| The amyloid hypothesis of Alzheimer's disease suggests that the age-dependent accumulation and deposition of Abeta in brain is pivotal. Abeta peptides are derived from amyloid precursor protein (APP) by the proteases beta- and gamma-secretase. Understanding the regulation of APP trafficking and metabolism by its cytoplasmic C-terminus and neuronal adaptor protein interactions is crucial to understanding its normal function in brain and developing novel therapeutic strategies based on the amyloid hypothesis.; The adaptor protein X11alpha interacts with the conserved -GYENPTY- sequence in the APP C-terminus to inhibit Abeta secretion. Utilizing either endogenous or overexpressed levels of beta- and gamma-secretase activities in human embryonic kidney 293 cells, X11alpha inhibited beta- but not gamma-cleavage of APP and APPswe. However, gamma-cleavage of a second substrate, NotchDeltaE, was unaffected by X11alpha overexpression, suggesting that X11alpha may be a relatively specific gamma-secretase inhibitor. Utilizing a cell-free system, X11alpha did not impair gamma-cleavage of C99, suggesting inhibition of Abeta secretion is mediated by impaired trafficking of APP to sites of active gamma-secretase complex.; We hypothesized that the -YENP- motif essential for endocytosis of cell surface APP is also the critical motif for X11alpha interaction. Consistent with this hypothesis, X11alpha did not interact with or modulate metabolism of endocytic-defective mutations of APPswe. In contrast, X11alpha retarded maturation, prolonged the half-life, and inhibited Abeta secretion from cells coexpressing either APPswe or APPsweY743A. X11alpha impaired trafficking of APPswe in both secretory and endocytic pathways and may inhibit secretion of Abeta generated in either pathway.; The APPsweY743A mutation increased the half-life of APPswe, resulted in accumulation of C-terminal fragments, and increased secretion of APPs, APPsbetaswe, Abeta40, and Abeta42 into conditioned media. Since this mutation does not impair X11alpha and Fe65 interaction, we discovered novel effects of these adaptor proteins when coexpressed with APPsweY743A. For example, coexpression of either X11alpha or Fe65 blocked the accumulation of C-terminal fragments induced by Y743A possibly by inhibiting their generation (with X11alpha) or by promoting their gamma-cleavage (with Fe65). Tyrosine743 plays a crucial role in regulating APP trafficking and processing. Together these results examine the mechanisms underlying adaptor protein mediated changes in APP metabolism. |
