In vivo monitoring of EGF receptor dimerization and activation

描述（由申请人提供）：受体酪氨酸激酶的表皮生长因子受体（EGFR）家族成员对于许多重要细胞过程（包括细胞增殖、分化和迁移）的适当调节至关重要。这些受体的异常行为与一系列疾病有关，包括癌症。理解EGFR介导的信号转导的重要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，主要是由于缺乏现有的方法来研究体内EGFR二聚化的动态过程。作为现有生化方法的补充，我们将应用新的二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）的二聚化和活化。双砷标记线性四半胱氨酸基序是一种成熟的蛋白质位点特异性标记方法。耶鲁大学Schepartz实验室最近的工作表明，当四半胱氨酸基序以二分的方式显示时，也可以实现有效的标记，其中半胱氨酸对在一级序列中彼此远离，但当靶蛋白处于折叠或组装状态时紧密相关。该提案概述了通过三个特定目的在监测活细胞中EGFR二聚化和活化的背景下利用二分四半胱氨酸展示。最初（目标1），我们进行了一系列实验，以验证扩展的二分展示方法的EGFR。接下来（目的2），我们问这种新的系统，监测蛋白质二聚体在体内是否可以用来回答长期存在的问题，EGFR信号转导的机制。最后（目的3），我们扩展了这个平台的基础上，一个敏感的，在细胞内，高通量筛选的激动剂和拮抗剂的EGFR家族。公共卫生相关性：拟议的研究将有助于我们了解EGFR活性的基本基础。这将导致对异常EGFR行为如何导致癌症的更好理解，并可能对靶向EGFR治疗或检测癌症的新策略产生影响。拟议的研究还涉及开发EGFR抑制剂的高通量筛选，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的联合疗法。

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.