Salvador Soriano

It has long been assumed that the C-terminal motif, NPXY, is the internalization signal for β-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 (ΔC), 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, Aβ, was tightly correlated with APP internalization, such that Aβ secretion was unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell lines. Remarkably, secretion of the Aβ42 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 Aβ species. It has long been assumed that the C-terminal motif, NPXY, is the internalization signal for β-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 (ΔC), 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, Aβ, was tightly correlated with APP internalization, such that Aβ secretion was unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell lines. Remarkably, secretion of the Aβ42 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 Aβ species. β-amyloid precursor protein Alzheimer's disease low density lipoprotein receptor Chinese hamster ovary wild type N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine enzyme-linked immunosorbent assay analysis of variance. APPs, secreted N1-terminal ectodomain of APP APP1 is a transmembrane protein with homology to glycosylated cell surface receptors (1Kang J. Lemaire H.-G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.-H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3916) Google Scholar), can reside at the cell surface (2Shivers B.D. Hilbich C. Multhaup G. Salbaum M. Beyreuther K. Seeburg P.H. EMBO J. 1988; 7: 1365-1370Crossref PubMed Scopus (387) Google Scholar, 3Breen K. Bruce M. Anderson B. J. Neurosci. Res. 1991; 28: 90-100Crossref PubMed Scopus (221) Google Scholar, 4Jung S.S. Nalbantoglu J. Cashman N.R. J Neurosci. Res. 1996; 46: 336-348Crossref PubMed Scopus (42) Google Scholar) and is reinternalized via clathrin-coated pits (5Nordstedt C. Caporaso G.L. Thyberg J. Gandy S.E. Greengard P. J. Biol. Chem. 1993; 268: 608-612Abstract PubMed Google Scholar, J. 1996; PubMed Google Scholar) to the C. A. Nature. PubMed Scopus Google Scholar, PubMed Scopus Google APP to to the cell surface J. Biol. PubMed Scopus Google Scholar, J. 1996; PubMed Google However, APP can also secreted the secreted APP ectodomain and Aβ, the protein of in Alzheimer's disease in PubMed Scopus Google Aβ to the by Aβ is from the precursor is an important of of Aβ, by amino acids at the and from cells normal C. C. A. Nature. PubMed Scopus Google Scholar, M. J. B. PubMed Scopus Google Scholar, C. M. M. J. M. M. P. G. 1996; PubMed Scopus Google in in 1993; PubMed Scopus Google Scholar) to pathogenic and a for in with Biol. 1996; Scholar), with type P. J. M. G. M. 1996; PubMed Scopus Google Scholar), and 1996; PubMed Scopus Google of Aβ in is However, cell lines expressing wild type APP can and Aβ internalization of APP from the cell surface J. Biol. Chem. PubMed Google Scholar, J. Biol. Chem. 1996; PubMed Scopus Google Although mutations in APP can Aβ secretion the J. Biol. Chem. 1996; PubMed Scopus Google Scholar, M. C. K. P. C. Nature. PubMed Scopus Google Scholar, 1993; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar), all wild type the for Aβ to of APP from the cell To the specific of APP to Aβ42 in has not been endocytosis in the cytoplasmic C-terminal NPXY sequence similar to that in the of the low density lipoprotein receptor and the protein J. EMBO J. 1988; 7: PubMed Scopus Google Scholar) is also in the tyrosine in NPXY is for endocytosis J. Biol. Chem. PubMed Google Scholar, Anderson PubMed Scopus Google Scholar), has long been assumed that the tyrosine in the APP NPXY is the signal for APP endocytosis. the an amino of the NPXY was a to endocytosis, the internalization in Biol. 1993; PubMed Scopus Google the to the APP signal from an receptor was to the GYENPTY (amino acids with a by