Retention of APP in the ER leads to the formation of SDS-stable homodimers
Although many aspects of APP dimerization have been studied, the precise cellular localization of its generation remains unclear. To identify the compartment where APP dimerization is initiated, we first generated chimeric proteins, using APP isoform 695 with a C-terminal myc tag, followed by either a -Lys-Lys-Ala-Ala (KKAA) or -Lys-Lys-Phe-Phe (KKFF) dilysine signal, designated APP ER or APP Golgi as illustrated in Fig. 1a. It has been reported that the sequence context of dilysine signals determines the efficiency of ER retention, shown for CD4-receptor chimeras fused to either retention motif . Specifically, the KKAA chimeras were strictly localized to the ER, unlike KKFF constructs which shuttle between ER-and cis-Golgi compartment, due to the double phenylalanines (-FF) acting as an ER exit determinant [20–22]. We tested the oligomerization potential of APP trafficking mutants, both for KPI and non-KPI isoforms, in stably transfected CHO-K1 mass cultures, expressing comparable levels of the respective APP695 constructs. To determine whether the addition of the KKXX motifs to the cytoplasmic tail of APP would prevent the protein from being transported to the cell surface, we compared retention mutants to APP695 wt, by first employing surface biotinylation experiments (Fig. 1b). Protein expression was analyzed using SDS-PAGE and subsequent Western blotting of total cell lysates (Fig. 1b, left panel). In the biotinylation, no APP full length was detected for any of the retention mutants, indicating a strong intracellular retention mediated by double-lysine motifs (Fig. 1b right panel), whereas APP695 wt appeared in its fully glycosylated form at the plasma membrane (Fig. 1b right panel, APPfl mature). In the lysate controls, APP695 migrated as a set of two bands (~110–130 kDa) representing immature, mostly ER glycosylated APP, and the mature protein that has undergone complex O-glycosylation in the late-Golgi compartment. Surprisingly, for APP ER, two distinct migrating bands appeared in the lysate controls (Fig. 1b, left panel), of which the faster migrating band (~110 kDa) represents immature APP (before cis-Golgi-specific carbohydrate modifications) consistent with permanent ER retention. Additionally, we found an explicit slower migrating band for APP ER, co-migrating at the apparent molecular weight of a putative APP dimer (~220 kDa). This was particularly interesting, because in our experimental conditions we treated all lysate samples with loading buffer, containing SDS and β-mercaptoethanol (BME) prior to electrophoresis, but most importantly, the samples were not heat denatured at the same time (Fig. 1b, left panel, +BME/−95°C). Since the upper band did not disappear upon SDS treatment, we assume that stronger forces, other than hydrophobic associations, must be involved in APP dimer formation, such as intermolecular disulfide bonds. This notion was strengthened by the fact that retention of APP695 in the ER seemed to be sufficient to visualize SDS-stable homodimers, most likely generated via disulfide bonds between cysteine residues located in the E1 ectodomain. However, APP Golgi (containing the “leaky” KKFF motif) did not reveal an upper high-molecular-weight species around ~220 kDa, although it migrated exclusive as immature APP (Fig. 1b, Input) and was absent from the cell surface (Fig. 1b, PD NeutrAvidin, right panel), equivalent to APP ER. We hypothesize that APP bearing the ER exit determinant (-FF) shuttles permanently between the cis-Golgi compartment and the ER, resulting in less APP molecules at steady state in the ER, making it difficult to capture dimerization with our experimental conditions. However, this does not exclude that disulfide-associated APP dimers also exist in other compartments, but under normal cellular conditions this likely represents a sub-population of APP dimers, among others, held together by hydrophobic interactions, normally dissolved by the presence of SDS. Since it was recently proposed that APP can form dimers at the cell surface [9, 11, 19], we intended to include the plasma membrane as a putative source of dimerization. To do so, we transiently co-expressed a dominant negative (D/N) mutant of adaptor protein 180 (AP180) (Fig. 1b, right panel), inhibiting clathrin-mediated endocytosis [25, 26], thereby retaining APP at the plasma membrane and increasing its surface exposure. Indeed, mature APP wt was strongly increased at the cell surface and in the lysates controls upon co-expression of AP180 D/N (Fig. 1b left and right panel, lane four). Nevertheless, we failed to monitor any SDS-stable dimers when retaining APP at the surface, possibly because under normal cellular conditions the amount of covalent APP dimers reaching the surface is below detection limit on Western blots. Nevertheless, we demonstrate that stable APP dimer formation starts early in the secretory pathway, in the ER. Interestingly, when we reversed the experimental conditions and heat denatured the samples, without adding BME, we detected even higher-molecular-weight species, likely representing oligomerized APP (data not shown). These data indicate for the first time that the actual APP dimerization most probably occurs in the ER and that intermolecular disulfide bond(s) might be involved in APP dimerization. Furthermore, this