Abstract
The protein interaction domain of the neuronal protein X11 binds to the YENPTY motif within the cytoplasmic domain of beta-amyloid precursor protein (betaAPP). Amyloid-beta protein (Abeta), the major constituent of the amyloid deposited in brain of Alzheimer's disease patients, is generated by proteolytic processing of betaAPP, which occurs in part following betaAPP internalization. Because the YENPTY motif has a role in the internalization of betaAPP, the effect of X11 binding on betaAPP processing was studied in mouse neuroblastoma N2a, human embryonic kidney 293, monkey kidney COS-1, and human glial U251 cell lines transfected with wild type or mutated betaAPP cDNAs. Secretion of soluble betaAPP via alpha-secretase activity increased significantly in cells transfected with betaAPP variants containing mutations that impair interaction with X11 when compared with cells transfected with wild type cDNA. Cotransfection of betaAPP and X11 caused retention of cellular betaAPP, decreased secretion of sbetaAPPalpha, and decreased Abeta secretion. Thus, betaAPP interaction with the protein interaction domain of X11 stabilizes cellular betaAPP and thereby participates in the regulation of betaAPP processing pathways.
Highlights
Amyloid- protein (A),1 deposited in the brain of patients with Alzheimer’s disease (AD), Down’s syndrome, and sporadic and hereditary cerebral amyloid angiopathy and in the brain of elderly individuals is a proteolytic peptide of a larger -amyloid precursor protein (APP)
1 The abbreviations used are: A, amyloid- protein; AD, Alzheimer’s disease; APP, -amyloid precursor protein; sAPP␣, soluble APP cleaved by ␣-secretase; sAPP, soluble APP cleaved by -secretase; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PDZ, postsynaptic density protein, disc-large, zo-1; PI/PTB, phosphotyrosine interaction/phosphotyrosine binding; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
Mutations That Inhibit Binding of X11 to the YENPTY Motif of APP Affect the Secretion of Soluble APP—We have previously analyzed the binding sites on APP for the X11 PI/PTB domain, using site-directed mutagenesis of the cytoplasmic domain of APP [24]
Summary
A, amyloid- protein; AD, Alzheimer’s disease; APP, -amyloid precursor protein; sAPP␣, soluble APP cleaved by ␣-secretase; sAPP, soluble APP cleaved by -secretase; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PDZ, postsynaptic density protein, disc-large, zo-1; PI/PTB, phosphotyrosine interaction/phosphotyrosine binding; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. The PI/PTB domain was first identified as the component of the adaptor protein Shc (Src homology 2/collagen homology) that binds to activated and tyrosine-phosphorylated receptors [29, 30] This domain was further found in several unrelated regulatory proteins [31], suggesting a general role for this domain in protein-protein interactions and signal transduction. Association of PI/PTB domain-containing proteins with the coated pit-mediated internalization signal of APP may affect the patterns of APP trafficking, generation of A as well as of soluble APP, and normal physiologic function. The effect of X11 binding on APP processing was studied in different cell lines transiently or permanently transfected with the wild type or mutated APP cDNAs. We demonstrate that mutations in APP that impair interactions with X11 result in increased proteolysis by an ␣-secretase. Overexpression of both X11 and APP results in accumulation of cell-associated APP and decreased secretion of A
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