Lipid Rafts and EGF Receptor Function

描述（由申请人提供）：EGF受体是一种质膜酪氨酸激酶，参与细胞生长的控制。在结合EGF后，EGF受体二聚化并通过其细胞内酪氨酸激酶的活化来转导其信号。EGF受体与低密度、富含胆固醇的膜结构域（称为筏）相关，其可能代表组织和调节信号转导的机制。在之前的研究中，我们发现胆固醇水平的改变会影响EGF受体结合和激酶活性，这表明EGF受体与筏的结合可能会调节受体功能。此外，我们开发了一种单分子方法，荧光强度聚类分析（FICA），使我们能够定义EGF受体在活细胞质膜上大小增加的簇之间的分布。使用这种方法，我们表明，胆固醇消耗增强受体聚集，这表明筏在这个过程中的参与。我们还表明，EGF刺激导致含有4个或更多受体的簇的形成。然而，受体单体仍然存在于用饱和剂量的EGF刺激的细胞中。从一个新的酶互补试验，评估EGF受体二聚化的结果也表明，EGF受体簇解离在高浓度的EGF。我实验室的长期研究目标是了解EGF受体的激活机制以及膜微区对这一过程的可能贡献。本研究的主要目的是：1）确定EGF受体胞外区近膜部分的结构特征，这些结构特征参与介导高亲和力EGF结合、信号转导和受体与脂筏的结合; 2）阐明EGF受体聚集的分子基础，以及EGF和膜胆固醇水平的变化;和3）确定二聚体解离对EGF受体活化过程的贡献。这些研究将使用传统的诱变分析，以及我们的新方法，亮度分析，FICA，和我们的新的酶互补试验。EGF受体在多种人类肿瘤中过度表达或组成性激活，目前的几种治疗方法都是基于抑制EGF受体的功能。为了充分了解信号转导过程，并最大限度地提高我们靶向EGF受体的治疗能力，有必要建立一个连贯的模型，该受体的激活机制，并了解受体的膜环境如何有助于EGF受体功能的整体调节。在这项授权中提出的实验专门解决这一需要，通过检查一个新的模型激活的表皮生长因子受体，并通过进一步阐明的潜在参与富含胆固醇的膜微区在调节表皮生长因子受体介导的信号。

DESCRIPTION (provided by applicant): The EGF receptor is a plasma membrane tyrosine kinase that is involved in the control of cell growth. Upon binding EGF, the EGF receptor dimerizes and transduces its signal via the activation of its intracellular tyrosine kinase. The EGF receptor associates with low density, cholesterol-enriched membrane domains, called rafts, that may represent a mechanism for organizing and regulating signal transduction. In the previous grant, we showed that alterations in cholesterol levels affect both EGF receptor binding and kinase activities, suggesting that the association of the EGF receptor with rafts may modulate receptor function. In addition, we developed a single-molecule method, fluorescence intensity cluster analysis (FICA), that allows us to define the distribution of the EGF receptor among clusters of increasing size on the plasma membrane in live cells. Using this method, we showed that cholesterol depletion enhances receptor clustering, suggesting the involvement of rafts in this process. We have also shown that EGF stimulation leads to the formation of clusters containing 4 or more receptors. However, receptor monomers still exist in cells stimulated with saturating doses of EGF. Results from a new enzyme complementation assay that assesses EGF receptor dimerization also suggest that EGF receptor clusters dissociate at high concentrations of EGF. The long term goal of the research in my laboratory is to understand the mechanism of activation of the EGF receptor and the possible contribution of membrane microdomains to this process. The specific aims of this new proposal are to: 1) Define the structural features of the membrane proximal portion of the extracellular domain of the EGF receptor that are involved in mediating high affinity EGF binding, signal transduction and association of the receptor with lipid rafts; 2) Elucidate the molecular basis of EGF receptor clustering in response to EGF and changes in membrane cholesterol levels; and, 3) Determine the contribution of dimer dissociation to the process of EGF receptor activation. These studies will be accomplished using traditional mutagenesis analysis as well as our new method for brightness analysis, FICA, and our new enzyme complementation assay. The EGF receptor is over-expressed or constitutively activated in a variety of human tumors and several current therapies are based on inhibiting the function of the EGF receptor. To fully understand the process of signal transduction and to maximize our ability to target the EGF receptor for therapeutic purposes, it is necessary to establish a coherent model for the mechanism of activation of this receptor and to understand how the membrane environment of the receptor contributes to the overall regulation of EGF receptor function. The experiments proposed in this grant specifically address this need by examining a new model for activation of the EGF receptor and by further elucidating the potential involvement of cholesterol-rich membrane microdomains in modulating EGF receptor-mediated signaling.