reveals that APP probably exists in a monomer–dimer equilibrium, and that covalent disulfide bonds represent one mechanism of how APP dimerization might be achieved. Additionally, we employed two additional APP antibodies against the C-or N-terminus and confirmed the upper band as specific for APP (data not shown). To further assess the involvement of disulfide bonds in APP dimer formation, we applied stronger denaturing conditions to determine up to which temperatures the dimers remain stable in the presence of the reducing agent BME (Fig. 1c). We established a temperature curve, mixing equal amounts of lysates from APP ER overexpressing CHO-K1 cells with SDS sample buffer (5% BME final conc. in samples), prior to heat samples for 10 min at the indicated temperatures (4–85°C, Fig. 1c). Immunoblotting with 9E10 antibody revealed that dimers persisted up to 50°C, whereas higher temperatures led to a progressive decline, finally resulting in the total disappearance of the upper band at temperatures over 60°C. Quantification of immunoblots clearly showed a corresponding increase of ~50% in monomers vice versa (Fig. 1d). These results indicated that under standard detergent lysis conditions, APP forms disulfide-linked dimers which can only be fully disrupted by extended heating of protein samples in the presence of BME.
ER retention does not affect APP biosynthesis but increases its half-life
We next investigated whether it is possible to visualize the dimerization of unmodified wild-type APP695 (APP695 wt) without adding a C-terminal retention signal. To test this, we applied brefeldin A (BFA), a macrocyclic lactone antibiotic, inhibiting protein secretion at an early step in the secretory pathway [27–30]. It has been shown that BFA leads to rapid redistribution of the Golgi apparatus into the ER, inhibiting the anterograde movement of membrane traffic beyond the mixed ER/Golgi system, thereby retaining secretory and membrane proteins in the ER [27–30]. Cell cultures, overexpressing APP ER or APP wt were treated with 5 μg/ml BFA for different time periods (0–8 h) at 37°C, to block ER export. Subsequently, lysates were separated on a non-reducing SDS-PAGE, followed by immunoblotting with the APP specific polyclonal antibody CT15. Indeed, we found that addition of BFA to cell cultures caused the formation of an SDS-resistant high-molecular-weight band for APP wt, which co-migrated with the upper band of APP ER. A longer exposure of the same blot showed a beginning dimer formation already after 1 h treatment (Fig. 2a, longer exposure and Fig. 2b), with further increase after 8 h. Moreover, the difference between mature and immature APP becomes indistinct, due to the accumulation of particular immature APP in the ER. Interestingly, we also found a stabilizing BFA effect on pre-existing dimers, generated by APP ER (Fig. 2a, c), suggesting that even APP dimerization can be increased when ER exit was further inhibited by the application of BFA. However, the strong increase in monomers for APP ER was due to the employed antibody (CT15), also detecting the fraction of endogenous APP from CHO cells. In contrast, treatment of cell cultures, overexpressing APP ER, with increasing concentrations of BFA (10–30 μg/ml) for 1 h, led to an inverted monomer–dimer equilibrium (Fig. 2d–f) with a 50% decline in monomers, accompanied by a twofold increase in dimers (Fig. 2e, f). Strikingly, this effect was now visible with 9E10 antibody, exclusively detecting overexpressed APP, since endogenous APP from CHO cells obscured this previously (compare Fig. 2a). Nevertheless, this effect was highly reproducible and not dose-dependent. Taken together, these results implicated that not all APP molecules retained in the ER, have necessarily transformed into a dimeric state so far. Thus, extended exposure of APP to an oxidative surrounding had a considerable influence on the monomer–dimer balance, favoring dimerization. To investigate a potential influence of ER-retention on the half-life and biosynthesis of APP, we performed a pulse-chase analysis (Fig. 2g). Following a 15-min pulse with 150 μCi of [35S]-methionine/cysteine, we found no difference in the generation of immature APP between APP695 wt, versus APP695 ER (0 min). At time zero, APP consists predominantly of immature N-glycosylated species in both cell lines. However, fully mature APP appeared after 15 min exclusively in APP695 wt cells, and becomes stabilized after 30 min, thus reflecting traffic through the Golgi compartment. After a 1-h chase period, the APP wt level was dramatically reduced (Fig. 2h, half-life APP wt 45 min) and hardly detectable after 4 h. In contrast, cells overexpressing the AP ER retention mutant did not generate any O-glycosylated, mature APP during the chase period. However, turnover of APP ER is significantly prolonged, and ~50% of total APP can still be detected even after 5.5 h (Fig. 2h). Thus, the half-life of APP ER is more than sevenfold longer than in APP695 wt cells. Note that different chase time points for the APP ER mutant were chosen after an initial pulse-chase experiment already suggesting a prolonged half-life (data not shown). We also confirmed that retention in the ER severely limits APP metabolism, leading to decreased secretion of Aβ and APPsα/β into the culture medium (data not shown), since access to secretases is prevented [31, 32].