tyrosine in the to the A. J. Biol. Chem. PubMed Scopus Google However, because the receptor the and the of the APP signal with to the is this to of the in that the of the signal Aβ42 in has not been to identify the specific amino acids required for APP endocytosis and to APP tyrosines with endocytosis of J. A. PubMed Scopus Google Scholar, B. A. M. A. PubMed Scopus Google Scholar), we mutations of all the APP cytoplasmic domain tyrosines of the amino acids and in the GYENPTY the of these mutations in cell lines and that amino acids to APP endocytosis and the of APP secreted we observed that the tyrosine in to APP endocytosis, the tyrosine in this the were Y743A cell lines APP turnover. we a the of APP endocytosis and Aβ in Aβ42 and Aβ secretion suggesting that to the secretion of the pathogenic Aβ42 and the by cell cells been in to APP to cells C. A. Nature. PubMed Scopus Google B. K. B. J. Biol. Chem. PubMed Scopus Google Scholar, P. C. M. M. J. C. Nature. PubMed Scopus Google Scholar, M. C. C. J. B. A. PubMed Scopus Google Scholar) and a for APP and J. 1996; PubMed Google Scholar, J. 1996; PubMed Google Scholar, J. Biol. Chem. PubMed Google Scholar, J. Biol. Chem. 1996; PubMed Scopus Google Scholar, A. J. Biol. Chem. PubMed Scopus Google Scholar, K. B. M. Alzheimer's and and of cells expressing APP or APP with C-terminal mutations and APP and we the tetrapeptide motif, (amino acids in APP751 or in APP695) in the APP the dominant APP we observed that mutation of Tyr743 in the NPXY APP endocytosis, this mutation significantly of we and parallel the secretion of APPs, Aβ, and Aβ42 in with C-terminal mutations that normal APP APP cell surface is in clathrin-coated (5Nordstedt C. Caporaso G.L. Thyberg J. Gandy S.E. Greengard P. J. Biol. Chem. 1993; 268: 608-612Abstract PubMed Google Scholar, J. 1996; PubMed Google Scholar), that APP a and a to cell surface receptors in Biol. 1993; PubMed Scopus Google receptor to is the role in endocytosis, NPXY has been assumed to the signal for APP endocytosis J. Biol. Chem. PubMed Google to the amino acids of this NPXY motif, an amino of NPXY also to endocytosis, the signal for this the J. Biol. Chem. PubMed Google been the motif, an sequence in the APP the for APP endocytosis and J. Biol. Chem. PubMed Google Scholar, A. J. Biol. Chem. PubMed Scopus Google Scholar, K. B. M. Alzheimer's and and C. A. J. Biol. PubMed Scopus Google Scholar, B. K. B. J. Biol. Chem. PubMed Scopus Google Although that the APP signal was within this motif, the specific amino acids APP endocytosis to endocytosis also been for the tyrosine the and for the in GYENPTY of APP A. J. Biol. Chem. PubMed Scopus Google we the to to APP endocytosis a for APP endocytosis J. 1996; PubMed Google we that mutations of or or a double mutation of and but not a mutation of the C-terminal in significantly APP endocytosis. mutations within the all reduced APP internalization that the amino is the signal for APP endocytosis. amino to a of the APP signal because the mutation reduced endocytosis to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic that to APP internalization in suggesting that these amino Tyr743 in NPXY, not of the APP amino acids from the amino acids from the was the in the A. J. Biol. Chem. PubMed Scopus Google Scholar), and this the tyrosine internalization in the Although ectodomain of APP a role in in cells B. K. B. J. Biol. Chem. PubMed Scopus Google Scholar), for the tyrosine subserving of APP has also been C. A. J. Biol. PubMed Scopus Google It that the tyrosine in APP751 and in APP695) with the protein with homology that with to APP to the in cells P. J. A. PubMed Scopus Google the role of Tyr743 in APP we that Tyr743 in the NPXY is not an APP signal and that cells APP with this mutation normal of Although endocytosis was not in cells with mutations of the NPXY tyrosine also observed