SPECTROSCOPIC STUDES OF EGF/RECEPTOR INTERACTIONS

该项目将利用独特的资源组合 范德比尔特的生物光谱学可用于探测结构和 与跨膜信号转导机制有关的动力学问题 EGF-EGF受体复合体。一系列光谱方法 包括线性和饱和转移电子顺磁共振 (EPR)光谱、荧光能量转移和荧光和 磷光各向异性衰变将在实验中使用 野生型小鼠EGF及其衍生的mEGF定点突变体 适当的光谱探头。 在本研究中，mEGF的特定定点突变体设计用于 将准备和表征光谱研究，并推出新的 将合成光谱探针。这些分子工具将是 应用于三个不同的研究领域：1)荧光能量 转移将被用来测量EGF分子之间的结合距离 在被占领的受体二聚体中。通过利用mEGF的定点突变体 派生在分子中的不同点，空间关系 受体二聚体相邻亚基中的两个分子将是 已映射。2)EGF与受体、配体解离的动力学 被占领受体单体的交换和被占领受体单体的交换 受体二聚体复合体将用EPR和荧光法进行研究 方法：研究方法。EPR将用于扩展我们对EGF的动力学研究 体外解离被占据的受体以研究其影响 受体自磷酸化的解离动力学 蛋白激酶C对受体的磷酸化及其相互作用 具有磷脂酶CGamma或其结构域的受体。EPR方法 也将被应用于调查EGF是否从 内吞作用后的受体。荧光方法将应用于 游离EGF与结合EGF的交换动力学研究 占位受体二聚体与占位交换动力学 占据受体二聚体之间的受体单体。3)旋转 被占据的EGF受体在膜制剂中的扩散 完整的细胞将使用饱和转移EPR和/或 磷光各向异性衰减测量。

This project will take advantage of a unique combination of resources in biological spectroscopy available at Vanderbilt to probe structural and kinetic questions related to the mechanism of transmembrane signaling by the EGF-EGF receptor complex. An array of spectroscopic approaches including linear and saturation transfer electron paramagnetic resonance (EPR) spectroscopy, fluorescence energy-transfer and fluorescence and phosphorescence anisotropy decay will be employed in experiments utilizing wild type murine EGF and site-directed mutants of mEGF derivitized with appropriate spectroscopic probes. For this study, specific site-directed mutants of mEGF designed for spectroscopic studies will be prepared and characterized, and new spectroscopic probes will be synthesized. These molecular tools will be applied to three distinguishable areas of study: 1) Fluorescence energy transfer will be used to measure the distance between EGF molecules bound in the occupied receptor dimer. By employing site-directed mutants of mEGF derivitized at different points in the molecule, the spatial relationship of the two molecules in adjacent subunits of the receptor dimer will be mapped. 2) Kinetics of dissociation of EGF from the receptor, of ligand exchange, and of exchange of occupied receptor monomers in the occupied receptor dimer complex will be investigated using EPR and fluorescence methods. EPR will be used to extend our kinetic studies of EGF dissociation from the occupied receptor in vitro to investigate the effects of dissociation kinetics of receptor autophosphorylation, of phosphorylation of the receptor by protein kinase C, and of the interaction of the receptor with phospholipase Cgamma or domains thereof. EPR methods will also be applied to investigate whether EGF is released from the receptor following endocytosis. Fluorescence methods will be applied to investigation of the kinetics of the exchange of free EGF with EGF bound to the occupied receptor dimer and the kinetics of exchange of occupied receptor monomers between occupied receptor dimers. 3) The rotational diffusion of the occupied EGF receptor in membrane preparations and in intact cells will be investigated using saturation transfer EPR and/or phosphorescence anisotropy decay measurements.