Synergistic Effects of Munc18a and X11 Proteins on Amyloid Precursor Protein Metabolism

Chi S Ho,Vlad Marinescu,Michelle L Steinhilb,James R Gaut,R Scott Turner,Edward L Stuenkel

doi:10.1074/jbc.m201823200

Abstract

X11 proteins have been shown to modulate metabolism of the amyloid precursor protein (APP) and to reduce the secretion of beta-amyloid peptides (Abeta) that are associated with Alzheimer's disease. Whereas X11alpha interacts with APP via its phosphotyrosine-binding domain, recent reports indicate that additional regulatory interactions involve the N terminus of X11. Here we report that the syntaxin-1a-binding protein Munc18a, which interacts with the Munc18a-interacting domain (MID) at the N terminus of X11, strongly regulates the actions of X11 on APP metabolism. When co-expressed with X11alpha, Munc18a potentiated the retention of APP and suppression of Abeta secretion by X11alpha. As a result, the constitutive release of Abeta40 was nearly abolished. Experiments using N terminus deletion mutants of X11alpha/beta and the MID-deficient X11gamma revealed that the majority of the regulatory effect by Munc18a occurred independent of a direct interaction of Munc18a with X11, although the presence of X11 was required. Munc18a expression induced a small increase in beta-secretase activity, whereas it also intensified the reduction in Abeta40 secretion by X11alpha. These data indicate that Munc18a in concert with X11 acts to suppress gamma-secretase processing. We conclude that Munc18a acts through direct and indirect interactions with X11 proteins and powerfully regulates APP metabolism and Abeta secretion.

Highlights

X11 proteins have been shown to modulate metabolism of the amyloid precursor protein (APP) and to reduce the secretion of ␤-amyloid peptides (A␤) that are associated with Alzheimer’s disease
1 The abbreviations used are: A␤, ␤-amyloid peptide; APP, amyloid precursor protein; APPs, secreted N-terminal ectodomain of APP; APPs-␤, secreted ␤-secretase cleavage product of APP; APPsw, double missense mutation of APP (K651N/M652L, positions based on APP751) identified in a Swedish kindred of familial Alzheimer’s disease; GST, glutathione Stransferase; HEK293 cells, human embryonic kidney 293 cells; His6, epitope tag with a string of six histidine residues; Munc18a-interacting domain (MID), Munc18a interacting domain; PDZ, conserved binding motif initially found in postsynaptic density-95 (PSD-95), Drosophila Disks-large (Dlg), and epithelial tight junction protein zona occludens-a (ZO-1); phosphotyrosine-binding domain (PTB), phosphotyrosinebinding domain; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonic acid; ANOVA, analysis of variance
Previous studies have shown that X11␣ and X11␤ proteins increase the cellular APP level, increase APPs release, and inhibit A␤ secretion [13, 15, 16, 27,28,29]

Summary

The abbreviations used are

A␤, ␤-amyloid peptide; APP, amyloid precursor protein; APPs, secreted N-terminal ectodomain of APP; APPs-␤, secreted ␤-secretase cleavage product of APP; APPsw, double missense mutation of APP (K651N/M652L, positions based on APP751) identified in a Swedish kindred of familial Alzheimer’s disease; GST, glutathione Stransferase; HEK293 cells, human embryonic kidney 293 cells; His, epitope tag with a string of six histidine residues; MID, Munc18a interacting domain; PDZ, conserved binding motif initially found in postsynaptic density-95 (PSD-95), Drosophila Disks-large (Dlg), and epithelial tight junction protein zona occludens-a (ZO-1); PTB, phosphotyrosinebinding domain; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonic acid; ANOVA, analysis of variance. A major pathway for A␤ production involves internalization of APP, as directed by an ENPTY sequence at the C terminus of APP, following its delivery to the plasma membrane [7]. This internalization motif has been found to interact with several phosphotyrosine-binding domain (PTB) containing proteins such as Fe65 and X11, phosphorylation within this motif is not required for binding (8 –12). The interaction between the PTB domains of Fe65 or X11 proteins and the C terminus of APP has been shown to effect the distribution and turnover of APP, and the secretion of A␤ [13,14,15,16]. The MID domain was sufficient and essential for a direct Munc18a interaction, we found that a portion of the synergistic effect was mediated via indirect interactions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION