Illuminating Dynamic Receptor Clustering in the Epidermal Growth Factor Receptor

描述（申请人提供）：表皮生长因子（EGF）受体（EGFR）家族成员的失调与肿瘤发生和肿瘤生长相关。由于其在癌症发展中的相关性，EGFR是癌症药物发现中的重要靶标，并且已经开发了几种EGFR靶向疗法。然而，他们的临床成功往往是适度的，加强了对EGFR信号通路更完整的理解的需要。该建议侧重于未充分理解的作用，大规模的EGFR联营公司（“集群”）在信号启动和转导，特别是。虽然早已知道配体诱导的二聚化在受体信号传导中起关键作用，但越来越多的证据表明，需要增强这种教科书模型以解释质膜中受体的异质性横向分布。受体在“微区”或“纳米团簇”中的局部富集可能强烈影响协同受体 相互作用，并通过局部浓度效应改变局部EGFR缔合平衡。传统的荧光显微镜下的大规模受体组织的基本机制的实验研究仍然具有挑战性，由于该方法的限制方面的吞吐量，空间和时间分辨率，和最大的观察时间。等离子体耦合显微镜（PCM）是一种新型的基于非荧光的方法，其使用贵金属纳米颗粒（NP）之间的电磁相互作用来研究亚衍射极限距离（但超出荧光共振能量转移（FRET）的空间障碍）上的受体聚集。NP不会闪烁或漂白，非常明亮，并且可以在常规的宽视场显微镜中成像。因此，PCM有助于监测EGFR聚集，而不受同时在许多单个细胞中观察时间的限制。这种竞争性的更新建立在前一个资助周期中开发的基于等离子体偶联的工具的基础上，并概述了一个有力的研究计划，以阐明动态EGFR聚集的结构起源。然后，PCM将被应用于测试的假设，受体聚集调节信号的模式和强度，并阐明信号强度和结果的空间调节的机制。所获得的洞察力将提高广泛的受体的空间调节机制的理解。贵金属纳米颗粒不仅是用于表征质膜中EGFR簇集的极好的光学标记，而且它们还代表了在共价连接至EGF后恢复和增强负EGFR信号传导的潜在治疗工具。这一假设在本提案中得到了实验检验。如果成功的话，这种策略将为克服癌症中的凋亡逃避提供一种新的方法。

DESCRIPTION (provided by applicant): Dysregulation of members of the epidermal growth factor (EGF) receptor (EGFR) family is associated with oncogenesis and tumor growth. Due to its relevance in cancer development, EGFR is an important target in cancer drug discovery, and several EGFR targeted therapies have already been developed. Their clinical success has, however, often been modest, reinforcing the need for a more complete understanding of the EGFR signaling pathway. This proposal focuses on the insufficiently understood role of large-scale EGFR associates ("clusters") in signaling initiation and transduction, in particular. While i has long been known that ligand induced dimerization plays a critical role in receptor signaling, there is growing evidence that this textbook model needs to be augmented to account for the heterogenous lateral distribution of the receptor in the plasma membrane. The local enrichment of the receptors in ''micro-domains" or ''nanoclusters'' could strongly affect cooperative receptor interactions and shift the local EGFR association equilibria through a local concentration effect. The experimental investigation of the fundamental mechanisms underlying the large-scale receptor organization with conventional fluorescence microscopy remains challenging, due to the method's limitation with regard to throughput, spatial and temporal resolution, and maximum observation time. Plasmon Coupling Microscopy (PCM) is a novel non-fluorescence based approach that uses electromagnetic interactions between noble metal nanoparticles (NPs) to investigate receptor clustering on subdiffraction limit distances (but beyond the spatial barrier o Fluorescence Resonance Energy Transfer, FRET). NP's do not blink or bleach, are very bright, and can be imaged in a conventional widefield microscope. Consequently, PCM facilitates the monitoring of EGFR clustering without limitations in observation time in many individual cells simultaneously. This competitive renewal builds upon the plasmon coupling based tools developed in the previous funding cycle and outlines a vigorous research plan to elucidate the structural origin of dynamic EGFR clustering. PCM will then be applied to test the hypothesis that receptor clustering regulates the mode and strength of signaling and to elucidate the mechanisms underlying a spatial regulation of signaling intensity and outcome. The obtained insight will improve the understanding of spatial regulation mechanisms for a broad range of receptors. Noble metal NPs are not only superb optical labels for characterizing EGFR clustering in the plasma membrane, but they also represent potential therapeutic tools to restore and enhance negative EGFR signaling after covalent attachment to EGF. This hypothesis is experimentally tested in this proposal. If successful, this strategy would provide a new approach for overcoming apoptosis evasion in cancer.