Mechanisms of transmembrane signalling by tetraspanins

本计画将利用核磁共振技术研究人类四跨膜蛋白的结构与相互作用。我们已经发现，四跨膜蛋白的C-末端结合的串联PDZ蛋白的syntenin，并打算阐明这些相互作用的结构基础，以了解它们如何影响受体信号和内吞作用。PDZ结构域磷酸化和稳定末端延伸的新的调节作用将被研究，以建立信号传导机制的模型。细胞外结构域已被表达用于其脂质相互作用位点的NMR分析和蛋白质对接研究，为定义其作为细胞表面受体的功能提供了基础。四跨膜蛋白已被表达为功能完整的全长蛋白质，生产将扩大规模，以分析其体外结构和结合特性。特别是，我们试图确定的寡聚状态和结构特性的蛋白质在自由和配体结合状态使用混合胶束溶解的完整受体。与合作在体内细胞生物学研究，这将提供一个更好的理解四跨膜蛋白信号的结构机制。

In this project the structures and interactions of the human tetraspanin proteins will be investigated by heteronuclear magnetic resonance spectroscopy. We have discovered that tetraspanin C-termini are bound by the tandem PDZ proteins of syntenin, and intend to elucidate the structural basis of these interactions in order to understand how they influence receptor signaling and endocytosis. The novel regulatory effects of PDZ domain phosphorylation and stabilizing terminal extensions will be investigated in order to build a model of the signaling mechanism. The extracellular domain has been expressed for NMR analysis of its lipid interaction sites and protein docking studies, providing a basis for defining its function as an cell surface receptor. Tetraspanins have been expressed as functionally intact full length proteins, and production will be scaled up to analyse their structural and binding properties in vitro. In particular, we seek to determine the oligomeric state and structural properties of the proteins in the free and ligand bound states using mixed micelles to solubilize the intact receptor. Together with collaborative in vivo cell biological studies this will provide a better understanding of the structural mechanism of tetraspanin signaling.