Calpain activity regulates the cell surface distribution of amyloid precursor protein. Inhibition of calpains enhances endosomal generation of beta-cleaved C-terminal APP fragments.
In murine L cells, treatment with calpeptin or calpain inhibitor III increased Abeta42, but not Abeta40, secretion in a dose-dependent fashion. This correlated with an increase in the levels of amyloid precursor protein (APP) carboxyl-terminal fragments (CTFs). Immunoprecipitation with novel mAbs directed against the carboxyl-terminus of APP or specific for the beta-cleaved CTF showed that generation of both alpha- and beta-cleaved CTFs increase proportionately following inhibition of calpains. Pulse-chase metabolic labeling confirmed that inhibiting calpains increases the production of alpha- and beta-cleaved APP metabolites. Immunolabeling showed greater betaCTF signal in calpeptin-treated cells, primarily in small vesicular compartments that were shown to be predominantly endosomal by colocalization with early endosomal antigen 1. A second mAb, which recognizes an extracellular/luminal epitope found on both APP and betaCTFs, gave more cell surface labeling of calpeptin-treated cells than control cells. Quantitative binding of this antibody confirmed that inhibiting calpains caused a partial redistribution of APP to the cell surface. These results demonstrate that 1) calpain inhibition results in a partial redistribution of APP to the cell surface, 2) this redistribution leads to an increase in both alpha- and beta-cleavage without changing the ratio of alphaCTFs/betaCTFs, and 3) the bulk of the betaCTFs in the cell are within early endosomes, confirming the importance of this compartment in APP processing.
Mathews
Paul M
PM
Nathan Kline Institute, Orangeburg, New York 10962, USA. mathews@nki.rfmh.org
Jiang
Ying
Y
Schmidt
Stephen D
SD
Grbovic
Olivera M
OM
Mercken
Marc
M
Nixon
Ralph A
RA
D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004151:Dipeptides / Q000037:antagonists & inhibitors / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D011992:Endosomes / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014407:Tumor Cells, Cultured
D016564:Amyloid beta-Protein Precursor; D000911:Antibodies, Monoclonal; D004151:Dipeptides; C071482:calpeptin; D002154:Calpain