Receptor Endocytosis as a Mechanism of Biased Agonism at CXCR3

G Protein Coupled Receptors (GPCRs) are the largest class of receptors in the human genome. They are expressed in virtually every cell type and are implicated in numerous physiological processes. GPCR signaling is highly specific due to biased agonism, in which different ligands, receptors, or cellular systems selectively activate different signaling pathways. The chemokine system, a subfamily of GPCRs primarily present on leukocytes, serves as an endogenous example of biased agonism due to the significant promiscuity between chemokine ligands and receptors. For example, the chemokine receptor CXCR3 and its ligands CXCL9, CXCL10, and CXCL11 have clearly demonstrated bias in vitro and in vivo. GPCRs are conventionally understood to signal exclusively from the plasma membrane. Therefore, the ligand‐induced internalization of receptors is thought to desensitize G Protein‐dependent signaling. However, recent research has shown that numerous GPCRs signal from intracellular membranes, including the endoplasmic reticulum, the golgi, and the endosome. This novel finding has led to the discovery that the subcellular location of a GPCR can affect its functional output. The objective of this study was to evaluate the contribution of receptor endocytosis to CXCR3's biased agonism. We hypothesize that CXCR3 signals from the early endosome, and as CXCL9, CXCL10, and CXCL11 promote different patterns of internalization, endocytic signaling is a mechanism for the functional selectivity of its endogenous ligands. We performed BRET and confocal microscopy on C‐terminal phosphodeficient mutants of CXCR3 to measure their internalization in response to stimulation with CXCL9, CXCL10, or CXCL11. While stimulation with CXCL9 did not induce significant internalization of WT CXCR3 or any CXCR3 mutants, stimulation with CXCL10 or CXCL11 promoted β‐arrestin mediated internalization of CXCR3 that was decreased in the phosphodeficient mutants. Internalization was almost completely eliminated at the CXCR3‐L344X truncation mutant. We also measured the phosphorylation of extracellular‐signal related kinase 1/2 (ERK 1/2) in response to stimulation with CXCL9, CXCL10, or CXCL11. These results showed that the CXCR3 mutants promoted more pERK 1/2 than WT CXCR3 when stimulated with CXCL10 or CXCL11. Notably, CXCR3‐L344X promoted the most pERK 1/2 out of all studied receptors. This negative correlation between receptor internalization and pERK1/2 could suggest that endocytosis has an inhibitory effect on ERK signaling. To further assess the contribution of endocytic signaling to CXCR3's signaling effects, we will inhibit receptor endocytosis through treatment with Barbadin, a selective pharmacological inhibitor of the β‐arrestin/AP2 complex required for endocytosis, or through overexpression of mutant dynamin, a protein responsible for scission of clathrin‐coated endocytic vesicles. We will evaluate how inhibiting endocytosis impacts CXCR3's ligand‐induced G Protein signal, ERK response, transcriptional reporter activation, and chemotaxis. We expect that inhibiting endocytosis will decrease the extent of bias between the three ligands, thus implicating location bias as a factor in CXCR3's functional selectivity.

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