DSL ligand endocytosis in Notch activation

描述(申请人提供)：Notch途径是一个保守的信号系统，在整个胚胎发育过程中广泛使用，继续在成人的组织和干细胞维持中发挥作用。考虑到依赖于Notch的大量细胞过程，信号的增减与发育综合征、中风、阿尔茨海默氏症和癌症有关也就不足为奇了。Notch被确定为一个潜在的治疗靶点，以及它在操纵胚胎和成人干细胞方面的重要性，强调了清楚地了解调节这一关键信号系统的机制的必要性。结合和激活Notch的配体是完整的膜蛋白，信号传递依赖于细胞与细胞之间的直接接触。配体诱导的Notch信号涉及一系列蛋白水解性切割事件，以释放作为转录因子的Notch胞内域，从而允许Notch受体直接在信号转导中发挥作用。Notch配体和激活的蛋白水解酶已经被确定，然而，Notch配体和受体细胞之间的相互作用如何激活信号仍不清楚。尽管很明显，配体细胞的内吞作用对于Notch细胞中信号的激活是至关重要的，但内吞作用触发信号的机制仍然不清楚。这些实验将解决清楚了解Notch信号机制的一个主要障碍，因为它们将区分在与Notch相互作用之前是否需要配体内吞来进行配体循环，或者实际上是否在与Notch结合之后需要配体内吞来影响下游信号的蛋白水解性激活。本申请概述了一项研究计划，该计划结合分子、生化和细胞方法，使用哺乳动物细胞培养，以获得对配体细胞用于激活信号的分子机制和内吞机制的全面了解。我们的初步研究表明，配体细胞使用依赖于内吞蛋白的网织蛋白介导的内吞作用来促进Notch的蛋白水解性激活。我们现在建议获得一个完整的分子描述的笼状蛋白包被的内吞结构，以获得对配体内吞如何在Notch激活中发挥作用的机制的深入了解。Epsin参与了在与Notch相互作用之前产生活性配体的循环过程。我们将通过以下两种方法直接验证这一假说：第一，确定配基循环是否需要胞外蛋白；第二，确定信号活性是否需要配基循环。鉴于epins将泛素化的内吞货物招募到笼蛋白包裹的凹坑，而Notch配体必须被泛素化才能激活信号，我们将确定配体泛素化是否对与epsin的相互作用和形成功能上不同的笼蛋白包裹的内吞结构至关重要，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是通过一种分子上不同形式的网状蛋白介导的配体内吞作用来激活的，这代表了一种信号受体内吞激活的全新模型。

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.