Lipid Rafts and EGF Receptor Function

描述(申请人提供)：EGF受体是一种质膜酪氨酸激酶，参与控制细胞生长。在与EGF结合后，EGF受体通过激活其细胞内酪氨酸激酶来二聚化和转导其信号。EGF受体与低密度、富含胆固醇的膜域相关，称为RAFT，它可能代表了一种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同时影响EGF受体结合和激酶活性，提示EGF受体与RAFT的联系可能调节受体功能。此外，我们发展了一种单分子方法，荧光强度聚类分析(FICA)，它允许我们定义EGF受体在活细胞质膜上不断增大的簇中的分布。使用这种方法，我们发现胆固醇耗竭增强了受体聚集，表明RAFT参与了这一过程。我们还表明，EGF刺激会导致包含4个或更多受体的簇的形成。然而，在饱和剂量的EGF刺激的细胞中，受体单体仍然存在。一种新的评估EGF受体二聚化的酶互补试验的结果也表明，EGF受体簇在高浓度的EGF下解离。本实验室研究的长期目标是了解EGF受体激活的机制以及膜微域在这一过程中的可能贡献。这一新建议的具体目的是：1)确定EGF受体胞外结构域膜近端的结构特征，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受体聚集响应EGF和膜胆固醇水平变化的分子基础；以及3)确定二聚体解离在EGF受体激活过程中的作用。这些研究将使用传统的突变分析以及我们的亮度分析新方法FICA和我们的新的酶互补分析方法来完成。EGF受体在多种人类肿瘤中过度表达或结构性激活，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的过程，并最大限度地提高靶向EGF受体治疗的能力，有必要建立一个连贯的模型来研究EGF受体的激活机制，并了解受体的膜环境如何在EGF受体功能的整体调控中发挥作用。这项资助中提出的实验通过研究EGF受体激活的新模型并进一步阐明富含胆固醇的膜微域在调节EGF受体介导的信号中的潜在参与，专门解决了这一需求。

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.