APP dimer formation is initiated in the endoplasmic reticulum and differs between APP isoforms

Abstract

The amyloid precursor protein (APP) is part of a larger gene family, which has been found to form homo- or heterotypic complexes with its homologues, whereby the exact molecular mechanism and origin of dimer formation remains elusive. In order to assess the cellular location of dimerization, we have generated a cell culture model system in CHO-K1 cells, stably expressing human APP, harboring dilysine-based organelle sorting motifs [KKAA-endoplasmic reticulum (ER); KKFF-Golgi], accomplishing retention within early secretory compartments. We show that APP exists as disulfide-bonded dimers upon ER retention after it was isolated from cells, and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. In contrast, strong denaturing and reducing conditions, or deletion of the E1 domain, resulted in the disappearance of those dimers. Thus we provide first evidence that a fraction of APP can associate via intermolecular disulfide bonds, likely generated between cysteines located in the extracellular E1 domain. We particularly visualize APP dimerization itself and identified the ER as subcellular compartment of its origin using biochemical or split GFP approaches. Interestingly, we also found that minor amounts of SDS-resistant APP dimers were located to the cell surface, revealing that once generated in the oxidative environment of the ER, dimers remained stably associated during transport. In addition, we show that APP isoforms encompassing the Kunitz-type protease inhibitor (KPI) domain exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-mediated cell aggregation of Drosophila Schneider S2-cells was isoform independent. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER.

Palmitoylated APP Forms Dimers, Cleaved by BACE1

“…BiFC assays were performed as described by Isbert et al . [17]. APP-GFP(1–10) and APP-GFP(11) expression plasmids contained two non-fluorescence portions of GFP fused separately to the N-terminus of APP.…”

“…APP homodimerization initiates in the ER [17], but APP dimers are also found in the Golgi and in the cell surface [18–20]. Likewise, APP palmitoylation is initiated in the ER, but pal APP is detected in lipid rafts, which are cholesterol-rich microdomains in Golgi and post-Golgi compartments [4].…”

The Amyloid Precursor Protein Represses Expression of Acetylcholinesterase in Neuronal Cell Lines

“…Effects of Overexpression of APP 695 (⌬CuBD)-The E1 domain has been linked to a number of APP-interacting proteins and also as containing critical residues in mediating APP dimerization (36,45,46). Indeed, studies of the APP interactome frequently list APP/APLP (APP-like protein) as an interacting partner for APP itself (54).…”

“…Although the APP C terminus is the predominant region for proteinprotein interactions, other regions are also involved, e.g. via the extracellular E1 region with reelin (42), fibulin-1 (43), and integrin ␤1 (44,45) and also in dimerization of APP (46). Within the E1 domain, there are subdomains, including the His-rich copper-binding domain (CuBD) (36,47), which has an important role in mediating APP dimerization (48).…”

The physiological functions of the β-amyloid precursor protein APP

“…APP dimers in cis orientation originate in the endoplasmic reticulum (Isbert et al 2012), whereas APP dimers in trans orientation might form at the cell surface. Therefore, interactions in cis seem to aVect APP processing, whereas interactions in trans may promote cell adhesion.…”