SPECTROSCOPIC STUDIES OF EGF RECEPTOR INTERACTIONS

描述：该项目将结合三个实验室的资源来 研究表皮生长因子相互作用的分子细节 以及它的受体。在一大类具有内在蛋白的受体中 激酶活性，是EGF/EGF受体系统中最重要的 深入研究成员，既是因为它在监管方面的重要性 细胞增殖，因为它是这种生长的模型 班级。尽管如此，有关机制的基本问题 EGF/EGF受体相互作用触发的信号转导仍然存在 悬而未决。建议的研究将旨在开发一种 深入了解EGF发生时最早发生的一些事件 与其质膜受体结合。表皮生长因子的定点突变 将被用来为自旋标记物和 在已知三维空间中的选定位置处的光学探测器 荷尔蒙的结构。这些标记的EGF将用于EPR和 旋光分子的荧光/荧光光谱研究 被占领的受体复合体的动力学。这些研究将针对 假设与高水平的EGF结合的受体亚群 亲和力与细胞骨架相互作用。新的自旋标记和 磷光亲和探针法测定蛋白激酶结构域的ATP结合位点 的受体将被合成并应用于EPR和磷光 分别对受体的旋转物种进行了研究 其存在于EGF结合之前和饱和的不同阶段。这些 研究将解决另一种假设，即高亲和力受体 是一个在没有配体的情况下是二聚体的亚群。荧光 将使用共振能量转移来解决基本问题 每个激活的受体二聚体结合的EGF数目的问题。已停止 表皮生长因子结合的A431细胞的流动荧光各向异性实验 解离将直接测量相对人口和 OFF率与高亲和力受体和低亲和力受体相关。这个 将测定与EGF结合的佛波酯的动力学效应，并 与EPR和EPR观察到的受体旋转群体相关 磷光各向异性衰减。

DESCRIPTION: This project will combine resources of three laboratories to study molecular details of the interaction of epidermal growth factor (EGF) and its receptor. Among the large class of receptors with intrinsic protein kinase activity, the EGF / EGF receptor system is one of the most intensively studied members, both because of its importance in regulating cellular proliferation and because it serves as a model for this growing class. Nonetheless, fundamental questions concerning the mechanisms of signal transduction triggered by EGF / EGF receptor interactions remain unresolved. The studies proposed will be directed toward developing an in-depth understanding of some of the earliest events that occur when EGF binds to its plasma membrane receptor. Site-directed mutagenesis of EGF will be employed to engineer unique reaction sites for spin-labels and optical probes at selected locations in the known three-dimensional structure of the hormone. These labelled EGF's will be employed in EPR and fluorescence/phosphore scence spectroscopic investigations of the rotational dynamics of the occupied receptor complex. These studies will address the hypothesis that the subpopulation of receptors which bind EGF with high affinity interacts with the cytoskeleton. New spin-labeled and phosphorescent affinity probes for the ATP binding site of the kinase domain of the receptor will be synthesized and employed in EPR and phosphorescence investigations, respectively, of the rotational species of the receptor which exist prior to EGF binding and at various stages of saturation. These studies will address the alternative hypothesis that high affinity receptors are a subpopulation that are dimeric in the absence of ligand. Fluorescence resonance energy transfer will be employed to address the fundamental question of the number of EGF's bound per activated receptor dimer. Stopped flow fluorescence anisotropy experiments on A431 cells of EGF association / dissociation will directly measure relative populations and on- and off-rates asocciated with high and low affinity classes of receptors. The kinetic effects of phorbol esters of EGF binding will be determined and correlated with rotational populations of receptors observed using EPR and phosphorescence anisotropy decay.