Homophilic Interactions of the Amyloid Precursor Protein (APP) Ectodomain Are Regulated by the Loop Region and Affect β-Secretase Cleavage of APP

Abstract

We found previously by fluorescence resonance energy transfer experiments that amyloid precursor protein (APP) homodimerizes in living cells. APP homodimerization is likely to be mediated by two sites of the ectodomain and a third site within the transmembrane sequence of APP. We have now investigated the role of the N-terminal growth factor-like domain in APP dimerization by NMR, biochemical, and cell biological approaches. Under nonreducing conditions, the N-terminal domain of APP formed SDSlabile and SDS-stable complexes. The presence of SDS was sufficient to convert native APP dimers entirely into monomers. Addition of an excess of a synthetic peptide (APP residues 91-116) containing the disulfide bridge-stabilized loop inhibited crosslinking of pre-existing SDS-labile APP ectodomain dimers. Surface plasmon resonance analysis revealed that this peptide specifically bound to the N-terminal domain of APP and that binding was entirely dependent on the oxidation of the thiol groups. By solution-state NMR we detected small chemical shift changes indicating that the loop peptide interacted with a large protein surface rather than binding to a defined pocket. Finally, we studied the effect of the loop peptide added to the medium of living cells. Whereas the levels of ␣-secretory APP increased, soluble ␤-cleaved APP levels decreased. Because A␤40 and A␤42 decreased to similar levels as soluble ␤-cleaved APP, we conclude either that ␤-secretase binding to APP was impaired or that the peptide allosterically affected APP processing. We suggest that APP acquires a loop-mediated homodimeric state that is further stabilized by interactions of hydrophobic residues of neighboring domains.The amyloid precursor protein (APP) 3 of Alzheimer disease is one of the best studied type I cell surface glycoproteins with a proposed regulatory role in cell adhesion in the peripheral and central nervous system (1). APP is physiologically processed by either ␣-or ␤-secretases, which release the soluble ectodomain (sAPP␣ or sAPP␤) from the cell surface (2-5). Alternatively, the membrane-bound fragment APP-C-terminal fragment, which is derived from ␤-secretase cleavage, is further processed by the ␥-secretase into the cytoplasmic domain of APP and amyloid ␤-peptides (A␤) of varying lengths (6 -9). Recently, elucidation of the ␥-secretase cleavage mechanism has revealed that a conserved GXXXG motif in the transmembrane sequence of APP mediates homophilic helix-helix interactions and has an important role in the processing of A␤40/42 into shorter A␤ species, i.e. A␤38, A␤37, A␤35, and A␤34 (7).Unraveling structural features of APP by NMR spectroscopy and x-ray crystallography has led to the identification of specific regions that function as subdomains (see Fig. 1A). The extracellular N-terminal domain of APP has been dissected into the E1 region consisting of the N-terminal growth factor-like domain (GFLD) followed by the copper-binding domain (CuBD) (10). An acidic region (residues 190 -264) precedes the carbohydrate domain, which contains...

Structure and biochemical analysis of the heparin-induced E1 dimer of the amyloid precursor protein

“…This points to a possible divergence in structure and function also between vertebrate and nonvertebrate APPs. Altogether, different interdomain interaction properties of distinct APP family proteins could be an explanation for functional differences of these proteins (31,37).…”

“…The ordered dimerization of APP has also been implicated in cell-cell interactions (29). It has been reported that dimerization depends on zinc binding (18,30), heparin binding (16), or the simultaneous interaction of several sites within the APP molecule (31) including its transmembrane domain (29) and that it is functionally associated with the concurrent dimerization of the β-site APP cleaving enzyme (28,(31)(32)(33). Recently, Gralle et al corroborated the physiological importance of APP dimerization using single molecule FRET methods (27).…”

Amyloid beta 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization

“…Recently, we and others have shown that APP forms dimers via at least three dimerization sites: two different sites in the ectodomain (21,22) and a third dimerization site formed by three consecutive GxxxG motifs as part of the APP-TMS (5). Here, we found that sulindac sulfide and derived compounds are able to reduce helix-helix interaction of the third dimerization site in a native bacterial membrane.…”

Role of Amyloid- Glycine 33 in Oligomerization, Toxicity, and Neuronal Plasticity

“…Peptides were synthesized as described previously (Beyermann et al, 1996;Kaden et al, 2008) and verified by matrix-associated laser desorption ionization-mass spectrometry (MALDI-MS; Ultraflex-II TOF/TOF, Bruker Daltonics). Briefly, peptides were synthesized at automated synthesizer (ABI 433a; Applied Biosystems) on Trt-Tentagel resin [Rapp-Polymere (0.25 mmol/g, 0.5 g) using N-(9-fluorenyl)methoxycarbonyl(Fmoc)-strategy (double couplings with 9 equivalents of Fmoc-aa/2-(1H-benzotriazole-1-yl)-1,1,3,3-tetra-methyluronium hexafluorophosphate/6 equivalents of N, N-diisopropylethylamine].…”