Biasing CXCR3 Signaling to Modulate the Inflammatory Response

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ABSTRACT CXCR3 is a chemokine receptor (CKR) that plays a central role in inflammation through its regulation of T cell migration and function. Despite the established clinical relevance of CKRs in disease, there are only three FDA approved drugs that target the entire chemokine system, which consists of approximately twenty receptors and fifty ligands that regulate nearly every aspect of inflammation. Reasons for this difficulty in CKR drug develop- ment include the potential redundancy between multiple cognate chemokine ligands for a given CKR and a lack of knowledge regarding how the signaling pathways activated by CKRs regulate immune cell function and in- flammation. Thus, there is a critical unmet need for drugs targeting the chemokine system. This puts into context work from my group on the chemokine receptor CXCR3. We have shown that the cognate ligands of CXCR3, CXCL9, 10 and 11, act as biased agonists, generating quantitatively and qualitatively distinct signals from one another through their interactions with heterotrimeric G proteins and β-arrestin adapter proteins. In the previous project period, we have identified small-molecule G protein- and β-arrestin-biased CXCR3 agonists that (1) dif- ferentially activate signaling pathways downstream of CXCR3, and (2) have distinct effects in a mouse model of T-cell-mediated inflammatory skin disease. These findings suggest distinct roles for G proteins and β-arrestin in promoting the CXCR3-mediated inflammatory response. The long-term goal of our research is to determine the mechanisms underlying biased agonism to develop novel therapies targeting CKRs in inflammation. The overall objective of this proposal is to determine how biased agonists promote different effector conformations that lead to distinct patterns of signaling resulting in changes in T cell function and inflammation. Our central hypothesis is that β-arrestin-biased agonists induce unique receptor and β-arrestin conformations that favor “location bi- ased” endosomal signaling that promotes a T cell transcriptional response that results in different patterns of inflammation. To address our objective, first, we will determine how the receptor:ligand complex promotes biased responses through allosteric regulation of effectors. We have found that CXCR3 biased agonists promote differ- ent β-arrestin-mediated effects, which we will explore further by using receptor mutants. Then, we will determine how endosomal signaling and location bias contribute to CXCR3 biased signaling. We have found that some CXCR3 biased agonists promote transcription that can be prevented by inhibiting receptor endocytosis. Lastly, we will determine how CXCR3 G protein- and β-arrestin-mediated signaling pathways contribute to T cell function and the inflammatory response. We will use CXCR3 mutants to test the contributions of specific signaling path- ways to T cell chemotaxis in vitro and inflammation in vivo. This project explores an innovative approach to study CXCR3 signaling that will provide an understanding of CXCR3 regulation of the inflammatory response by the selective activation of G proteins and β-arrestins. The research is significant as it will lay the groundwork for future research on biased agonists as CXCR3 therapeutics and serve as a model for targeting other CKRs.