normal Aβ secretion from cell lines J. J. Biol. Chem. PubMed Google Scholar, C. A. C. J. Biol. Chem. PubMed Scopus Google Although not an signal, reduced the rapid turnover of Although the APP half-life was in Y743A APP was from to glycosylated APP to and suggesting that the was normal for these cell lines. we that Tyr743 in a signal for that cells expressing C-terminal reduced of Aβ secretion J. Biol. Chem. PubMed Google Scholar, K. B. M. Alzheimer's and and Aβ was by cells expressing mutations that reduced APP endocytosis. Y738A, N740A, and cell lines all reduced Aβ secretion by in Aβ secretion was observed for Y709A, G737A, or cell lines that also normal APP endocytosis. the of the for the of Aβ by we Aβ42 secretion by we were to that in parallel with the of Aβ secretion, the Y738A, N740A, and cell lines also secreted that of APP in the to Aβ42 However, J. Biol. Chem. 1996; PubMed Scopus Google Scholar, C. A. C. J. Biol. Chem. PubMed Scopus Google Scholar, B. J. A. PubMed Scopus Google Scholar, G. J. M. P. J. Biol. Chem. PubMed Scopus Google Scholar, J. P. J. PubMed Scopus Google Scholar) and cells J. Biol. Chem. 1996; PubMed Scopus Google Scholar, B.D. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar) to Aβ42 in the of the C. A. C. J. Biol. Chem. PubMed Scopus Google Scholar, J. P. J. PubMed Scopus Google B.D. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. A. Multhaup G. Masters C.L. Beyreuther K. A. PubMed Scopus Google Scholar, B. Masters C.L. K. Beyreuther K. PubMed Scopus Google Scholar), we were to that to significantly to the of Aβ42 secreted from cell lines. However, is not the Aβ in the secreted by the that Aβ42 not because the level of Aβ42 the was the we not Aβ in for Aβ, and Aβ42 in cell or in or of C-terminal required to or not a in endocytosis can the Aβ species. that that mutations that APP internalization reduced Aβ secretion, the mutations APP to is the amino acids in the endocytosis, is to that by with such or in G. P. PubMed Scopus Google in APP was assessed in cells or M. B. J. Biol. Chem. PubMed Scopus Google Scholar, J. A. Scholar) that the of APP with these in and that Aβ is by of this to APP in the because of reduced by M. B. J. Biol. Chem. PubMed Scopus Google Scholar), because of reduced of APP at the cell with in an of the because of in cells increased secretion J. A. Scholar), an to of APP at or the cell for cells Greengard P. J. Biol. Chem. PubMed Scopus Google of with APP to within B.D. A. J. Biol. Chem. PubMed Scopus Google an NPXY APP and the receptor protein by of the and has also been that APP and M. B. J. J. Biol. Chem. PubMed Scopus Google that the dominant signal for APP endocytosis in the that in the APP to APP and that of APP to and Aβ of the by cells APP secreted for or the of of Aβ and secretion for APP1 is a transmembrane protein with homology to glycosylated cell surface receptors (1Kang J. Lemaire H.-G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.-H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3916) Google Scholar), can reside at the cell surface (2Shivers B.D. Hilbich C. Multhaup G. Salbaum M. Beyreuther K. Seeburg P.H. EMBO J. 1988; 7: 1365-1370Crossref PubMed Scopus (387) Google Scholar, 3Breen K. Bruce M. Anderson B. J. Neurosci. Res. 1991; 28: 90-100Crossref PubMed Scopus (221) Google Scholar, 4Jung S.S. Nalbantoglu J. Cashman N.R. J Neurosci. Res. 1996; 46: 336-348Crossref PubMed Scopus (42) Google Scholar) and is reinternalized via clathrin-coated pits (5Nordstedt C. Caporaso G.L. Thyberg J. Gandy S.E. Greengard P. J. Biol. Chem. 1993; 268: 608-612Abstract PubMed Google Scholar, J. 1996; PubMed Google Scholar) to the C. A. Nature. PubMed Scopus Google Scholar, PubMed Scopus Google APP to to the cell surface J. Biol. PubMed Scopus Google Scholar, J. 1996; PubMed Google However, APP can also secreted the secreted APP