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J. Biol. Chem., Vol. 281, Issue 46, 35176-35185, November 17, 2006
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1
From the
Department of Neuroscience and the ¶Department of Neurology, Georgetown University Medical Center, Washington, D. C. 20057-1464 and Neurogenetics Branch, NINDS Porter Neuroscience Research Center, National Institutes of Health, Bethesda, Maryland 20892-3705
Received for publication, March 7, 2006 , and in revised form, July 6, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, affect amyloid precursor protein (APP) processing and A
production. Dab1 is another adaptor protein that interacts with APP as well as with members of the apoE receptor family. We examined the effect of Dab1 on APP and apoEr2 processing in transfected cells and primary neurons. Dab1 interacted with APP and apoEr2 and increased levels of their secreted extracellular domains and their cytoplasmic C-terminal fragments. These effects depended on the NPXY domains of APP and apoEr2 and on the phosphotyrosine binding domain of Dab1 but did not depend on phosphorylation of Dab1. Dab1 decreased the levels of APP -C-terminal fragment and secreted A. Full-length Dab1 or its phosphotyrosine binding domain alone increased surface levels of APP, as determined by surface protein biotinylation and live cell staining. A ligand for apoEr2, the extracellular matrix protein Reelin, significantly increased the interaction of apoEr2 with Dab1. Surprisingly, we also found that Reelin treatment significantly increased the interaction of APP and Dab1. Moreover, Reelin treatment increased cleavage of APP and apoEr2 and decreased production of the -C-terminal fragment of APP and A. Together, these data suggest that Dab1 alters trafficking and processing of APP and apoEr2, and this effect is influenced by extracellular ligands. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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peptide, found in plaques of Alzheimer disease. The cytoplasmic domain of APP contains an NPTY sequence that serves as a binding motif for adaptor proteins that possess a phosphotyrosine binding domain (PTB), such as members of the Fe65, X11, JIP, and Dab protein families. Such adaptor proteins play critical roles in tyrosine kinase-mediated signal transduction, protein trafficking and localization, phagocytosis, cell fate determination, and neuronal development (1). Most importantly for Alzheimer disease, interactions between these cytoplasmic proteins and APP also lead to altered processing of APP.
The effects of several of these adaptor proteins on APP trafficking and processing have been studied. The Fe65 family (FE65, FE65L1 (2), and FE65L2 (3)) is expressed at high levels in neurons (4). Co-expression of FE65 and APP promotes secreted APP and A secretion in H4 cells and Madin-Darby canine kidney cells (2). However, co-expression of FE65 and APP in HEK293 cells stabilizes immature APP and inhibits APPs formation and A secretion (5). The X11 family (X11,-, and -
; also called Mint1, -2, and -3) are important in neuronal synaptic function (6). X11 slows cellular APP processing and reduces A40 and A42 secretion, perhaps through preventing trafficking of APP to subcellular compartments containing active -secretase (7). X11 reduces A levels and amyloid plaque formation in the brains of transgenic APP mice (8). Similar to X11 and -, JIP-1b interaction with APP stabilizes immature APP and inhibits APPs, A40, and A42 secretion in vitro (9).
In contrast, the effects of the Dab family (Dab1 and Dab2) on APP processing are less well studied. Dab1 is important for nervous system development in mammals (10). Dab1 and Dab2 possess a PTB domain and have been shown to interact with the cytoplasmic domains of APP and apoE receptors (apoEr2, VLDLr, and low density lipoprotein receptor-related protein) (11, 12). The extracellular domains of apoEr2 and VLDLr interact with extracellular matrix protein Reelin during neuronal positioning (13). Reelin induces Dab1 tyrosine phosphorylation (13). If any of these components (Dab1, apoE receptors, or Reelin) is knocked out, a similar phenotype of impaired neuronal migration is observed.
In this study, we demonstrate that Dab1 increases cell surface expression of APP and apoEr2, increases -cleavage of APP and apoEr2, and decreases APP -CTF and A in transfected cells and in primary neurons. We found that Reelin treatment significantly increases co-immunoprecipitation of Dab1 with both APP and apoEr2. Moreover, Reelin, like Dab1, increases cleavage of APP and apoEr2 and decreases production of the -CTF of APP and A.
