Amyloid Precursor Protein Signaling

描述(申请人提供)：研究将测试这样一种假设，即β-淀粉样前体蛋白(APP)通常作为信号转导细胞表面受体发挥作用，而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。有人认为，APP与受体Notch相似，有两条可供选择的信号通路--一条来自膜上驻留的受体，另一条是在蛋白质跨膜序列中被γ-分泌酶切割后，通过受体胞质区域的核移位而传播的。研究将使用不同的细胞系，包括霓虹灯-神经元细胞系COS7细胞，以及神经生长因子分化的PC12细胞，神经细胞系。瞬时和稳定的转基因将被用来操纵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β-淀粉样前体蛋白在细胞内的转运。APP-GaI4/VP1 6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，为评估APP胞浆结构域的核转位提供一种定量手段，并为APP突变和促进或抑制胞质结构域核访问的药物操作提供一种分析方法。突变将针对CDK5磷酸化位点、可能的核定位信号、可能的PEST基序、Dab1/Fe65结合位点和可能的泛素化位点，并将评估其对亚细胞定位和核易位的影响。将评估激动型APP单抗、β-淀粉样多肽、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.