ectodomain and Aβ, the protein of in Alzheimer's disease in PubMed Scopus Google Aβ to the by Aβ is from the precursor is an important of of Aβ, by amino acids at the and from cells normal C. C. A. Nature. PubMed Scopus Google Scholar, M. J. B. PubMed Scopus Google Scholar, C. M. M. J. M. M. P. G. 1996; PubMed Scopus Google in in 1993; PubMed Scopus Google Scholar) to pathogenic and a for in with Biol. 1996; Scholar), with type P. J. M. G. M. 1996; PubMed Scopus Google Scholar), and 1996; PubMed Scopus Google of Aβ in is However, cell lines expressing wild type APP can and Aβ internalization of APP from the cell surface J. Biol. Chem. PubMed Google Scholar, J. Biol. Chem. 1996; PubMed Scopus Google Although mutations in APP can Aβ secretion the J. Biol. Chem. 1996; PubMed Scopus Google Scholar, M. C. K. P. C. Nature. PubMed Scopus Google Scholar, 1993; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar), all wild type the for Aβ to of APP from the cell To the specific of APP to Aβ42 in has not been APP endocytosis in the cytoplasmic C-terminal NPXY sequence similar to that in the of the low density lipoprotein receptor and the protein J. EMBO J. 1988; 7: PubMed Scopus Google Scholar) is also in the tyrosine in NPXY is for endocytosis J. Biol. Chem. PubMed Google Scholar, Anderson PubMed Scopus Google Scholar), has long been assumed that the tyrosine in the APP NPXY is the signal for APP endocytosis. the an amino of the NPXY was a to endocytosis, the internalization in Biol. 1993; PubMed Scopus Google the to the APP signal from an receptor was to the GYENPTY (amino acids with a by tyrosine in the to the A. J. Biol. Chem. PubMed Scopus Google However, because the receptor the and the of the APP signal with to the is this to of the in that the of the signal Aβ42 in has not been to identify the specific amino acids required for APP endocytosis and to APP tyrosines with endocytosis of J. A. PubMed Scopus Google Scholar, B. A. M. A. PubMed Scopus Google Scholar), we mutations of all the APP cytoplasmic domain tyrosines of the amino acids and in the GYENPTY the of these mutations in cell lines and that amino acids to APP endocytosis and the of APP secreted we observed that the tyrosine in to APP endocytosis, the tyrosine in this the were Y743A cell lines APP turnover. we a the of APP endocytosis and Aβ in Aβ42 and Aβ secretion suggesting that to the secretion of the pathogenic Aβ42 and the by cell lines. cells been in to APP to cells C. A. Nature. PubMed Scopus Google B. K. B. J. Biol. Chem. PubMed Scopus Google Scholar, P. C. M. M. J. C. Nature. PubMed Scopus Google Scholar, M. C. C. J. B. A. PubMed Scopus Google Scholar) and a for APP and J. 1996; PubMed Google Scholar, J. 1996; PubMed Google Scholar, J. Biol. Chem. PubMed Google Scholar, J. Biol. Chem. 1996; PubMed Scopus Google Scholar, A. J. Biol. Chem. PubMed Scopus Google Scholar, K. B. M. Alzheimer's and and of cells expressing APP or APP with C-terminal mutations and APP and we the tetrapeptide motif, (amino acids in APP751 or in APP695) in the APP the dominant APP we observed that mutation of Tyr743 in the NPXY APP endocytosis, this mutation significantly of we and parallel the secretion of APPs, Aβ, and Aβ42 in with C-terminal mutations that normal APP APP cell surface is in clathrin-coated (5Nordstedt C. Caporaso G.L. Thyberg J. Gandy S.E. Greengard P. J. Biol. Chem. 1993; 268: 608-612Abstract PubMed Google Scholar, J. 1996; PubMed Google Scholar), that APP a and a to cell surface receptors in Biol. 1993; PubMed Scopus Google receptor to is the role in endocytosis, NPXY has been assumed to the signal for APP endocytosis J. Biol. Chem. PubMed Google to the amino acids of this NPXY motif, an amino of NPXY also to endocytosis, the signal for this the J. Biol. Chem. PubMed Google been the motif, an sequence in the APP the for APP endocytosis