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MATERIALS AND METHODS |
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Cell Lines and Culture Conditions—COS7 and HEK293 were maintained in Opti-MEM (Invitrogen) with 10% fetal bovine serum (FBS, Invitrogen) in a 5% CO2 incubator. COS7 cells were transiently transfected with 0.5-1 µg of plasmid in FuGENE 6 (Roche Applied Science) according to the manufacturer's protocol and cultured 24 h in Dulbecco's modified Eagle's medium containing 10% FBS. For co-transfections, cells were similarly transfected with 0.5-1 µg of each plasmid in FuGENE 6 (Roche Applied Science) and cultured for 24 h in Dulbecco's modified Eagle's medium with 10% FBS. After 24 h the cells were transferred to Opti-MEM serum-free media (Invitrogen) and treated with indicated compounds. The Reelin-conditioned and control media were prepared and concentrated 10 times as described (15). Conditioned media were incubated at a 1:10 dilution in serum-free media on primary neuronal cells for 30 min.
Antibodies—We used antibodies anti-HA (Abcam), anti-FLAG (Sigma), anti-c-Myc (Abcam), and anti-Dab1 (from Dr. Andre Goffinet). Antibody 5810 (against the N terminus of apoEr2) was the kind gift of Dr. Uwe Beffert. For analysis of APP, we used 6E10 (identifying sAPP) (Signet), 22C11 (Chemicon), and c1/6.1 (recognizing the C terminus of APP), provided by Dr. Anne Cataldo.
Quantification of APP and ApoEr2 Proteolytic Fragments— Secreted fragments were identified by Western blot analysis of conditioned media (sAPP, 6E10 antibody; sapoEr2, 5810 antibody). CTF were measured by Western blots of cell lysates (APP -CTF, C1/6.1; APP CTF, 6E10; apoEr2 CTF, HA antibodies). A40 and A42 levels in the conditioned media were determined by ELISA, using 1A10 (anti-A40) or 1C3 (anti-A42) as capture antibodies and 12B2, which recognizes both mouse and human A as the detection antibody (Immuno-Biological Laboratories) (16).
Primary Neuronal Cell Culture—Primary mouse embryonic cortical neurons were prepared from embryonic day 16 (E16) Swiss-Webster mice as described (17). Brain cortices were chopped and trypsinized for 10 min at 37 °C. After trypsinization, 0.4 µg/ml trypsin inhibitor, 0.025% DNase, and 12 mM MgSO4 were added and mixed until tissue was thoroughly homogenized. Cells were then transferred to Neurobasal medium containing B27 serum supplement, 1 mM glutamine, gentamycin, and Ara-C. Neurons (2 x 106 cells) were seeded on 50 µg/ml poly-D-lysine-coated 12-well tissue culture plates. For analysis of the APP-Dab1 interaction, cells were seeded on 35-mm tissue culture plates. Cells at 10 days in vitro were treated with either control or Reelin-containing medium for 30 min. For Dab1 knock-out primary neurons, brain cortices from mouse E16 embryos of Dab1+/- crosses were dissociated separately, and neurons were seeded from each embryo into the pre-coated 24-well tissue cultures. For experiments on the effects of Dab1, neurons derived from individual embryos were used as follows: Dab1+/+ (n = 12), Dab1+/- (n = 9), and Dab1-/- (n = 8).
Reelin Treatment of APPsw-TG Primary Neurons—APPsw-TG mice (Tg2576) were obtained from Taconic. APPsw-TG (E16.5) cortices were dissected in Hanks' balanced saline solution (HBSS) and were dissociated with 0.25% trypsin at 37 °C for 15 min. Cells were washed twice with HBSS and dissociated further with DNase with trypsin inhibitors (Roche Applied Science) with a glass pipette. The dissociated cells were washed again in HBSS twice and resuspended in Neurobasal defined media (Invitrogen/Invitrogen) with 1% penicillin/streptomycin/glutamine (Invitrogen) and 4% B-27 supplement. Primary neurons were plated on poly-L-lysine-coated 12-well dishes and grown at 37 °C for 4-5 days. The media were changed for all neurons 1 h before stimulation, and then primary neurons were treated with either control or Reelin-conditioned media (10x) for 18 h. The supernatants were collected, and the levels of secreted A40 were measured by ELISA (BIOSOURCE). Wild-type (n = 3) and APPsw-TG (n = 3) cortices were used, and three cultures from each animal were treated with Reelin or control media.
