Project Abstract CXCL12 stimulation of the chemokine receptors CXCR4 and ACKR3 drives cellular migration during development, immune system mobility, and inflammatory responses. The trio plays a major role in cancers where they promote metastasis and tumor proliferation. Thus, both receptors are promising targets for therapeutics, with ongoing clinical trials of compounds targeting CXCR4 and ACKR3 ligands in development. Despite the common agonist, CXCR4 and ACKR3 have decidedly different responses. While CXCR4 signals through both G proteins and β-arrestins like other classical G protein-coupled receptors (GPCRs), ACKR3 only couples with arrestins. . CXCR4 and ACKR3 are structurally alike and bind CXCL12 in a similar manner, yet the receptors appear to activate by different mechanisms. CXCR4 is extremely sensitive to changes to the chemokine interaction, with single-point mutations leading the high-affinity agonist to act as an antagonist. Conversely, ACKR3 is promiscuous, with nearly all ligands tested acting as agonists. How these differences translate into the biased signaling is an open question. I propose to investigate the how the CXCL12 signal is interpreted by these receptors by resolving how the ligand affects the receptor conformation and the secondary interactions with kinases and β-arrestins. The central hypothesis of this proposal is that these interactions will be different for the classical and atypical receptors and the results of the presented experiments will provide insights into receptor-level biased signaling. This hypothesis will be pursued by three specific aims. Aim 1: Identify the structural rearrangements of ACKR3 induced by CXCL12 binding by determining the high resolution crystal structure of the complex. The resulting structure will reveal the specific interactions through an atypical receptor that are induced by CXCL12 binding and structural basis of activation. Aim 2: Determine how CXCL12-activated ACKR3 is phosphorylated and interacts with β-arrestin. This aim will determine what kinases phosphorylate the ACKR3, where those phosphates are incorporated and how the pattern of modification alters β-arrestin interactions and signaling. Aim 3: Determine how β-arrestin interacts with CXCL12-stimulated CXCR4 by imaging the complex with electron microscopy and ultimately resolving the structure of the CXCL12:CXCR4:β-arrestin complex. Together, these studies will present an unparalleled view into how receptor-mediated biased agonism is manifested by classical and atypical GPCRs. Determining how these receptors respond the natural stimulus will ultimately facilitate future drug development.