and J. Biol. Chem. PubMed Google Scholar, A. J. Biol. Chem. PubMed Scopus Google Scholar, K. B. M. Alzheimer's and and C. A. J. Biol. PubMed Scopus Google Scholar, B. K. B. J. Biol. Chem. PubMed Scopus Google Although that the APP signal was within this motif, the specific amino acids APP endocytosis to endocytosis also been for the tyrosine the and for the in GYENPTY of APP A. J. Biol. Chem. PubMed Scopus Google we the to to APP endocytosis a for APP endocytosis J. 1996; PubMed Google we that mutations of or or a double mutation of and but not a mutation of the C-terminal in significantly APP endocytosis. mutations within the all reduced APP internalization that the amino is the signal for APP endocytosis. amino to a of the APP signal because the mutation reduced endocytosis to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic that to APP internalization in suggesting that these amino Tyr743 in NPXY, not of the APP amino acids from the amino acids from the was the in the A. J. Biol. Chem. PubMed Scopus Google Scholar), and this the tyrosine internalization in the Although ectodomain of APP a role in in cells B. K. B. J. Biol. Chem. PubMed Scopus Google Scholar), for the tyrosine subserving of APP has also been C. A. J. Biol. PubMed Scopus Google It that the tyrosine in APP751 and in APP695) with the protein with homology that with to APP to the in cells P. J. A. PubMed Scopus Google the role of Tyr743 in APP we that Tyr743 in the NPXY is not an APP signal and that cells APP with this mutation normal of Although endocytosis was not in cells with mutations of the NPXY tyrosine also observed normal Aβ secretion from cell lines J. J. Biol. Chem. PubMed Google Scholar, C. A. C. J. Biol. Chem. PubMed Scopus Google Although not an signal, reduced the rapid turnover of Although the APP half-life was in Y743A APP was from to glycosylated APP to and suggesting that the was normal for these cell lines. we that Tyr743 in a signal for that cells expressing C-terminal reduced of Aβ secretion J. Biol. Chem. PubMed Google Scholar, K. B. M. Alzheimer's and and Aβ was by cells expressing mutations that reduced APP endocytosis. Y738A, N740A, and cell lines all reduced Aβ secretion by in Aβ secretion was observed for Y709A, G737A, or cell lines that also normal APP endocytosis. the of the for the of Aβ by we Aβ42 secretion by we were to that in parallel with the of Aβ secretion, the Y738A, N740A, and cell lines also secreted that of APP in the to Aβ42 However, J. Biol. Chem. 1996; PubMed Scopus Google Scholar, C. A. C. J. Biol. Chem. PubMed Scopus Google Scholar, B. J. A. PubMed Scopus Google Scholar, G. J. M. P. J. Biol. Chem. PubMed Scopus Google Scholar, J. P. J. PubMed Scopus Google Scholar) and cells J. Biol. Chem. 1996; PubMed Scopus Google Scholar, B.D. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar) to Aβ42 in the of the C. A. C. J. Biol. Chem. PubMed Scopus Google Scholar, J. P. J. PubMed Scopus Google B.D. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. A. Multhaup G. Masters C.L. Beyreuther K. A. PubMed Scopus Google Scholar, B. Masters C.L. K. Beyreuther K. PubMed Scopus Google Scholar), we were to that to significantly to the of Aβ42 secreted from cell lines. However, is not the Aβ in the secreted by the that Aβ42 not because the level of Aβ42 the was the we not Aβ in for Aβ, and Aβ42 in cell or in or of C-terminal required to or not a in endocytosis can the Aβ species. that that mutations that APP internalization reduced Aβ secretion, the mutations APP to is the amino acids in the endocytosis, is to that by with such or in G. P. PubMed Scopus Google in APP was assessed in cells or M. B. J. Biol. Chem. PubMed Scopus Google Scholar, J. A. Scholar) that the of APP with these in and that Aβ is by of this to APP in the because of reduced by M. B. J. Biol. Chem. PubMed Scopus Google