Inhibition of Receptor-mediated Endocytosis Demonstrates Generation of Amyloid β-Protein at the Cell Surface*

Sequential cleavages of the amyloid β-protein precursor (APP) by the β- and γ-secretases generate the amyloid β-protein (Aβ), which plays a central role in Alzheimer's disease. Previous work provided evidence for involvement of both the secretory and endocytic pathways in Aβ generation. Here, we used HeLa cells stably expressing a tetracycline-regulated dominant-negative dynamin I (dyn K44A), which selectively inhibits receptor-mediated endocytosis, and analyzed the effects on the processing of endogenous APP. Upon induction of dyn K44A, levels of mature APP rose at the cell surface, consistent with retention of APP on the plasma membrane. The α-secretase cleavage products of APP were increased by dyn K44A, in that α-APPs in medium and the C83 C-terminal stub in the membrane both rose. The β-secretase cleavage of APP, C99, also increased modestly. The use of specific γ-secretase inhibitors to study the accumulation of α- and β-cleavage products independent of their processing by γ-secretase confirmed that retention of APP on the plasma membrane results in increased processing by both α- and β-secretases. Unexpectedly, endogenous Aβ secretion was significantly increased by dyn K44A, as detected by three distinct methods: metabolic labeling, immunoprecipitation/Western blotting, and enzyme-linked immunosorbent assay. Levels of p3 (generated by sequential α- and γ-cleavage) also rose. We conclude that endogenous Aβ can be produced directly at the plasma membrane and that alterations in the degree of APP endocytosis may help regulate its production. Our findings are consistent with a role for the γ-secretase complex in the processing of numerous single-transmembrane receptors at the cell surface.