Amyloid Precursor-like Protein 1 Influences Endocytosis and Proteolytic Processing of the Amyloid Precursor Protein

Abstract

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid ␤ peptide (A␤). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by ␣-secretase and slightly reduced ␤-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and thereby increases APP ␣-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for ␣-secretase cleavage.The amyloid precursor protein (APP) 2 is one of a large number of membrane proteins that are proteolytically converted to their soluble counterparts. This process is referred to as ectodomain shedding and is an important way of regulating the biological activity of membrane proteins (1, 2). The shedding of APP may occur through two different protease activities termed ␣-and ␤-secretase, which cleave APP within its ectodomain close to its transmembrane domain (for a review, see Ref.3) (see Fig. 1A). APP cleavage by ␣-or ␤-secretase is a key regulatory process in the generation of the amyloid ␤ peptide (A␤). Generation and subsequent deposition of A␤ are assumed to be the first events in the pathogenesis of Alzheimer disease. ␤-Secretase has been identified as the aspartyl protease BACE1 and cleaves APP at the N terminus of the A␤ peptide domain, thus catalyzing the first step in A␤ peptide generation (4). After the initial cleavage of APP by BACE1, the remaining C-terminal APP fragment is cleaved by ␥-secretase within its transmembrane domain at the C terminus of the A␤ domain, leading to the secretion of the A␤ peptide (5). In contrast to ␤-secretase, ␣-secretase cleaves within the A␤ sequence and thereby precludes the generation of the A␤ peptide. ␣-Secretase is a member of the ADAM family of proteases. Candidate ␣-secretases are ADAM10, ADAM17 (TACE), or ADAM9 (6). ␣-Secretase cleavage of APP is assumed to take place at or close to the cell surface, whereas ␤-secretase cleavage takes place after endocytosis of wild-type APP into endosomes. Endocytosis of APP requires t...

Subcellular localization and dimerization of APLP1 are strikingly different from APP and APLP2

“…By investigating the impact of APP:APLP coexpression on APP processing we observed no effect on α-secretase activity, as the sAPPα levels in the APPoverexpressing cells remained constant. This contradicts observations by Neumann et al, according to whom coexpression with APLP1 led to an increased shedding of APP by α-secretase (Neumann et al, 2006). Most likely these differences are due to the different cell types and different APP isoforms.…”

“…Besides the retention of APLP1, endocytosis may be influenced by heterointeraction as previously suggested by others (Neumann et al, 2006). However, this is less likely, as APP homodimerization was shown to occur already in the ER (Scheuermann et al, 2001), and cell surface levels of APLP1 are increased relating to total protein levels.…”

The Novel Membrane Protein TMEM59 Modulates Complex Glycosylation, Cell Surface Expression, and Secretion of the Amyloid Precursor Protein

“…GST-TMEM59-CT was cloned in pGEX5.1. Peak12/FLAG-TNF␣-HA, peak12/HA-SEAP, peak12/GFP (transfection control), and peak12/luciferase (negative control) have been described (29,36,39,40). The pcDNA3.1/wtPrP construct was cloned as described (41).…”

Ubiquilin 1 Modulates Amyloid Precursor Protein Trafficking and Aβ Secretion

“…HEK293-AP-APP cells overexpressing alkaline phosphatase (AP) and APP695 fusion protein and Bcl-XL/CrmA were grown as previously described (23). AP ectodomain was fused to the N terminus of full-length APP 695 lacking signal peptide (24). PS1/ PS2 double knock-out mouse embryonic fibroblasts (PSDKO-MEF) were maintained in OPTI-MEM media containing 10% fetal bovine serum.…”