Amyloid Precursor Protein Signaling

描述（由申请人提供）：研究将验证β -淀粉样蛋白前体蛋白（APP）正常作为信号转导细胞表面受体的假设，APP在阿尔茨海默病中的病理功能反映了该受体功能的扰动。APP类似于受体Notch，具有两种可选择的信号通路——一种是由膜驻留受体传播的，另一种是通过受体细胞质区域的核易位传播的，随后在蛋白质的膜跨越序列内进行γ分泌酶裂解。研究将使用多种细胞系，包括COS7细胞（一种神经元-神经元细胞系）和ngf分化的PC12细胞（一种神经元细胞系）。瞬时和稳定的转染将被用来操纵APP及其相互作用蛋白的表达。绿色荧光蛋白（GFP）和myc表位标签将被用来跟踪转染后β -淀粉样蛋白前体蛋白的细胞内运输。APP- gai4 / vp16和APP- gai4融合蛋白将在Gal4启动子控制下表达荧光素酶的报告细胞系中表达，这为评估APP细胞质结构域的核易位提供了定量手段，并为APP突变和促进或抑制细胞核进入细胞质结构域的药理学操作提供了分析方法。突变将靶向一个CDK5磷酸化位点、一个假定的核定位信号、一个假定的PEST基元、一个Dab1/Fe65结合位点和假定的泛素化位点，并评估对亚细胞定位和核易位的影响。我们将评估激动性APP单克隆抗体、β -淀粉样肽、LRP和α -2巨球蛋白对APP信号的影响。

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.