APP wild-type homodimers are initially formed in the ER but occur also at the cell surface
We have shown that retention of APP695 in the ER, either by a C-terminal ER retention motif, or blocking its export with BFA, led to the formation of SDS stable homodimers (Fig. 1). To verify that APP dimerization occurs early in the secretory pathway, we attempt to use a different approach. We employed a GFP tagging system for the intracellular detection of APP695, which is based on the auto-assembly capacity of two non-fluorescent portions of GFP, termed GFP 1–10 and GFP 11. When brought together by the association of two interacting partner proteins, individually fused to the fragments, they restore a fully fluorescent GFP, termed bimolecular fluorescence complementation (BiFC) . This enables us to visualize APP–APP interactions and their subcellular localization under physiological conditions . Therefore we fused the two complementary GFP fragments separately to the C-termini of HA-APP695  and co-expressed both constructs in neuroblastoma N2a cells (Fig. 3a). The green fluorescence in cells co-expressing the BiFC constructs clearly indicates an interaction of APP molecules under physiological conditions. We observed a punctate vesicular staining, possibly resembling Golgi or endosomal structures and also an ER staining, as indicated by co-staining with the ER marker protein Grp78 (overlay). These data corroborate, together with our data based on the ER retention signal, that APP dimerization occurs in the endoplasmic reticulum (ER) before its trafficking through the Golgi to the cell surface. Due to its rapid travel along the secretory pathway, we observed the same outcome on SDS-PAGE, only when we mediated APP retention in the ER (Figs. 1, 2). By contrast, no specific GFP fluorescence was detected when single halves of GFP fused to APP (APP-GFP 1–10 and APP-GFP 11) were individually expressed in N2a cells (Fig. 3b). To rule out that the non-detectable GFP signals in the negative controls might result from no expression of transfected plasmids, we performed immunostainings of HA-APP (Fig. 3b, right part), clearly revealing the presence of the APP fusion proteins. Moreover, this confirmed that the split GFP does not perturb the true APP localization in the cell, since expression patterns of negative controls were comparable to those of APP BiFC in the positive controls. Additionally, the expression levels of the APP-GFP fusion proteins in the positive and negative controls were also confirmed by Western blotting (Fig. 3c).These results suggest that APP dimers are initially formed in the ER. However, it cannot be ruled out that dimerization might occur at later stages of the transport. Having established that APP695 wt can form homodimers at an early stage in the secretory pathway, this does not tell us whether these dimers are caused by disulfide bonds or other interactions. We therefore wanted to show that disulfide bridged APP homodimers are in fact generated in the ER, but do also occur naturally in the cell. The APP ER retention construct has proven as a reliable tool to visualize stable dimer formation in the ER, nevertheless since it does not traffic beyond ER, we cannot use this construct to show the existence of disulfide linked APP dimers in other cellular compartments, e.g., at the plasma membrane. We employed a different approach to demonstrate that dimers generated in the ER can indeed reach the cell surface. We first slowed down anterograde ER-to-Golgi transport [27–30] by pre-treating cells overexpressing APP 695 wt with 10 μg/ml BFA for 1 h to allow efficient dimer formation in the ER (Fig. 3d). Next, cells were either directly lysed, or BFA was washed out and cultures were incubated for an additional 30 min at 37°C with medium containing chlorpromazine (CPZ, 10 μM). Chlorpromazine prevents assembly of the adaptor protein-2 (AP-2) on clathrin-coated pits and thus causes a loss of endocytosis . Subsequently, cells were lysed or optionally subjected to a surface biotinylation (Fig. 3e). As expected, upon BFA treatment APP disappeared from the cell surface, verified by biotinylation (Fig. 3e). Furthermore, we were able to trigger APP695 wt dimer formation, as