Signaling Mechanisms and Mobility of the EGF Receptor

小行星9304393 该项目的重点是表皮生长因子（EGF）通过其受体（EGF-R）的结合调节。 将通过使用定量光学显微镜在单细胞水平上研究EGF的荧光衍生物（f-EGF）与EGF-R结合的时间过程。 初步数据表明，结合的f-EGF培养的A431细胞进行了一个双相的时间过程，这是不符合普遍接受的模型EGF/EGF-R，其中两个EGF-R的亲和力类被假定为存在于固定的密度在细胞表面上。 已开发出与单个细胞结合数据一致的三元复合物模型。 有人提出，肌动蛋白/EGF-R的相互作用，通过特定的结合域调节受体的亲和力状态。 最近的研究表明EGF-R是一种肌动蛋白结合蛋白。 HL-33，一种与EGF-R竞争肌动蛋白结合位点的肽，将被用作这种相互作用的探针。 初步的f-EGF与单个A431细胞的结合数据也表明，结合的动力学在细胞之间是可变的。 还将通过定量单细胞荧光成像来检查这种可变的时间和主要信号输入对随后的单个细胞响应的程度的后果。 预测的EGF-R与肌动蛋白相互作用的结果是，一些单独的EGF-R将被固定在质膜上，并且固定的受体将优先具有高亲和力。 测量单个EGF受体在刺激和未刺激细胞上的迁移率形成肌动蛋白调节模型的重要测试。 单一受体将通过生物素化的抗受体Fab'片段和荧光链霉亲和素-乳胶纳米颗粒或链霉亲和素-胶体金颗粒作为可视化探针来标记。 单个EGF-R的横向迁移率将在EGF转染之前和期间进行跟踪，并将在用HL-33竞争肽显微注射的单个细胞中进行检查。 将检查单个EGF受体运动和受体的亲和力状态之间的相关性。 将单个EGF-R的横向迁移率与单个LDL受体的横向迁移率进行比较。 (The LDL受体系统是迄今为止唯一描述了单个受体运动的系统;在该系统中，三种类型的横向运动被表征为包括随机、定向和无移动性。 这项研究的目的是更好地了解已知调节细胞生长的细胞表面蛋白质，表皮生长因子受体和激活它的信号分子，表皮生长因子（EGF）之间的分子相互作用。 这两种分子对单个培养细胞的相互作用将使用荧光标记的EGF和定量光学显微镜进行研究。 EGF受体系统的这项研究结果将大大增加有关生长因子信号细胞分裂机制的分子细节的知识。 ***

9304393 Gross This project focuses on the regulation of binding of epidermal growth factor (EGF) by its receptor (EGF-R). The time course of binding of a fluorescent derivative of EGF (f-EGF) to the EGF-R will be studied at the level of single cells by the use of quantitative optical microscopy. Preliminary data indicates that the binding of f-EGF to cultured A431 cells proceeds over a biphasic time course that is not consistent with the commonly accepted model of EGF/EGF-R in which two EGF-R affinity classes are postulated to exist at fixed densities on the cell surface. A ternary complex model has been developed that is consistent with the individual cell binding data. It is proposed that actin/EGF-R interaction through specific binding domains modulates the affinity state of the receptor. Recent studies have shown that the EGF-R is an actin-binding protein. Microinjection of HL-33, a peptide that competes for the actin binding site with the EGF-R, will be used as a probe of this interaction. Preliminary f-EGF binding data to individual A431 cells also indicates that the kinetics of binding are variable between cells. The consequences of this variable timing and degree of primary signal input on subsequent individual cell responses will also be examined by quantitative, single-cell fluorescence imaging. A consequence of the predicted interaction of the EGF-R with actin is that some individual EGF-Rs will be immobilized in the plasma membrane, and that the immobile receptors will be preferentially of the high-affinity class. Measurement of the mobilities of individual EGF receptors on stimulated and unstimulated cells forms an important test of the actin-regulation model. Single receptors will be labeled by biotinylated anti- receptor Fab' fragments and fluorescent streptavidin-latex nanoparticles or streptavidin-colloidal gold particles as visualization probes. The lateral mobility of individual EGF-Rs will be followed before and during EGF challen ge, and will be examined in individual cells microinjected with the HL-33 competing peptide. Correlations between individual EGF receptor movements and the affinity state of the receptor will be examined. The lateral mobilities of individual EGF-Rs will be compared to those for individual LDL receptors. (The LDL receptor system is thus far the only one in which individual receptor movements have been described; in this system three classes of lateral movement were characterized including random, directed, and no mobility.) %%% The goal of this research is to better understand the molecular interactions between a cell surface protein that is known to regulate cell growth, the epidermal growth factor receptor, and the signal molecule that activates it, epidermal growth factor (EGF). The interaction of these two molecules on individual cultured cells will be studies using fluorescently labeled EGF and quantitative optical microscopy. The results of this study of the EGF receptor system will add much to knowledge about molecular details of the mechanism by which growth factors signal cells to divide. ***