DSL ligand endocytosis in Notch activation

描述（由申请人提供）：Notch 通路是一种保守的信号系统，在整个胚胎发育过程中广泛使用，在成人组织和干细胞的维持中继续发挥作用。鉴于大量的细胞过程依赖于Notch，信号传导的增强和减弱与发育综合征、中风、阿尔茨海默病和癌症相关也就不足为奇了。 Notch 被确定为潜在的治疗靶点，及其在操纵胚胎和成体干细胞中的重要性，强调需要清楚地了解调节这一关键信号系统的机制。结合并激活 Notch 的配体是完整的膜蛋白，信号传导依赖于细胞间的直接接触。配体诱导的Notch信号传导涉及一系列蛋白水解裂解事件，以释放充当转录因子的Notch胞内结构域，从而允许Notch受体直接在信号转导中发挥作用。 Notch配体和激活蛋白酶已被鉴定，然而，目前尚不清楚Notch配体和受体细胞之间的相互作用如何激活信号传导。尽管很明显配体细胞的内吞作用对于Notch细胞中信号传导的激活至关重要，但内吞作用触发信号传导的机制仍然难以捉摸。这里提出的实验将解决清楚了解Notch信号传导机制的主要障碍，因为它们将区分在与Notch相互作用之前配体循环是否需要配体内吞作用，或者实际上在与Notch结合后是否需要配体内吞作用以影响下游信号传导的蛋白水解激活。该申请概述了一项研究计划，该计划结合了分子、生物化学和细胞方法，利用哺乳动物细胞培养来全面了解配体细胞激活信号传导的分子机制和内吞机制。我们的初步研究表明，配体细胞使用网格蛋白介导的内吞作用的epsin依赖性模式来促进Notch的蛋白水解激活。我们现在建议获得网格蛋白包被的内吞结构的完整分子描述，以深入了解配体内吞作用如何在Notch激活中发挥作用。 Epsin 在与 Notch 相互作用之前参与回收以产生活性配体。我们将通过首先确定配体回收是否需要epsin，然后确定信号传导活性是否需要配体回收来直接检验这一假设。鉴于epsins将泛素化的内吞货物募集到网格蛋白包被的凹坑中，并且Notch配体必须被泛素化才能激活信号传导，我们将确定配体泛素化是否对于与epsin的相互作用以及功能上不同的网格蛋白包被的内吞结构的形成至关重要，以及配体和Notch细胞之间的相互作用是否刺激配体泛素化。我们的假设是，Notch 通过网格蛋白介导的配体内吞作用的分子独特形式被激活，这代表了信号受体内吞激活的全新模型。 公共健康相关性：Notch 通路是在整个胚胎发育过程中反复使用并在成人中继续发挥作用的少数信号系统之一。与遗传性发育综合征、癌症、中风和阿尔茨海默病的联系强调了定义 Notch 信号传导分子基础的必要性。在这方面，Notch 信号传导的成功治疗靶向需要全面了解调节该通路的机制。 Notch 信号传导机制的知识也将有助于胚胎和成体干细胞的临床和商业应用的操作。

DESCRIPTION (provided by applicant): The Notch pathway is a conserved signaling system used extensively throughout embryonic development that continues to function in the maintenance of tissues and stem cells in adults. Given the large repertoire of cellular processes dependent on Notch, it is not surprising that both gains and losses in signaling have been linked to developmental syndromes, stroke, Alzheimers disease and cancer. The identification of Notch as a potential therapeutic target, along with its importance in manipulating embryonic and adult stem cells, underscore the need to clearly understand the mechanisms regulating this key signaling system. Ligands that bind and activate Notch are integral membrane proteins and signaling is dependent on direct cell-to-cell contact. Notch signaling induced by ligand involves a series of proteolytic cleavage events to release the Notch intracellular domain that functions as a transcription factor, thereby allowing the Notch receptor to function directly in signal transduction. Notch ligands and activating proteases have been identified, however, it is still unclear how interactions between Notch ligand and receptor cells activate signaling. Although it is clear that endocytosis by the ligand cell is critical for activation of signaling in the Notch cell, the mechanisms whereby endocytosis triggers signaling have remained elusive. The experiments proposed here will address a major obstacle to obtaining a clear understanding of Notch signaling mechanisms, since they will discriminate as to whether ligand endocytosis is required for ligand recycling prior to interactions with Notch, or whether in fact ligand endocytosis is required following binding to Notch to effect proteolytic activation for downstream signaling. This application outlines a research plan that combines molecular, biochemical and cellular approaches using mammalian cell culture to obtain a comprehensive understanding of the molecular machinery and endocytic mechanism used by ligand cells to activate signaling. Our preliminary studies indicate that ligand cells use an epsin-dependent mode of clathrin-mediated endocytosis to promote proteolytic activation of Notch. We now propose to obtain a complete molecular description of the clathrin-coated endocytic structure to gain mechanistic insight into how ligand endocytosis functions in Notch activation. Epsin has been implicated in recycling to produce an active ligand prior to interaction with Notch. We will directly test this hypothesis by determining first whether epsin is required for ligands to recycle and second, whether ligand recycling is required for signaling activity. Given that epsins recruit ubiquitinated endocytic cargo to clathrin- coated pits and Notch ligands must be ubiquitinated to activate signaling, we will determine if ligand ubiquitination is critical for interactions with epsin and the formation of a functionally distinct clathrin-coated endocytic structure, and whether interactions between ligand and Notch cells stimulate ligand ubiquitination. Our hypothesis that Notch is activated via a molecularly distinct form of clathrin-mediated ligand endocytosis represents a fundamentally new model for endocytic activation of a signaling receptor. PUBLIC HEALTH RELEVANCE: The Notch pathway is one of a few signaling systems that is used over and over again throughout embryonic development that continues to function in the adult. Links to inherited developmental syndromes, cancer, stroke and Alzheimer disease underscore the need to define the molecular basis of Notch signaling. In this regard, successful therapeutic targeting of Notch signaling requires a comprehensive understanding of the mechanisms regulating this pathway. Knowledge of Notch signaling mechanisms will also contribute to the manipulation of embryonic and adult stem cells for clinical and commercial applications.