Structural Basis for Chemokine Function

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DESCRIPTION (provided by applicant): The goal of this project is to understand the structural and functional requirements for chemokine signaling in vivo. Chemokines are small secreted proteins that mediate inflammation, stem cell homing and early embryonic development by providing directional cues to migrating cells. The 50 known chemokines induce chemotaxis by specifically activating members of a group of 20 G protein-coupled receptors, which are integral membrane proteins of the seven transmembrane type. A chemokine concentration gradient maintained by interactions with extracellular matrix glycosaminoglycans (GAG) is also needed to induce chemotactic responses in vivo, and, for some chemokines, formation of homodimeric structures is also a functional requirement. Stromal cell-derived factor-1 (SDF1) and its cognate receptor CXCR4 comprise a chemokine signaling system that is exploited by metastatic cancers and HIV/AIDS. HIV-1 gains entry to T cells through specific binding to CXCR4, a process that is inhibited by the chemokine SDF1. Cancer cells that express CXCR4 and escape the primary tumor environment travel the circulatory and lymphatic systems homing in on a select group of tissues that constitutively produce high levels of SDF1, including bone marrow, lung and lymph nodes. A complete model for chemokine function at a molecular level will include GPCR activation, GAG binding and dimer formation. We intend to combine NMR spectroscopy with mutagenesis studies and functional assays to characterize each of the specific binding interactions required for SDF1-CXCR4 signaling. In Aim 1, we will determine whether SDF1 functions as a monomer or dimer by designing mutants that form only one or the other species and testing their activities in vivo. Experiments in Aim 2 will determine the structure of an SDF1-GAG complex, and explore the relationship between GAG binding and dimerization. In Aim 3 we will determine the structure of a complex between SDF1 and a soluble fragment of CXCR4, and pursue NMR studies of the full-length CXCR4 protein solubilized in detergent micelles. These studies will enable the construction of a unified model for SDF1 activity in vivo that accounts for all its known functional interactions.