SPECTROSCOPIC STUDIES OF EGF RECEPTOR INTERACTIONS

EGF 受体相互作用的光谱研究

• 批准号: 6591953
• 负责人: JAMES V STAROS
• 金额: $ 7.74万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6591953
• 关键词: affinity labeling; biological signal transduction; cell membrane; electron spin resonance spectroscopy; epidermal growth factor; fluorescence spectrometry; growth factor receptors; high performance liquid chromatography; molecular site; protein tyrosine kinase; receptor binding; site directed mutagenesis; stop flow technique

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.

描述：该项目将结合三个实验室的资源 研究表皮生长因子 (EGF) 相互作用的分子细节 及其受体。 具有内在蛋白的一大类受体之一 EGF/EGF受体系统是最具激酶活性的系统之一 深入研究成员，既因为其在监管方面的重要性 细胞增殖，因为它可以作为细胞增殖的模型 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 未解决。 拟议的研究将旨在开发一种 深入了解 EGF 发生时发生的一些最早事件 与其质膜受体结合。 EGF 的定点诱变 将用于设计自旋标签的独特反应位点和 光学探针位于已知三维空间中的选定位置 激素的结构。 这些标记的 EGF 将用于 EPR 和 旋转的荧光/磷光光谱研究 占据的受体复合物的动力学。 这些研究将解决 假设与 EGF 结合的受体亚群具有高 亲和力与细胞骨架相互作用。 新的自旋标记和 用于激酶结构域 ATP 结合位点的磷光亲和探针 的受体将被合成并用于 EPR 和磷光 分别研究受体的旋转种类 它存在于 EGF 结合之前并处于不同的饱和阶段。 这些 研究将解决另一种假设，即高亲和力受体 是在没有配体的情况下呈二聚体的亚群。 荧光 将采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 数量的问题。 已停止 EGF关联A431细胞流式荧光各向异性实验 / 解离将直接测量相对群体以及上和下 解离速率与受体的高亲和力和低亲和力类别相关。 这 将确定佛波酯与 EGF 结合的动力学效应 与使用 EPR 观察到的受体轮换群体相关 磷光各向异性衰减。