seen previously (compare to Fig. 2a). In contrast, removal of BFA rapidly restored protein secretion from the ER to the Golgi and the cell surface [29, 30], and APP reappeared at the plasma membrane, established by CTF generation and fully glycosylated species, both in lysates and biotinylation experiments (Fig. 3d, e). Strikingly, chlorpromazine (CPZ) treatment led to a marked re-establishment of mature APP at the surface, compared to vehicle control (Fig. 3e), and BFA/CPZ treatment caused the formation of SDS-resistant high-molecular-weight species, migrating as a doublet (Fig. 3d). We assume that the slight size shift of the upper bands represents different glycosylated APP dimers, indicating transport along the secretory route. However, this higher “mature” dimer was absent in APP ER-or BFA-treated APP695 cells, suggesting that dimers were initially generated in the ER, but can be effectively transported through the secretory pathway, reaching the cell surface. This was particularly visible upon blocking endocytosis, thus preventing newly generated dimers from being instantly re-internalized. Note that heating the samples (+95°C) with BME resulted in the disappearance of the upper band(s), supporting our proposed model that APP dimers can associate via covalent intermolecular disulfide bonds.
APP homodimerization is dependent on the extracellular cysteine-rich E1 domain of the protein
Findings so far, by us and others, strongly argue for a mechanism that drives APP dimerization by its N-terminal cysteine-rich E1-domain [9, 11]. To further dissect the mechanisms by which APP homodimerization is accomplished and to exclude a potential interaction via its cytosolic part, we co-expressed C-terminal adaptor proteins in order to compete for binding, possibly perturbing or diminishing homodimerization (Fig. 4a). The intracellular domain of APP contains one YENPTY motif, which has been shown to interact with several adaptor proteins including FE65, X11, JIP, Shc, Tip60, and Dab1 [34, 35]. Specifically, we overexpressed increasing amounts of either Fe65 or X11α in CHO cells stably transfected with APP ER (Fig. 4a), thus providing a putative sterical hindrance for APP dimers. We observed a slight increase in APP monomers as well as dimers with co-expression of Fe65 and a more severe effect for X11α. This is consistent with previous findings that both adapter proteins stabilize either APP intracellular fragments or increase the half-life of full length APP [36, 37]. Additionally, we confirmed a direct binding of Fe65 to APP wt and APP ER by co-immunoprecipitation experiments and successfully co-purified equal amounts of flag-tagged Fe65 (data not shown). These results showed that Fe65 can interact efficiently with the APP ER mutant via its cytosolic exposed C-terminal part, while the APP N-terminus remains directed into the ER lumen where disulfide-bridged dimerization can occur. We next set out to ascertain that the ~220-kDa band indeed represented an APP homodimer and not any heterodimer of APP with another protein of the same putative molecular mass. Therefore, we generated an additional APP695 construct with a C-terminal HA tag and co-expressed APP695 HA wt or its ER retained counterpart (APP695 HA ER) in CHO cells, stably overexpressing APP695 myc ER (Fig. 4b). The overexpressed APP-myc and APP-HA fusion proteins were probed with mouse anti-myc or rat anti-HA primary antibodies vice versa on different immunoblots. In fact, we confirmed that the high-molecular-weight band represented APP homodimers, since Western blots with either myc-or HA-antibody, independently revealed a band at ~220 kDa in cells co-expressing HA-tagged and myc-tagged APP species. Individually, the APP HA and APP myc proteins migrate as distinct and separable bands under either reducing or non-reducing conditions (Fig. 4b). However, in the presence of reducing and denaturing conditions (+95°C), dimers were converted to monomers, which increased simultaneously (Fig. 4b, upper right blot). Interestingly, co-transfection of APP HA wt into cells already expressing APP myc ER revealed a slightly weaker band around ~220 kDa with HA antibody (Fig. 4b, bottom blot, third lane), in addition to