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 chemokine ligands for a given CKR and a lack of knowledge of how the signaling pathways activated by CKRs regulate immune cell function and inflammation. 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 chemokine 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) differentially 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 pro- moting 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 CXCR3 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 promote unique receptor and β-arrestin conformations that favor “location-biased” endosomal signaling that promotes a unique transcriptional response in T cells and inflamma- tion. To address our objective, first, we will determine how the receptor:ligand complex promotes biased re- sponses through allosteric regulation of effectors. We have found that CXCR3 biased agonists promote different 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...