Mutagenesis identifies new signals for beta-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including A beta 42

It has long been assumed that the C-terminal motif, NPXY, is the internaliz ation signal for beta-amyloid precursor protein (APP) and that the NPXY tyr osine (Tyr(743) by APP751 numbering, Tyr(682) in APP695) is required for AP P endocytosis. To evaluate this tenet and to identify the specific amino ac ids subserving APP endocytosis, we mutated all tyrosines in the APP cytopla smic domain and amino acids within the sequence GYENPTY (amino acids 737-74 3). Stable cell lines expressing these mutations were assessed for APP endo cytosis, secretion, and turnover. Normal APP endocytosis was observed for c ells expressing Y709A, G737A, and Y743A mutations. However, Y738A, N740A, a nd P741A or the double mutation of Y738A/P741A significantly impaired APP i nternalization to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic domain (Delta C), arguing that the dominant sig nal for APP endocytosis is the tetrapeptide YENP, Although not an APP inter nalization signal, Tyr(743) regulates rapid APP turnover because half-life increased by 50% with the Y743A mutation alone. Secretion of the APP-derive d proteolytic fragment, A beta, was tightly correlated with APP internaliza tion, such that A beta secretion was unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell line s. Remarkably, secretion of the A beta 42 isoform was also reduced in paral lel with endocytosis from internalization-deficient cell lines, suggesting an important role for APP endocytosis in the secretion of this highly patho genic A beta species.