the doublet representing both maturation states of APP. This band most likely represents a dimer composed of APP myc ER and APP HA wt, suggesting that APP695 ER could retain a portion of wt APP within the ER, due to homodimer formation. The most feasible interpretation of our approach is that these high-molecular-weight bands represent APP myc/APP HA “heterodimers”, indicating that APP forms homodimers within the same cellular compartment (cis-dimers). Finally, these findings also minimized the possibility that APP happens to “heterodimerize” with any other unrelated protein of the same molecular weight. Moreover, this is in agreement with our BiFC approach (Fig. 3), already demonstrating a specific interaction of two APP695 molecules, by restoring a functional fluorescent GFP. It has been shown by co-immunoprecipitation experiments and FRET analysis that deletion of the E1 domain impedes APP homodimerization [9, 11] as well as heterointeraction with APLPs [9, 11]. Accordingly, we hypothesized that APP lacking the cysteine-bearing E1 domain is no longer capable of dimer formation in the ER. This region of APP encompasses the growth factor-like domain (GFLD) and a copper-binding domain (CuBD) [38, 39], which contain together 12 cysteine residues, possibly promoting stable covalently linked dimers or higher multimers. We generated CHO-K1 cell lines, overexpressing APP695 deletion constructs, lacking the N-terminal growth factor-like and copper-binding domains (∆E1), thereby omitting all of the cysteines and compared them with cells expressing the corresponding full-length constructs (Fig. 4b). Indeed, the KKAA motif particularly retained APP∆E1 ER in the cell, confirmed by surface biotinylation (Fig. 4c, right panel) and the lack of Golgi-specific carbohydrate modifications (Fig 4 c, left panel). Strikingly, the generated deletion constructs were incapable of forming stable high-molecular-weight species, regardless of ER retention or not (Fig. 4c), indicating that the E1 domain is essential for generating SDS-resistant APP homodimers (compare illustration in Fig. 4e). Note that less mature APP∆E1 was detected at the surface compared to APP695 wt, likely due to less free primary amino groups for biotin attachment, present in the N-terminal truncated APP construct. It has been reported that APP contains a region within the E1 domain, encompassing residues 99–110, with an active heparin binding capacity  that might form a loop, stabilized by a disulfide bond between Cys-98 and Cys-105. Based on findings that a synthetic peptide corresponding to the loop residues was able to interfere with APP dimerization , we generated an ER-retained APP loop double mutant exchanging Cys-98 and Cys-105 for serine residues (APP ER 98-105ser). We compared cell extracts from CHO-K1 cells transiently transfected with the serine mutant to lysates of our stably expressing APP 695 wt and APP695 ER cells (Fig. 4d). Western-blot analysis clearly demonstrated that the APP ER 98-105ser mutant failed to form homodimers in an oxidizing environment when sulfhydryl groups were exchanged by hydroxyl groups, compared to APP ER, which contains the respective cysteine residues. Thus, we conclude that these cysteines located within the APP heparin-binding site are sufficient to mediate APP homointeraction, and that binding of two N-terminal domains of APP was entirely dependent on the oxidation of the thiol groups. However, at this point we cannot exclude the possibility that cysteine mutants other than those analyzed would have had similar effects on APP dimer formation. Nevertheless, this indicates for the first time that cysteines, located within the E1 domain, might also contribute to cis-dimerization between two adjacent APP molecules, via intermolecular disulfide bonds, as well as stabilizing the APP structure via intramolecular S–S bonds (see illustration in Fig. 4e). We confirmed the necessity of the E1 domain for APP N-terminal interactions, by generating APP truncated at the β-secretase site , C99 wt or C99 ER (data not shown). Thereby the entire extracellular part, including the central E2 domain, was absent and Western-blot analysis revealed no SDS-stable dimers for C99 wt or the C99 ER.
