Amyloid Precursor Protein Signaling

描述（由申请人提供）：研究将检验以下假设：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，而阿尔茨海默病中APP的病理功能反映了该受体功能的扰动。有人提出，APP类似于受体Notch，具有两种替代信号传导途径-一种从膜驻留受体传播，另一种通过在蛋白质的跨膜序列内的γ-分泌酶切割后受体的胞质结构域的核转位传播。研究将使用各种细胞系，包括COS 7细胞（一种神经元细胞系）和NGF分化的PC 12细胞（一种神经元细胞系）。瞬时和稳定转染将用于操纵APP和与其相互作用的蛋白质的表达。将使用绿色荧光蛋白（GFP）和myc表位标签来跟踪转染后β-淀粉样前体蛋白的细胞内运输。APP-GaI 4/VP 16和APP-GaI 4融合蛋白将在报告细胞系中表达，所述报告细胞系在Gal 4启动子的控制下表达荧光素酶，提供评估APP胞质结构域的核易位的定量手段，并提供促进或抑制胞质结构域的核进入的APP突变和药理学操作的测定。突变将靶向CDK 5磷酸化位点、推定的核定位信号、推定的PEST基序、Dab 1/Fe 65结合位点和推定的泛素化位点，并评估对亚细胞定位和核转位的影响。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信号传导的影响。

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.