Notch Signaling in Mammalian Cells

描述（由申请人提供）：Notch信号传导通过指导神经祖细胞分化为神经元和神经胶质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首先被鉴定为神经源性基因，然而通过Delta/Serrate/Lag 2（DSL）配体激活Notch受体调节多种神经元和非神经元细胞类型的分化。此外，Notch信号传导与某些癌症、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未明确。为了生物化学和分子表征配体-Notch相互作用和下游信号传导事件，我们开发了配体结合和细胞共培养测定，其中Notch信号传导系统的组分可以容易地操纵和研究。重要的是，这些共培养系统测量由配体诱导的Notch信号传导，其比广泛使用的Notch的组成型活性形式更能反映生理Notch信号传导。我们的研究已经确定了一种非经典的Notch信号通路，该通路在Notch亚型和下游效应子CSL中都与“核心”Notch信号通路不同（CBF-1，Su（H），Lag-1），由配体活化，此外，我们已经鉴定了DSL配体和Notch受体蛋白中的序列，受体特异性，并已证明糖基转移酶边缘差异调节配体诱导的Notch信号转导，如首次描述的果蝇。最后，我们发现DSL家族成员Delta 3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以前发现的逻辑扩展。提出了三个目标：（1）定义CSL非依赖性Notch信号传导的要求和下游靶标;（2）表征不同配体-受体组合对Notch信号传导的影响;以及（3）表征Delta 3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，并提供有关该信号系统如何调节胚胎和成人中多种细胞过程的线索。

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.