Heterointeraction between APP and its homologue APLP1 is initiated and increased in the ER, depending on the APP E1 domain
Various experimental approaches have demonstrated that APP is capable of forming heterodimers with its homologues, APLP1 and APLP2 [9, 11], both intracellular in cis- and intercellular in trans-manner . Since we could define the ER as the cellular compartment providing ideal conditions for triggering APP695 homointeractions via disulfide-bridge formation, we investigated whether this mechanism would also contribute to heterodimerization between APP and APLP1. As a first biochemical approach, we performed a cell surface biotinylation in our CHO-K1 cells stably overexpressing either APP695 wt or APP ER, transiently co-transfected the human APLP1 cDNA (Fig. 5a). Co-expression of APLP1 resulted in a significant reduction of APP695 ER homodimers (Fig. 5a, upper panel, lane 4) representing a decrease of more than 60% (Fig. 5b), accompanied by the simultaneous formation of APP/APLP1 heterocomplexes in the ER (Fig. 5a lower panel, lane 4 and Fig. 5c). At the same time, we monitored an increase of ~50% in APP monomers (Fig. 5b), indicating that a portion of APP homodimers was converted back to monomers, whereas the rest appeared as heterodimers with APLP1. Despite equal expression of APLP1 in both cell lines (Fig. 5a, bottom panel), we found less mature APLP1 at the surface when co-expressed with APP695 ER (Fig. 5a, bottom panel, lane 2 and 4), corresponding to a 50% reduction when compared to APP695 wt (Fig. 5d, bottom panel, lane 2 and 4). This indicates that the interaction of APP and APLP1 occurs already in the ER, and that APP can potentially retain a portion of APLP1 in the ER as a consequence of forming stable heterocomplexes. Interestingly, we found less C-terminal fragments for APLP1 when co-expressed with APP ER, suggesting impaired generation due to ER retention. Note that only fully mature proteins appear at the cell surface whereas secretase derived metabolites (CTFs) are only visible in the lysate controls of APP695 wt and APLP1. To provide further evidence for the interaction between APP and its homologue APLP1, we performed co-immunoprecipitation studies in our stable CHO-K1 cells upon co-expression of human APLP1 (Fig. 6). Lysate controls and co-purified proteins (IP APLP1 or APP) clearly indicate a strong interaction between both proteins in the ER. However, APP homodimerization in the ER is reduced upon APLP1 co-expression (Fig. 6b lysate WB 9E10, lane 4), compared to APP695 ER alone (compare Fig. 6b). At the same time, we observed again the formation of a distinct higher-molecular-weight band for APLP1 in cell lysates, only when co-expressed with APP695 ER, as seen previously (Fig. 5a, bottom blot). In line with the biotinylation approach (Fig. 5a), this band most likely represents APP/APLP1 heterocomplexes, since it co-migrates with the size of APP homodimers (~220 kDa) and both homologues have about the same molecular mass. Furthermore, specific interaction between both proteins was confirmed in APP-and APLP1-directed co-immunoprecipitation experiments (Fig. 6, lower panels). Most interestingly, the amount of co-purified proteins (immature form) was increased when APP was retained in the ER. Taken together, our findings clearly demonstrate a heterocomplex formation between APP and APLP1 as early as in the ER, most likely via disulfide-bond formation between the respective cysteine-bearing E1 domains of both proteins.
Intracellular cis-dimerization of APP770 in the ER is diminished by the KPI domain, but can be induced by a constitutive cysteine bond at the transmembrane domain
All studies investigating APP dimerization so far have used exclusively the neuronal APP isoform 695 or APLP1 [9, 11, 13, 17]. However, most tissues and glial cells express APP with an additional KPI domain, a 57-amino-acid insert, showing homology to the Kunitz family of serine protease inhibitors . Therefore, we wanted to explore whether APP harboring an additional KPI domain in its extracellular part would display the same dimerization pattern as seen for APP695, since it possesses six further cysteine residues, additional to 12 located within the E1 domain. We generated CHO-K1 cells with stable overexpression of APP770 wt or the respective ER retention mutant (APP770 ER), or we transiently overexpressed APP 751 in CHO-K1 cells to include all different isoforms (Fig. 7a). Surprisingly, when we compared lysates of cells expressing APP695 ER to cells transfected with the longer isoforms APP770 ER and APP751 ER, we did not observe an explicit slower migrating band representing SDS-stable APP-homodimers (Fig. 7a). However, APP770 ER and APP751 ER also appear as single bands representing immature APP, whereas the fully glycosylated form was absent. Moreover, the absence of CTFs confirmed ER retention of all APP isoforms (Fig. 7a, bottom panel) compared to wild-type constructs. Additionally, we generated APP of all three isoforms with a C-terminal HA tag and transiently overexpressed them in a different cell line (HEK 293T) to assure