Scholar), because of reduced of APP at the cell with in an of the because of in cells increased secretion J. A. Scholar), an to of APP at or the cell for cells Greengard P. J. Biol. Chem. PubMed Scopus Google of with APP to within B.D. A. J. Biol. Chem. PubMed Scopus Google an NPXY APP and the receptor protein by of the and has also been that APP and M. B. J. J. Biol. Chem. PubMed Scopus Google that the dominant signal for APP endocytosis in the that in the APP to APP and that of APP to and Aβ of the by cells APP secreted for or the of of Aβ and secretion for cells been in to APP to cells C. A. Nature. PubMed Scopus Google B. K. B. J. Biol. Chem. PubMed Scopus Google Scholar, P. C. M. M. J. C. Nature. PubMed Scopus Google Scholar, M. C. C. J. B. A. PubMed Scopus Google Scholar) and a for APP and J. 1996; PubMed Google Scholar, J. 1996; PubMed Google Scholar, J. Biol. Chem. PubMed Google Scholar, J. Biol. Chem. 1996; PubMed Scopus Google Scholar, A. J. Biol. Chem. PubMed Scopus Google Scholar, K. B. M. Alzheimer's and and of cells expressing APP or APP with C-terminal mutations and APP and we the tetrapeptide motif, (amino acids in APP751 or in APP695) in the APP the dominant APP we observed that mutation of Tyr743 in the NPXY APP endocytosis, this mutation significantly of we and parallel the secretion of APPs, Aβ, and Aβ42 in with C-terminal mutations that normal APP endocytosis. APP cell surface is in clathrin-coated (5Nordstedt C. Caporaso G.L. Thyberg J. Gandy S.E. Greengard P. J. Biol. Chem. 1993; 268: 608-612Abstract PubMed Google Scholar, J. 1996; PubMed Google Scholar), that APP a and a to cell surface receptors in Biol. 1993; PubMed Scopus Google receptor to is the role in endocytosis, NPXY has been assumed to the signal for APP endocytosis J. Biol. Chem. PubMed Google to the amino acids of this NPXY motif, an amino of NPXY also to endocytosis, the signal for this the J. Biol. Chem. PubMed Google been the motif, an sequence in the APP the for APP endocytosis and J. Biol. Chem. PubMed Google Scholar, A. J. Biol. Chem. PubMed Scopus Google Scholar, K. B. M. Alzheimer's and and C. A. J. Biol. PubMed Scopus Google Scholar, B. K. B. J. Biol. Chem. PubMed Scopus Google Although that the APP signal was within this motif, the specific amino acids APP endocytosis to endocytosis also been for the tyrosine the and for the in GYENPTY of APP A. J. Biol. Chem. PubMed Scopus Google we the to to APP endocytosis a for APP endocytosis J. 1996; PubMed Google we that mutations of or or a double mutation of and but not a mutation of the C-terminal in significantly APP endocytosis. mutations within the all reduced APP internalization that the amino is the signal for APP endocytosis. amino to a of the APP signal because the mutation reduced endocytosis to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic that to APP internalization in suggesting that these amino Tyr743 in NPXY, not of the APP amino acids from the amino acids from the was the in the A. J. Biol. Chem. PubMed Scopus Google Scholar), and this the tyrosine internalization in the Although ectodomain of APP a role in in cells B. K. B. J. Biol. Chem. PubMed Scopus Google Scholar), for the tyrosine subserving of APP has also been C. A. J. Biol. PubMed Scopus Google It that the tyrosine in APP751 and in APP695) with the protein with homology that with to APP to the in cells P. J. A. PubMed Scopus Google the role of Tyr743 in APP we that Tyr743 in the NPXY is not an APP signal and that cells APP with this mutation normal of Although endocytosis was not in cells with mutations of the NPXY tyrosine also observed normal Aβ secretion from cell lines J. J. Biol. Chem. PubMed Google Scholar, C. A. C. J. Biol. Chem. PubMed Scopus Google Although not an signal, reduced the rapid turnover of Although the APP half-life was in Y743A APP was from to glycosylated APP to