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DSL ligand endocytosis in Notch activation

Notch 激活中的 DSL 配体内吞作用

基本信息

批准号：
8588941
负责人：
GERALDINE A WEINMASTER
金额：
$ 30.8万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-01 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8588941
关键词：
Accounting Actins Address Adult Alzheimer&apos s Disease Binding Biochemical Biological Models Cell Communication Cell Culture Techniques Cell physiology Cells Clathrin Clathrin Adaptors Coculture Techniques Defect Development Embryo Embryonic Development Endocytosis Event Excision Extracellular Domain Goals Inherited Integral Membrane Protein Knowledge Life Ligand Binding Ligands Link Maintenance Malignant Neoplasms Mammalian Cell Mediating Membrane Modeling Molecular Pathway interactions Pattern Peptide Hydrolases Proteolysis Receptor Cell Receptor Signaling Recruitment Activity Recycling Research Resistance Role Series Signal Transduction Stem cells Stroke Structure Study models Syndrome System Testing Tissues Transducers Ubiquitination adult stem cell base clinical application coated pit commercial application epsin insight notch protein novel polymerization public health relevance research study therapeutic target trafficking transcription factor ubiquitin-protein ligase

项目摘要

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.

描述（由申请人提供）：Notch通路是一种保守的信号传导系统，广泛用于整个胚胎发育过程，在成人组织和干细胞的维持中继续发挥作用。考虑到大量依赖于Notch的细胞过程，信号传导的获得和损失与发育综合征、中风、阿尔茨海默病和癌症有关也就不足为奇了。Notch作为一个潜在的治疗靶点的鉴定，沿着其在操纵胚胎和成体干细胞中的重要性，强调了清楚地理解调节这一关键信号系统的机制的必要性。结合并激活Notch的配体是整合的膜蛋白，并且信号传导依赖于直接的细胞与细胞接触。由配体诱导的Notch信号传导涉及一系列蛋白水解切割事件以释放作为转录因子起作用的Notch细胞内结构域，从而允许Notch受体直接在信号转导中起作用。已经鉴定了Notch配体和活化蛋白酶，然而，仍不清楚Notch配体和受体细胞之间的相互作用如何活化信号传导。虽然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是关键的，但是内吞作用触发信号传导的机制仍然是难以捉摸的。本文提出的实验将解决获得对Notch信号传导机制的清楚理解的主要障碍，因为它们将区分在与Notch相互作用之前配体再循环是否需要配体内吞，或者事实上在与Notch结合之后是否需要配体内吞以实现下游信号传导的蛋白水解激活。本申请概述了一项研究计划，结合分子，生物化学和细胞的方法，使用哺乳动物细胞培养，以获得全面的了解配体细胞激活信号的分子机制和内吞机制。我们的初步研究表明，配体细胞使用的网格蛋白介导的内吞作用，以促进蛋白水解激活的Notch的epsin依赖性模式。我们现在建议获得一个完整的分子描述网格蛋白包被的内吞结构，以获得机制的洞察如何配体内吞功能的Notch激活。在与Notch相互作用之前，Epsin参与再循环以产生活性配体。我们将直接测试这一假设，首先确定是否需要epsin的配体再循环和第二，是否需要配体再循环的信号传导活动。鉴于胰蛋白酶将泛素化的内吞货物募集到网格蛋白包被的凹坑，并且Notch配体必须被泛素化以激活信号传导，我们将确定配体泛素化是否对于与胰蛋白酶的相互作用和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，Notch是通过一个分子上不同形式的网格蛋白介导的配体内吞激活代表了一个全新的模型内吞激活的信号受体。