Developing a mouse model to study Notch furin processing

开发小鼠模型来研究 Notch 弗林蛋白酶加工

• 批准号: 6539300
• 负责人: GERALDINE A WEINMASTER
• 金额: $ 15.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-24 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6539300
• 关键词: active sites; alleles; biological models; biological signal transduction; cell type; chemical cleavage; dimer; enzyme activity; gene targeting; genetically modified animals; histogenesis; laboratory mouse; ligands; model design /development; mutant; prohormone convertase; proteolysis; tissue /cell culture

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.

描述（由申请人提供）：Notch细胞表面受体是 被邻近细胞表面表达的配体激活， 正确的细胞规格，图案和形态发生在几乎所有 迄今为止研究的后生动物。因此，Notch并不奇怪。 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Notch信令的模型已经在 该配体结合诱导Notch的蛋白水解以释放其细胞内的 域，然后通过直接相互作用充当信号换能器 和转录调节因子CSL的激活。诱导CSL依赖性 信号传导发生在配体与Notch的异二聚体形式结合后， 是通过弗林蛋白酶的蛋白水解过程产生的。除了 CSL依赖的Notch信号传导我们已经确定了另一条途径， 独立于CSL激活。生物素化研究表明， 除了异源二聚体Notch1之外，未切割的全长Notch1存在于Notch1的 细胞表面，这表明这种亚型也可能在配体中起作用。 信号转导的结合和激活。与这个想法相一致， 细胞培养中的功能分析表明，未切割的全长细胞 表面Notch1阻断肌细胞对配体的反应分化， 没有CSL激活。为了研究在完整的细胞中的CSL非依赖性Notch信号传导， 动物本申请概述了旨在产生“敲入”的实验 (Ki)缺失Notch1弗林蛋白酶切割位点的小鼠， 未切割的全长Notch1在纯合突变体的表面上表达 细胞这些Notch1突变体的生化和表型表征 动物将不仅记录非CSL依赖性的生物学相关性， Notch信号传导，并证实未切割的全长Notch作为一种信号传导途径， 受体，但它也应该允许识别细胞 由这条途径调节的类型和过程。