and suggesting that the was normal for these cell lines. we that Tyr743 in a signal for that cells expressing C-terminal reduced of Aβ secretion J. Biol. Chem. PubMed Google Scholar, K. B. M. Alzheimer's and and Aβ was by cells expressing mutations that reduced APP endocytosis. Y738A, N740A, and cell lines all reduced Aβ secretion by in Aβ secretion was observed for Y709A, G737A, or cell lines that also normal APP endocytosis. the of the for the of Aβ by we Aβ42 secretion by we were to that in parallel with the of Aβ secretion, the Y738A, N740A, and cell lines also secreted that of APP in the to Aβ42 However, J. Biol. Chem. 1996; PubMed Scopus Google Scholar, C. A. C. J. Biol. Chem. PubMed Scopus Google Scholar, B. J. A. PubMed Scopus Google Scholar, G. J. M. P. J. Biol. Chem. PubMed Scopus Google Scholar, J. P. J. PubMed Scopus Google Scholar) and cells J. Biol. Chem. 1996; PubMed Scopus Google Scholar, B.D. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar) to Aβ42 in the of the C. A. C. J. Biol. Chem. PubMed Scopus Google Scholar, J. P. J. PubMed Scopus Google B.D. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. A. Multhaup G. Masters C.L. Beyreuther K. A. PubMed Scopus Google Scholar, B. Masters C.L. K. Beyreuther K. PubMed Scopus Google Scholar), we were to that to significantly to the of Aβ42 secreted from cell lines. However, is not the Aβ in the secreted by the that Aβ42 not because the level of Aβ42 the was the we not Aβ in for Aβ, and Aβ42 in cell or in or of C-terminal required to or not a in endocytosis can the Aβ species. that that mutations that APP internalization reduced Aβ secretion, the mutations APP to Although is the amino acids in the endocytosis, is to that by with such or in G. P. PubMed Scopus Google in APP was assessed in cells or M. B. J. Biol. Chem. PubMed Scopus Google Scholar, J. A. Scholar) that the of APP with these in and that Aβ is by of this to APP in the because of reduced by M. B. J. Biol. Chem. PubMed Scopus Google Scholar), because of reduced of APP at the cell with in an of the because of in cells increased secretion J. A. Scholar), an to of APP at or the cell for cells Greengard P. J. Biol. Chem. PubMed Scopus Google of with APP to within B.D. A. J. Biol. Chem. PubMed Scopus Google an NPXY APP and the receptor protein by of the and has also been that APP and M. B. J. J. Biol. Chem. PubMed Scopus Google that the dominant signal for APP endocytosis in the that in the APP to APP and that of APP to and Aβ of the by cells APP secreted for or the of of Aβ and secretion for for and for and

Presenilin 1 (PS1) is required for the proteolytic processing of Notch and the beta-amyloid precursor protein (APP), molecules that play pivotal roles in cell-fate determination during development and Alzheimer's disease pathogenesis, respectively. In addition, PS1 interacts with beta-catenin and promotes its turnover through independent mechanisms. Consistent with this activity, we report here that PS1 is important in controlling epidermal cell proliferation in vivo. PS1 knockout mice that are rescued through neuronal expression of human PS1 transgene develop spontaneous skin cancers. PS1-null keratinocytes exhibit higher cytosolic beta-catenin and beta-catenin/lymphoid enhancer factor-1/T cell factor (beta-catenin/LEF)-mediated signaling. This effect can be reversed by reintroducing wild-type PS1, but not a PS1 mutant active in Notch processing but defective in beta-catenin binding. Nuclear beta-catenin protein can be detected in tumors. Elevated beta-catenin/LEF signaling is correlated with activation of its downstream target cyclin D1 and accelerated entry from G(1) into S phase of the cell cycle. This report demonstrates a function of PS1 in adult tissues, and our analysis suggests that deregulation of beta-catenin pathway contributes to the skin tumor phenotype.

