Mechanisms of transmembrane signalling by tetraspanins

本项目将利用异核磁共振波谱技术研究人类四联蛋白的结构和相互作用。我们已经发现四跨蛋白c -末端与syntenin的串联PDZ蛋白结合，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传导和内吞作用。为了建立信号机制的模型，我们将研究PDZ结构域磷酸化和稳定末端延伸的新调控作用。胞外结构域已被表达，用于核磁共振分析其脂质相互作用位点和蛋白质对接研究，为确定其作为细胞表面受体的功能提供了基础。四跨蛋白已被表达为功能完整的全长蛋白，其生产规模将扩大，以分析其体外结构和结合特性。特别是，我们试图确定在自由和配体结合状态下的蛋白质的寡聚状态和结构特性，使用混合胶束来溶解完整的受体。结合协作的体内细胞生物学研究，这将提供更好地理解四联蛋白信号传导的结构机制。

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.