Developing a mouse model to study Notch furin processing

描述(申请人提供)：Notch细胞表面受体是 由表达在相邻细胞表面的配体激活以指导 正确的细胞规格、图案化和形态发生几乎在 到目前为止研究过的后生生物。因此，诺奇并不令人惊讶 信号与某些癌症、遗传性人类综合征和 神经退行性疾病。一种Notch信令模型已在 哪种配体结合诱导Notch的蛋白降解以释放其细胞内 域，然后通过直接相互作用起到信号转换器的作用 和转录调控因子CSL的激活。CSL依赖的诱导 信号转导发生在配体与异二聚体形式的Notch结合之后 是通过用呋喃进行蛋白质分解处理而产生的。除了……之外 依赖CSL的Notch信号--我们发现了另一条发挥作用的途径 与CSL激活无关。生物素化研究表明，在 除异二聚体Notch1外，未切割的全长Notch1还存在于 细胞表面，提示该异构体也可能在配体中起作用 信号转导的结合和激活。与这个想法一致的是， 细胞培养中的功能分析表明，未分裂的全长细胞 表面Notch1阻断肌源性分化对配体的反应 缺少CSL激活。非CSL依赖的完整Notch信号的研究 这个应用程序概述了用来产生“敲打”的实验。 (Ki)Notch1呋喃裂解位点已被删除的小鼠 未切割的全长Notch1在纯合子突变体表面表达 细胞。这些Notch1突变体的生化和表型特征 动物不仅将证明CSL独立的生物学相关性 切入信号并确认未切割的全长切槽用作 这一途径的受体，但它也应该允许识别细胞 受这一途径调控的类型和过程。

DESCRIPTION (provided by applicant): The Notch cell surface receptor is activated by ligands expressed on the surface of neighboring cells to direct correct cell specification, patterning, and morphogenesis within virtually all metazoan organisms studied to date. Therefore it is not surprising that Notch signaling has been linked to certain cancers, inherited human syndromes and neurodegenerative diseases. A model for Notch signaling has been proposed in which ligand binding induces proteolysis of Notch to liberate its intracellular domain, which then functions as a signal transducer through direct interaction and activation of the transcriptional regulator CSL. Induction of CSL-dependent signaling occurs following ligand binding to a heterodimeric form of Notch that is generated through proteolytic processing by furin. In addition to CSL-dependent Notch signaling we have identified another pathway that functions independently of CSL activation. Biotinylation studies have indicated that in addition to heterodimeric Notch1, uncleaved full-length Notch1 exists at the cell surface, suggesting that this isoform might also function in ligand binding and activation of signal transduction. Consistent with this idea, functional analysis in cell culture indicates that uncleaved full-length cell surface Notch1 blocks myogenic differentiation in response to ligand, in the absence of CSL activation. To study CSL-independent Notch signaling in intact animals this application outlines experiments designed to generate "knock-in" (Ki) mice in which the Notch1 furin-cleavage site has been deleted so that only uncleaved full-length Notch1 is expressed on the surface of homozygous mutant cells. Biochemical and phenotypic characterization of these Notch1 mutant animals will not only document the biological relevance of CSL-independent Notch signaling and confirm that uncleaved full-length Notch serves as a receptor for this pathway, but it should also allow the identification of cell types and processes regulated by this pathway.