relatively little known about the role of GRKs in directing GPCR functional selectivity. To study the role of GRKs in biased signaling, we studied the GPCR CXCR3, a chemokine receptor primarily involved in leukocyte migration, which has three endogenous biased ligands, CXCL9, CXCL10, and CXCL11, and two synthetic biased ligands, VUF10661 and VUF11418. We found that, following ligand stimulation, GRK2 and GRK3 are recruited to CXCR3, with VUF10661 and CXCL11 being the most efficacious ligands. However, we also observed that GRK3 and GRK5 are able to traffic to endosomes following ligand stimulation with VUF10661, even though all ligands are able to promote receptor internalization. Given biased engagement of the GRKs observed at both the receptor and in endosomes, we next studied the functional consequences of ligand and location bias by creating GRK 2, 3, 5 and 6 constructs that are specifically targeted to the plasma membrane or the endosome. Using GRK2/3/5/6 KO cells, we determined that localization of the GRKs impacts receptor internalization, ERK activity, and transcription in a ligand specific manner. Together, we observed that the endogenous and synthetic ligands of CXCR3 demonstrate biased engagement of the GRKs at different subcellular locations and highlight the functional significance of these findings on downstream receptor signaling. These data provide evidence that the functional selectivity of biased signaling extends beyond β‐arrestins and G‐proteins to other effector proteins that interact with GPCRs.