In addition to its documented role in the proteolytic processing of Notch-1 and the beta-amyloid precursor protein, presenilin 1 (PS1) associates with beta-catenin. In this study, we show that this interaction plays a critical role in regulating beta-catenin/T Cell Factor/Lymphoid Enhancer Factor-1 (LEF) signaling. PS1 deficiency results in accumulation of cytosolic beta-catenin, leading to a beta-catenin/LEF-dependent increase in cyclin D1 transcription and accelerated entry into the S phase of the cell cycle. Conversely, PS1 specifically represses LEF-dependent transcription in a dose-dependent manner. The hyperproliferative response can be reversed by reintroducing PS1 expression or overexpressing axin, but not a PS1 mutant that does not bind beta-catenin (PS1Deltacat) or by two different familial Alzheimer's disease mutants. In contrast, PS1Deltacat restores Notch-1 proteolytic cleavage and Abeta generation in PS1-deficient cells, indicating that PS1 function in modulating beta-catenin levels can be separated from its roles in facilitating gamma-secretase cleavage of beta-amyloid precursor protein and in Notch-1 signaling. Finally, we show an altered response to Wnt signaling and impaired ubiquitination of beta-catenin in the absence of PS1, a phenotype that may account for the increased stability in PS1-deficient cells. Thus, PS1 adds to the molecules that are known to regulate the rapid turnover of beta-catenin.

Although an association between the product of the familial Alzheimer's disease (FAD) gene, presenilin 1 (PS1), and beta-catenin has been reported recently, the cellular consequences of this interaction are unknown. Here, we show that both the full length and the C-terminal fragment of wild-type or FAD mutant PS1 interact with beta-catenin from transfected cells and brains of transgenic mice, whereas E-cadherin and adenomatous polyposis coli (APC) are not detected in this complex. Inducible overexpression of PS1 led to increased association of beta-catenin with glycogen synthase kinase-3beta (GSK-3beta), a negative regulator of beta-catenin, and accelerated the turnover of endogenous beta-catenin. In support of this finding, the beta-catenin half-life was dramatically longer in fibroblasts deficient in PS1, and this phenotype was completely rescued by replacement of PS1, demonstrating that PS1 normally stimulates the degradation of beta-catenin. In contrast, overexpression of FAD-linked PS1 mutants (M146L and DeltaX9) failed to enhance the association between GSK-3beta and beta-catenin and interfered with the constitutive turnover of beta-catenin. In vivo confirmation was demonstrated in the brains of transgenic mice in which the expression of the M146L mutant PS1 was correlated with increased steady-state levels of endogenous beta-catenin. Thus, our results indicate that PS1 normally promotes the turnover of beta-catenin, whereas PS1 mutants partially interfere with this process, possibly by failing to recruit GSK-3beta into the PS1-beta-catenin complex. These findings raise the intriguing possibility that PS1-beta-catenin interactions and subsequent activities may be consequential for the pathogenesis of AD.