Lipid Rafts and EGF Receptor Function

描述(申请人提供)：小凹和脂筏与低相关 密度，耐洗涤剂的膜域。他们被认为参与了 在信号转导的过程中，由于各种信号成分 包括EGF受体，已被证明高度富含在这些 域名。我们已经证明，正常的功能需要完整的脂筏。 EGF刺激的PI代谢及胆固醇对EGF受体的调节作用 结合活性和激酶活性。这些发现表明，脂筏是 EGF受体介导的信号调节的重要贡献者。至 了解脂筏在EGF受体信号转导中的作用，四种特异性 目的是：1)分析EGF受体在脂质之间的分布 木筏和非木筏隔室中的完整细胞。2)确定域名(S) 负责将受体靶向于脂质的EGF受体 木筏。3)比较RAFT和RAFT上EGF受体的酶学性质 膜组分。以及，4)评价小窝蛋白-1的作用机制 导致EGF受体激酶活性的抑制。两个非侵入性， 基于荧光的方法、荧光光漂白回收和 荧光相关光谱，将被用来确定相对 快速扩散(非RAFT)和缓慢扩散(RAFT)GFP-EGF的比例 完整细胞中的受体。EGF受体和EGF受体之间的结构域交换 VSV-G蛋白是一种非RAFT蛋白，将用于识别EGF的部分 负责引导受体进入脂筏的受体。的影响 RAFT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来直接测量 RAFT和非RAFT膜组分的体外实验。这一机制是通过 小窝蛋白、小窝蛋白-1抑制EGF受体激酶活性 通过对这两个基因中建议的相互作用部位的突变进行研究 蛋白质和通过分析胆固醇在体内的潜在作用 小窝蛋白-1对EGF受体激酶的抑制作用加在一起，这些 研究将确定EGF受体如何以及在多大程度上划分为 脂筏。他们还将描述RAFT本地化对EGF的影响 受体功能。这些发现将为我们深入了解 脂筏对EGF受体介导的信号传导的调节作用。

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.