Notch Signaling in Mammalian Cells

描述(申请人提供)：Notch信号通过引导神经前体细胞分化为神经元和神经胶质，以及调节神经突起的形态发生和突触相互作用，影响神经系统的发育和维持。Notch最初被认为是一种神经源性基因，但Delta/Serrate/Lag2(DSL)配体激活Notch受体可调节多种神经细胞和非神经细胞的分化。此外，Notch信号与某些癌症、遗传性人类综合征和神经退行性疾病有关。虽然Notch在这些不同的过程中的作用是确定的，但配体诱导的Notch信号的分子机制还不是很清楚。为了从生化和分子水平表征配体-Notch相互作用和下游信号事件，我们发展了配体结合和细胞共培养实验，可以方便地操纵和研究Notch信号系统的组成部分。重要的是，这些共培养系统测量了配体诱导的Notch信号，比广泛使用的组成活性形式的Notch更能反映生理Notch信号。我们的研究已经发现了一条非规范的Notch信号通路，它在Notch异构体和下游效应因子CSL(CBF-1，SU(H)，Lag-1)上都不同于“核心”的Notch信号通路，由配体激活。此外，我们还在DSL配体和Notch受体蛋白中鉴定了赋予配体-受体特异性的序列，并证明了糖基转移酶条纹对配体诱导的Notch信号的差异性调节，就像首次在果蝇中描述的那样。最后，我们发现DSL家族成员Delta3并不激活Notch信号，而是抑制配体诱导的Notch信号。本申请中提出的实验是我们先前发现的合乎逻辑的扩展。提出了三个目标：(1)确定CSL非依赖的Notch信号的需求和下游靶点；(2)表征不同的配体-受体组合对Notch信号的影响；(3)表征Delta3对配体诱导的Notch信号的抑制作用。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年体内的各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.