整合素激活c-Src的机制及其在血栓中的应用研究

Integrin介导的c-Src活化和血栓的形成、肿瘤细胞的迁移相关联。一般情况下，c-Src由其SH3结构域和富含脯氨酸的蛋白序列结合而活化。Integrin利用其胞内C末端的RGT来激活c-Src。由于结构数据的缺失，RGT如何结合SH3，又如何激活c-Src，是有待于解决的问题。最近，我们报道了分辨率达2.0埃的SH3:RGT复合物晶体结构（Xiao et al, Blood, 2012）。结构显示，RGT结合在SH3的"N"-Src loop。体内外的功能实验表明，"N"-Src loop的突变，将影响RGT和SH3的结合以及integrin介导的c-Src活化。我们提出了新的非经典的c-Src的活化机制模型。本计划书的研究目标是，利用结构生物学手段，并结合斑马鱼模型，进一步阐明integin激活c-Src的调控机制，同时寻找和设计可以应用在临床上的小分子靶向药物。

The integrin β3-mediated c-Src priming and activation, via the SH3 domain, is consistently associated with diseases such as the formation of thrombosis and the migration of tumor cells. Conventionally, activation of c-Src is often induced by the binding of proline-rich sequences to its SH3 domain. Instead, integrin β3 utilizes R760GT762 for priming and activation. Due to the lack of structural information, it is not clear where RGT will bind to SH3, and under what mechanism this interaction can prime/activate c-Src. Recently, we reported a 2.0 ? X-ray crystal structure in which SH3 is complexed with the RGT peptide. Functional characteriazation showed that mutations in the "N"-Src loop (i.e. the binding site) will disrupt the interaction between the RGT peptide and SH3 domain. This has led to the hypothesis of a novel non-canonical c-Src activation mechanism. More importantly, the crystal structure of SH3:RGT complex has laid out the foundation that could lead to the discovery/design of small molecules targeting the thrombosis and cancer mestatasis. In this proposal, we propose to use the tools of structura biology, the zebrafish model and other biochemical assays to 1) furhter clarify the activation mechanism of c-Src by integin β3; 2) design and test the small molecules in the content of c-Src:β3 ineraction and platelet aggregation.