Structural Basis for Chemokine Function

描述(申请人提供)：本项目的目标是了解体内趋化因子信号的结构和功能要求。趋化因子是一种分泌的小蛋白，通过为迁移细胞提供定向信号，介导炎症、干细胞归巢和早期胚胎发育。已知的50种趋化因子通过特异性激活一组20G蛋白偶联受体的成员来诱导趋化作用，这些受体是七种跨膜类型的完整膜蛋白。通过与细胞外基质糖胺多聚糖(GAG)的相互作用维持的趋化因子浓度梯度也是体内诱导趋化反应所必需的，对于某些趋化因子来说，形成同源二聚体结构也是一种功能要求。基质细胞衍生因子-1(SDF1)及其同源受体CXCR4构成趋化因子信号系统，被转移性癌症和HIV/AIDS所利用。HIV-1通过与CXCR4特异性结合进入T细胞，这一过程被趋化因子SDF1抑制。表达CXCR4并逃离原发肿瘤环境的癌细胞通过循环系统和淋巴系统，聚集在一组选择性的组织上，这些组织构成了高水平的SDF1，包括骨髓、肺和淋巴结。一个完整的分子水平趋化因子功能模型将包括GPCR激活、GAG结合和二聚体形成。我们打算将核磁共振波谱与突变研究和功能分析相结合，以表征SDF1-CXCR4信号所需的每一种特定结合相互作用。在目标1中，我们将通过设计只形成一种或另一种物种的突变体并测试它们在体内的活性来确定SDF1是作为单体还是作为二聚体发挥作用。目标2中的实验将确定SDF1-Gag复合物的结构，并探索Gag结合和二聚化之间的关系。在目标3中，我们将确定SDF1与CXCR4的可溶性片段之间的络合物的结构，并对溶解在洗涤剂胶束中的全长CXCR4蛋白进行核磁共振研究。这些研究将为SDF1在体内的活性构建一个统一的模型，该模型解释了SDF1所有已知的功能相互作用。

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.