In vivo monitoring of EGF receptor dimerization and activation

描述(由申请人提供)：表皮生长因子受体(EGFR)受体酪氨酸激酶家族的成员对许多重要的细胞过程的适当调节至关重要，包括细胞的增殖、分化和迁移。这些受体的异常行为与包括癌症在内的一系列疾病有关。在理解EGFR介导的信号转导方面取得的重大进展依赖于结晶学方法。然而，关于这一基本机制的许多问题仍然存在，这主要是由于现有方法缺乏现有方法来研究体内EGFR二聚化的动态过程。作为对现有生化方法的补充，我们将应用新的两部分四半胱氨酸显示技术来监测活细胞中表皮生长因子受体(EGFR)的二聚化和激活。线性四半胱氨酸基序的生物标记法是一种成熟的蛋白质定点标记方法。耶鲁大学Schepartz实验室最近的工作表明，当四半胱氨酸基序以两部分方式显示时，也可以实现有效的标记，其中半胱氨酸对在初级序列中彼此远离，但当目标蛋白处于折叠或组装状态时，半胱氨酸对密切相关。这项提案概述了在通过三个特定目标监测活细胞中EGFR二聚化和激活的背景下，两部分四半胱氨酸展示的应用。首先(目标1)，我们进行了一系列实验，以验证二部分显示方法扩展到EGFR。接下来(目标2)，我们问这个监测体内蛋白质二聚化的新系统是否可以用来回答长期以来关于EGFR信号转导机制的问题。最后(目标3)，我们将该平台扩展为敏感的、细胞内的、高通量的EGFR家族激动剂和拮抗剂筛选的基础。公共卫生相关性：拟议的研究将有助于我们了解EGFR活动的基本基础。这将导致对EGFR异常行为如何导致癌症的更好理解，并可能对针对EGFR治疗或癌症检测的新策略产生影响。拟议的研究还包括开发高通量的EGFR抑制剂筛选，这可能导致发现新的、有效的EGFR抑制剂和/或创新地使用已知的抑制剂作为治疗癌症的高效和选择性联合疗法。

DESCRIPTION (provided by applicant): Members of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases are crucial to the proper regulation of a number of important cellular processes, including cell proliferation, differentiation, and migration. Abnormal behavior of these receptors is associated with a range of diseases, including cancer. Significant advances towards understanding EGFR-mediated signal transduction have relied on crystallographic methods. However, many questions about this fundamental mechanism still remain, largely due to the paucity of existing methods available to study the dynamic processes of EGFR dimerization in vivo. As a complement to existing biochemical methods, we will apply the novel bipartite tetracysteine display technique to monitor the dimerization and activation of epidermal growth factor receptor (EGFR) in live cells. Biarsenical labeling of linear tetracysteine motifs is a well established method for the site-specific labeling of proteins. Recent work from the Schepartz laboratory at Yale has shown that efficient labeling can also be achieved when the tetracysteine motif is displayed in a bipartite fashion, where the cysteine pairs are far away from each other in primary sequence, but closely associated when the target protein is in a folded or assembled state. This proposal outlines the utilization of bipartite tetracysteine display in the context of monitoring EGFR dimerization and activation in live cells through three specific aims. Initially (Aim 1), we conduct a series of experiments to validate the extension of the bipartite display methodology to the EGFR. Next (Aim 2), we ask whether this novel system for monitoring protein dimerization in vivo can be used to answer longstanding questions about the mechanism of EGFR signal transduction. Finally (Aim 3), we extend this platform to be the basis of a sensitive, in-cell, high-throughput screen for agonist and antagonists of the EGFR family. Public Health Relevance: The proposed research will contribute to our knowledge about the fundamental basis of EGFR activity. This will lead to an improved understanding of how aberrant EGFR behavior can lead to cancer and could have implications for new strategies to target EGFR for the treatment or detection of cancer. The proposed research also involves the development of a high-throughput screen for inhibitors of EGFR that may lead to the discovery of new, potent EGFR inhibitors and/or to the innovative use of known inhibitors as highly effective and selective combination therapies for the treatment of cancer.