the previous result. Indeed, we found no dimerization for the KPI domain containing isoforms upon ER retention (data not shown). These findings raised the possibility that the KPI domain might hinder cis-directed lateral homodimerization of APP within the cell, possibly due to different intramolecular folding, providing a sterical obstruction. We next wanted to investigate whether it is possible to force lateral cis-dimerization of APP770 in our cellular system. Therefore, we introduced a single cysteine substitution at the APP juxtamembrane region, previously described by Scheuermann and colleagues, to obtain covalently stabilized dimers through an intermolecular disulfide bond . Since substitution of the basic lysine at position 28 of Aβ numbering by a cysteine residue (K28C) was shown to be the most effective mutant in stabilizing APP695 dimers , we generated APP K28C mutants of both isoforms. After transfection of native CHO-K1 cells with the cysteine mutants, we detected a slower migrating band at ~220 kDa for both APP695 K28C and APP770 K28C under non-reducing conditions (Fig. 7b), co-migrating at the same molecular weight as dimers from APP695 ER. Most interestingly, this band disappeared after boiling the samples with β-mercaptoethanol (Fig. 7b, +BME, +95°C), as seen for APP695 ER dimers. These findings clearly indicated that dimerization of APP retained in the ER is mediated by disulfide-bond formation, since linking of two thiol groups could only be generated through oxidation in the ER. Furthermore, we demonstrated that it is possible to force lateral cis-dimerization of APP770. The K28C mutation seems to be potent enough to overcome a putative sterical hindrance of the KPI domain, by cross-linking two apposed transmembrane domains of APP770. Next, we questioned whether the artificial disulfide bond, potentially generated in the ER, might affect APP trafficking and processing through the secretory pathway. It has been reported previously by Eggert and colleagues that cysteine substitution at position 28 of Aβ caused a significant decrease in soluble cleavage products such as APPsα/β and Aβ-peptides . We therefore analyzed surface levels of APP695 K28C by surface biotinylation (Fig. 7c). Indeed, we could clearly show that the APP K28C mutant reached the plasma membrane comparable to APP695 wt (Fig. 7c, right panel), whereas trafficking of our ER retention mutant was efficiently inhibited. We therefore conclude that dimerization itself does not necessarily interfere with APP trafficking, nor seems dimerization a prerequisite for plasma membrane localization.
Trans-dimerization at the cell surface is independent of the APP isoform
As we have shown before, APP770 containing a KPI domain displayed a weakened tendency for intracellular cis-dimerization upon ER retention (Fig. 7a) compared to the neuronal isoform 695. However, in line with previous findings [9, 11], we have verified the E1-domain as the major dimerization interface of two APP molecules (Fig. 4). Consequently, we predicted that the presence of the KPI-domain should not affect intercellular trans-dimerization properties of APP770 (see illustration in Fig. 8d). To compare the homophilic trans-cellular dimerization properties of KPI-containing isoforms versus APP695, we employed a well-established cell aggregation assay using Drosophila Schneider (S2) cells, which lack endogenous expression of the APP homologue APPL . We have previously shown that expression of APP695 and APLPs in different pools of S2 cells caused significant clustering after mixing of the two cell populations, whereas cells expressing only GFP did not co-aggregate with APP/APLP-expressing cells . Thus, cell clustering provides a direct readout for the specific interaction of the two proteins, whereas unspecific interaction of APP/APLPs with endogenous S2 cell proteins was excluded . Therefore, we transfected S2 cells with cDNAs encoding all human APP isoforms (695, 751, 770), or APP695∆E1 and analyzed aggregation via immunocytochemical analysis, as shown in Fig. 8a. Despite a low transfection efficiency (15%), we observed almost equal clustering ratios for cells expressing either APP isoform (Fig. 8b), whereas cells expressing APP695∆E1 did not cluster specifically (Fig 8a, b), similar to untransfected S2 cells. Furthermore, confocal microscopy revealed accumulated APP immunoreactivity at cell–cell contact sites of homotypic clusters (Fig. 8c), in line with previously published data . Together, these results demonstrate that cell–cell interaction by ectodomain-mediated adhesion is independent of the presence of a KPI domain. However, APP dimerization specifically depends on E1 domain associations at both the cell surface and within the cell (compare to Fig. 4). In our studies, only APP695 was able to form lateral intracellular (in the ER) as well as intercellular trans-dimers (at the cell surface). However, the KPI domain of the longer isoforms hindered lateral cis-interaction but not homophilic trans-interaction (Fig. 8d, schematic drawing), because the E1 domains of two molecules can still dimerize.