Mathews PM, Jiang, Schmidt, Grbovic, Mercken, Nixon.
Calpain Activity Regulates the Cell Surface Distribution of Amyloid Precursor Protein. Inhibition of Calpains Enhances Endosomal Generation of beta-Cleaved C-Terminal APP Fragments.
J Biol Chem. 2002 Sep;277(39):36415-24.
Nathan Kline Institute and New York University School of Medicine, Orangeburg, New York 10962 and Johnson and Johnson Pharmaceutical Research and Development, Janssen Pharmaceutica, Turnhoutseweg 30, B-2340 Beerse, Belgium.
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.