Mechanism of Notch activation by Epsin-dependent ligand endocytosis in Drosophila

描述（由申请人提供）：该授权的主要目标是确定Delta/Serrate/Lag-2（DSL）超家族的跨膜配体激活细胞表面受体Notch的机制。DSL-Notch信号传导是细胞间通讯的一种重要和普遍的机制，在从海绵到人类的所有多细胞动物中保守。它对人类健康具有巨大的影响，因为DSL-Notch信号传导的遗传和环境干扰导致广泛的癌症，以及发育，免疫和神经系统疾病。因此，确定DSL配体如何激活Notch对于开发治疗人类疾病的诊断和治疗工具至关重要，这是NIH的中心使命。我们过去的工作有助于确定Notch信号转导的基本机制。使用果蝇作为模型系统，我们发现Notch是一种膜束缚的转录因子，其响应于配体而被切割，允许细胞内结构域进入细胞核并打开靶基因。在拟议的工作中，我们将解决仍然没有答案和关键的问题，如何配体结合诱导初始裂解负责激活受体。我们将建立在我们以前的发现，即激活信号接收细胞上的Notch，DSL配体必须在信号发送细胞中特异性地通过衔接蛋白Epsin进行内吞作用。这一发现，连同最近的结构和生物物理学研究，已经表明，配体内吞的Epsin诱导变构变化的Notch共结构域，暴露了一个否则掩埋的切割位点的活化蛋白酶。在拟议的研究中，我们将使用新的方法来操纵体内配体和受体的结构，以测试三个假设。首先，跨细胞间配体/受体桥产生的机械力负责使受体易于裂解的变构变化。第二，这种力是由配体施加的，因为它经历了Epsin依赖性内吞作用进入发送细胞。第三，该受体通过（i）诱导配体进入Epsin途径，和（ii）施加依赖于其自身内吞作用的相反力，在产生这种力中起积极作用。这些实验的结果将建立机械力模型和Epsin依赖性配体内吞作用在产生所需力中的作用，或导致配体激活Notch的基本机制的其他可检验的假设。我们开发的创新方法和试剂也将适用于信号转导和动物发育中的其他问题。

DESCRIPTION (provided by applicant): The main goal of this grant is to determine the mechanism of activation of the cell surface receptor Notch by transmembrane ligands of the Delta/Serrate/Lag-2 (DSL) superfamily. DSL-Notch signaling is an important and pervasive mechanism of intercellular communication, conserved in all multi-cellular animals, from sponges to man. It has enormous implications for human health, as genetic and environmental perturbations of DSL-Notch signaling cause a wide range of cancers, as well as developmental, immune, and neurological disorders. Thus, determining how DSL ligands activate Notch is critical for developing diagnostic and therapeutic tools to treat human disease, a central mission of the NIH. Our past work was instrumental in defining the basic mechanism of signal transduction by Notch. Using Drosophila as a model system, we discovered that Notch is a membrane tethered transcription factor that is cleaved in response to ligand, allowing the intracellular domain to enter the nucleus and turn on target genes. In the proposed work, we will address the still unanswered and crucial question of how ligand binding induces the initial cleavage responsible for activating the receptor. We will build on our previous discovery that to activate Notch on signal-receiving cells, DSL ligands must undergo endocytosis in signal-sending cells specifically by the adaptor protein Epsin. This finding, together with recent structural and biophysical studies, has suggested that ligand endocytosis by Epsin induces an allosteric change in the Notch codomain that exposes an otherwise buried cleavage site to the activating protease. In the proposed research we will use new approaches to manipulate ligand and receptor structure in vivo to test three hypotheses. First, that mechanical force generated across the intercellular ligand/receptor bridge is responsible for the allosteric change that renders the receptor susceptible to cleavage. Second, that this force is exerted by the ligand as it undergoes Epsin-dependent endocytosis into the sending cell. Third, that receptor plays an active role in generating this force by (i) inducing the ligand to enter the Epsin pathway, and (ii exerting an opposing force that depends on its own endocytosis. The results of these experiments will either establish the mechanical force model and the role of Epsin- dependent ligand endocytosis in generating the required force, or lead to other testable hypotheses for the basic mechanism by which ligand activates Notch. The innovative methods and reagents we generate will also be applicable to other problems in signal transduction and animal development.2