Notch Signaling in Mammalian Cells

描述（由申请人提供）：Notch信号通过引导神经祖细胞进入神经元和胶质细胞，以及调节神经突形态发生和突触相互作用来影响神经系统的发育和维持。Notch最初被认为是一个神经源性基因，然而Delta/Serrate/Lag2 （DSL）配体激活Notch受体可调节多种神经元和非神经元细胞类型的分化。此外，Notch信号还与某些癌症、遗传性人类综合症和神经退行性疾病有关。虽然Notch在这些不同过程中的作用是确定的，但配体诱导的Notch信号传导的分子机制尚不清楚。为了生物化学和分子表征配体-Notch相互作用和下游信号事件，我们开发了配体结合和细胞共培养试验，其中Notch信号系统的组成部分可以很容易地进行操作和研究。重要的是，这些共培养系统测量由配体诱导的Notch信号，这比广泛使用的组成活性形式的Notch更能反映生理Notch信号。我们的研究发现了一个非典型的Notch信号通路，它在Notch异构体和下游效应物CSL （CBF-1, Su(H), Lag-1）上都不同于“核心”Notch信号通路，并且被配体激活。我们已经在DSL配体和Notch受体蛋白中确定了赋予配体受体特异性的序列，并证明了糖基转移酶边缘对配体诱导的Notch信号传导的差异调节，这是首次在果蝇中描述的。最后，我们发现DSL家族成员Delta3不激活Notch信号，而是抑制配体诱导的Notch信号。在这个应用中提出的实验是我们先前发现的逻辑延伸。本文提出了三个目标：(1)定义与csl无关的Notch信号的要求和下游目标；(2)表征不同配体-受体组合对Notch信号传导的影响；(3)表征Delta3对配体诱导的Notch信号传导的抑制作用。使用这些方法获得的信息将扩展我们目前对Notch信号转导的分子机制的理解，并为该信号系统如何调节胚胎和成人的多种细胞过程提供线索。

DESCRIPTION (provided by applicant): Notch signaling influences development and maintenance of the nervous system through directing the specification of neural progenitors into neurons and glia, as well as regulating neurite morphogenesis and synaptic interactions. Notch was first identified as a neurogenic gene, yet activation of Notch receptors by Delta/Serrate/Lag2 (DSL) ligands regulates the differentiation of a wide variety of neuronal and non-neuronal cell types. Moreover, Notch signaling has been linked to certain cancers, inherited human syndromes and neurodegenerative diseases. Although a role for Notch in these different processes is certain, the molecular mechanisms of ligand-induced Notch signaling are not well defined. To biochemically and molecularly characterize ligand-Notch interactions and downstream signaling events we have developed ligand-binding and cell co-culture assays in which components of the Notch signaling system can be readily manipulated and studied. Importantly, these co-culture systems measure Notch signaling induced by ligand, which is more reflective of physiological Notch signaling than the widely used constitutively active forms of Notch. Our studies have identified a noncanonical Notch signaling pathway that differs from the "core" Notch signaling pathway both in the Notch isoform and downstream effector, CSL (CBF-1, Su(H), Lag-1), activated by ligand, in addition, we have identified sequences within both the DSL ligand and Notch receptor proteins that confer ligand-receptor specificity and have demonstrated that the glycosyltransferase fringe differentially modulates ligand-induced Notch signaling as first described for Drosophila. Finally, we have discovered that the DSL family member, Delta3, does not activate Notch signaling but rather functions to inhibit ligand-induced Notch signaling. The experiments proposed in this application are logical extensions of our previous findings. Three aims are proposed to (1) define the requirements and downstream targets of CSL-independent Notch signaling; (2) characterize the effects of different ligand-receptor combinations on Notch signaling; and (3) characterize the inhibitory effects of Delta3 on ligand-induced Notch signaling. Information obtained using these approaches will extend our current understanding of the molecular mechanisms of Notch signal transduction and provide clues as to how this signaling system regulates a diverse array of cellular processes both in the embryo and in the adult.