Amyloid Precursor Protein Signaling

淀粉样蛋白前体蛋白信号转导

• 批准号: 6530492
• 负责人: MARK ALLEN BOTHWELL
• 金额: $ 36.01万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-15 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6530492
• 关键词: Alzheimer's disease; PC12 cells; amyloid proteins; biological signal transduction; cell line; chimeric proteins; confocal scanning microscopy; gene expression; genetic transcription; green fluorescent proteins; intracellular transport; luciferin monooxygenase; protein localization; protein structure function; protein transport; receptor; tissue /cell culture; transcription factor; transfection

DESCRIPTION (provided by applicant): Studies will test the hypothesis that beta-amyloid precursor protein (APP) normally functions as a signal-transducing cell surface receptor and pathological functions of APP in Alzheimer's disease reflect perturbations of that receptor function. It is proposed that APP resembles the receptor Notch in possessing two alternative signaling pathways - one propagated from the membrane-resident receptor and another propagated by nuclear translocation of the cytoplasmic domain of the receptor following gamma-secretase cleavage within the membrane-spanning sequence of the protein. Studies will employ various cell lines including COS7 cells, a neon-neuronal cell line, and NGF-differentiated PC12 cells, a neuronal cell line. Transient and stable transfection will be employed to manipulate expression of APP and proteins with which it interacts. Green fluorescent protein (GFP) and myc epitope tags will be employed to follow intracellular trafficking of beta-amyloid precursor protein following transfection. APP-GaI4/VP1 6 and APP-GaI4 fusion proteins will be expressed in reporter cell lines that express luciferase under control of a Gal4 promoter, providing a quantitative means of assessing the nuclear translocation of the APP cytoplasmic domain, and providing an assay for APP mutations and pharmacological manipulations that promote or inhibit nuclear access of the cytoplasmic domain. Mutations will be targeted to a CDK5 phosphorylation site, to a putative nuclear localization signal, to a putative PEST motif, to a Dab1/Fe65 binding site, and to putative ubiquitinization sites, and the effects on subcellular localization and nuclear translocation will be assessed. Effects of agonistic APP monoclonal antibody, beta-amyloid peptide, LRP, and alpha-2 macroglobulin on APP signaling will be assessed.

描述（由申请人提供）：研究将检验以下假设：β-淀粉样蛋白前体蛋白（APP）通常在阿尔茨海默氏病中APP的信号转移细胞表面受体和病理功能作用，反映了该受体功能的扰动。提出，应用程序在拥有两个替代信号通路时类似于受体缺口 - 一种是从膜居住的受体传播的，另一个是通过在γ-分泌酶裂解后在膜上旋转酶裂解后的细胞质结构域传播的，而另一个是通过核易位的传播。蛋白质。研究将采用各种细胞系，包括COS7细胞，霓虹灯神经元细胞系和NGF分化的PC12细胞（神经元细胞系）。瞬态和稳定的转染将用于操纵与其相互作用的APP和蛋白质的表达。转染后，将使用绿色荧光蛋白（GFP）和MYC表位标签来跟踪β-淀粉样蛋白前体蛋白的细胞内运输。 App-gai4/vp1 6和App-Gai4融合蛋白将以在GAL4启动子控制下表达荧光素酶的记者细胞系中表达促进或抑制细胞质结构域的核通路的突变和药理操作。突变将针对CDK5磷酸化位点，推定的核定位信号，推定的害虫基序，dab1/fe65结合位点以及推定的泛素化位点，并评估对亚细胞定位和核转运的影响。激动剂APP单克隆抗体，β-淀粉样蛋白肽，LRP和Alpha-2大球蛋白对APP信号的影响。