Lipid Rafts and EGF Receptor Function

描述（由申请方提供）：小窝和脂筏相对较低 密度，耐洗涤剂的膜域。他们被认为与 在信号转导过程中，由于各种信号成分 包括EGF受体，已经被证明是高度富集在这些 域.我们已经证明了完整的脂筏是正常功能所必需的 EGF刺激的PI周转和胆固醇调节EGF受体 结合和激酶活性。这些发现表明，脂筏 EGF受体介导的信号传导的调节的重要贡献者。到 了解脂质筏在EGF受体信号传导中的作用，四种特异性 目的：1）分析EGF受体在脂质间的分布 完整细胞中的筏和非筏隔室。2)确定域 表皮生长因子受体，负责将受体靶向脂质 木筏3)筏上和筏上EGF受体酶学性质的比较 膜组分以及，4）评估小窝蛋白-1 抑制EGF受体激酶活性。两种非侵入性， 基于荧光的方法，荧光光漂白恢复和 荧光相关光谱法，将用于确定相对 快速扩散（非筏）和缓慢扩散（筏）GFP-EGF的比例 完整细胞中的受体。EGF受体与EGF受体之间的结构域交换 VSV-G蛋白，一种非筏蛋白，将用于鉴定EGF的部分， 负责将受体导入脂筏的受体。的影响 筏定位和胆固醇水平对EGF受体功能的影响 将通过评估受体结合和激酶活性直接测量， 筏和非筏膜部分在体外。有何机制 小窝蛋白，小窝蛋白-1抑制表皮生长因子受体激酶的活性， 通过诱变这两个相互作用的拟议位点进行研究， 蛋白质，并通过分析胆固醇在 caveolin-1介导的EGF受体激酶抑制。所有这些 研究将确定EGF受体如何以及在多大程度上分配到 脂筏他们还将描绘筏定位对EGF的影响， 受体功能这些发现将有助于深入了解 脂质筏调节EGF受体介导的信号传导。

DESCRIPTION (provided by applicant): Caveolae and lipid rafts are related low density, detergent-resistant membrane domains. They are thought to be involved in the process of signal transduction since a variety of signaling components including the EGF receptor, have been shown to be highly enriched in these domains. We have shown that intact lipid rafts are required for proper function of EGF-stimulated PI turnover and that cholesterol modulates EGF receptor binding and kinase activities. These findings suggest that lipid rafts are important contributors to the regulation of EGF receptor-mediated signaling. To understand the role of lipid rafts in EGF receptor signaling, four specific aims are proposed: 1) Analyze the distribution of EGF receptors between lipid rafts and the non-raft compartment in intact cells. 2) Identify the domains(s) of the EGF receptor that are responsible for targeting the receptor to lipid rafts. 3) Compare the enzymatic properties of EGF receptors in raft and on-raft membrane fractions. And, 4) Evaluate the mechanism through which caveolin-1 induces inhibition of EGF receptor kinase activity. Two non-invasive, fluorescence-based methods, fluorescence photobleaching recovery and fluorescence correlation spectroscopy, will be used to determine the relative proportion of rapidly diffusing (non-raft) and slowly diffusing (raft) GFP-EGF receptors in intact cells. Domain swapping between the EGF receptor and the VSV-G protein, a non-raft protein, will be used to identify portions of the EGF receptor responsible for directing the receptor into lipid rafts. The effect of raft localization and cholesterol levels on the function of the EGF receptor will be measured directly by assessing receptor binding and kinase activity in raft and non-raft membrane fractions in vitro. The mechanism through which the caveolar protein, caveolin- 1 inhibits EGF receptor kinase activity will be investigated by mutagenesis of the proposed sites of interaction in these two proteins and by analysis of the potential role played by cholesterol in caveolin-1 -mediated inhibition of the EGF receptor kinase. Together, these studies will determine how and to what extent the EGF receptor partitions into lipid rafts. They will also delineate the effect of raft localization on EGF receptor function. These findings will provide insight into the contribution of lipid rafts to the regulation of EGF receptor-mediated signaling.