Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42.
Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Abeta42.
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发表时间：
1999
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通讯作者：
R. Perez;S. Soriano;J. D. Hayes;B. Ostaszewski;W. Xia;D. Selkoe;X. Chen;G. Stokin;E. Koo
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作者：
R. Perez;S. Soriano;J. D. Hayes;B. Ostaszewski;W. Xia;D. Selkoe;X. Chen;G. Stokin;E. Koo
长期以来一直认为C-末端基序NPXY是β-淀粉样前体蛋白（APP）的内化信号，并且NPXY酪氨酸（APP 751编号为Tyr 743，APP 695中为Tyr 682）是APP内吞作用所需的。为了评估这一原则并鉴定辅助APP内吞作用的特定氨基酸，我们突变了APP胞质结构域中的所有酪氨酸和序列GYENPTY（氨基酸737-743）内的氨基酸。评估表达这些突变的稳定细胞系的APP内吞、分泌和周转。对于表达Y 709 A、G737 A和Y 743 A突变的细胞，观察到正常的APP内吞作用。然而，Y 738 A、N740 A和P741 A或Y 738 A/P741 A的双突变显著损害APP内化至与几乎缺乏整个APP胞质结构域（DeltaC）的细胞所观察到的水平相似的水平，这表明APP内吞作用的主导信号是四肽YENP。虽然不是APP内化信号，但Tyr 743调节APP的快速周转，因为仅Y 743 A突变的半衰期增加了50%。APP衍生的蛋白水解片段Abeta的分泌与APP内化密切相关，使得对于具有正常APP内吞作用的细胞Abeta分泌不变，但对于内吞作用缺陷的细胞系Abeta分泌显著降低。值得注意的是，分泌的Abeta 42亚型也减少了平行的内吞作用从内化缺陷的细胞系，这表明APP内吞作用在这种高致病性Abeta物种的分泌中的重要作用。
It has long been assumed that the C-terminal motif, NPXY, is the internalization signal for beta-amyloid precursor protein (APP) and that the NPXY tyrosine (Tyr743 by APP751 numbering, Tyr682 in APP695) is required for APP endocytosis. To evaluate this tenet and to identify the specific amino acids subserving APP endocytosis, we mutated all tyrosines in the APP cytoplasmic domain and amino acids within the sequence GYENPTY (amino acids 737-743). Stable cell lines expressing these mutations were assessed for APP endocytosis, secretion, and turnover. Normal APP endocytosis was observed for cells expressing Y709A, G737A, and Y743A mutations. However, Y738A, N740A, and P741A or the double mutation of Y738A/P741A significantly impaired APP internalization to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic domain (DeltaC), arguing that the dominant signal for APP endocytosis is the tetrapeptide YENP. Although not an APP internalization signal, Tyr743 regulates rapid APP turnover because half-life increased by 50% with the Y743A mutation alone. Secretion of the APP-derived proteolytic fragment, Abeta, was tightly correlated with APP internalization, such that Abeta secretion was unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell lines. Remarkably, secretion of the Abeta42 isoform was also reduced in parallel with endocytosis from internalization-deficient cell lines, suggesting an important role for APP endocytosis in the secretion of this highly pathogenic Abeta species.