Co-immunoprecipitations—Transfected COS7 cells were washed with PBS and lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 0.15 M NaCl, 1% Nonidet P-40, phosphatase inhibitors (Sigma), and protease inhibitors (Roche Applied Science). For immunoprecipitation, the lysates were incubated for 2 h at 4 °C with the anti-HA antibody or 6E10 antibody bound to protein G-Sepharose beads (Amersham Biosciences). The precipitates were then washed three times with lysis buffer and resuspended in SDS sample buffer. The samples were separated by SDS-PAGE on 4-15% polyacrylamide gels, transferred electrophoretically to nitrocellulose membranes, and blocked with 5% nonfat dry milk. The blots were incubated with antibodies at room temperature for 1 h. Horseradish peroxidase-conjugated secondary antibodies were visualized by ECL detection system and exposed to film.
Biotin Labeling of Cell Surface Proteins—COS7 cells were transiently transfected with APP and Dab1 constructs or apoEr2 and Dab1 constructs in FuGENE 6 (Roche Applied Science) and cultured for 24 h in Dulbecco's modified Eagle's medium containing 10% FBS. After 24 h, cells were washed twice with PBS, and surface proteins were labeled with Sulfo-NHS-SS-Biotin (Pierce) in PBS under gentle shaking at 4 °C for 30 min. 50 µl of quenching solution was added to cells at 4 °C, which were washed twice with TBS. Cells were collected in 500 µl of lysis buffer, disrupted by sonication on ice, incubated for 30 min on ice, and clarified by centrifugation (10,000 x g, 2 min). To isolate biotin-labeled proteins, lysate was added to immobilized NeutrAvidinTM gel (50 µl) and incubated 1 h at room temperature. Gels were washed five times with wash buffer and incubated 1 h with SDS-PAGE sample buffer, including 50 mM dithiothreitol. Eluants were analyzed by immunoblotting.
Live Cell Surface Staining—Transfected HEK293 cells were washed with PBS and incubated with primary antibody diluted in ECS (containing 145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, 5 mM glucose, 15 mM sucrose, 0.25 mg/l phenol red, and 10 µM D-serine (all from Sigma), adjusted to pH 7.4 with NaOH) for 8 min at room temperature. Cells were washed three times in ECS and then incubated with 22C11 antibody for 8 min, washed with ECS, and incubated with Alexa Fluor 488 (Molecular Probes) goat anti-mouse antibody for 8 min at room temperature. Final washes were carried out with ECS. After fixing the cells with 4% paraformaldehyde, cells were imaged on an Axioskop FS microscope (Zeiss, Germany) equipped with a x63, 0.9 NA, Achroplan, water-immersion objective or with a Nikon E600 microscope (Nikon, Japan) equipped with a x60, 1.0 NA.
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-galactosidase plasmid (to normalize transfection efficiency). Luciferase activity from cell lysates was determined in triplicate using the luciferase assay kit (Promega) by VICTOR2 (PerkinElmer Life Sciences). Results were normalized to -galactosidase expression levels by using a-galactosidase enzyme assay kit (Promega).
Statistical Analyses—Experiments were repeated a minimum of four times unless otherwise noted. All data were analyzed using analysis of variance with Graphpad Prism 4 software, using Tukey's multiple comparison test for post hoc analyses with significance determined as p < 0.05. Student's t test was performed on A measures from Reelin-treated primary neurons. Descriptive statistics were calculated with StatView 4.1 and displayed as an expressed mean ± S.E.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We tested whether the interaction of Dab1 with apoEr2 affected processing of apoEr2. ApoEr2 is cleaved similarly to APP, with an -secretase-like activity generating a secreted form of the receptor and a membrane-bound CTF (14). In addition, -secretase activity cleaves the apoEr2 CTF (14). COS7 cells were transfected with full-length apoEr2 alone or apoEr2 with Dab1 constructs lacking the C terminus of Dab1 or lacking the PTB domain. Secreted apoEr2 was measured in conditioned media, and apoEr2 CTF was measured in cell lysates. Full-length Dab1 significantly increased secreted apoEr2 and apoEr2 CTF (Fig. 1C). Dab1 expression did not alter cell apoEr2 levels (Fig. 1C). We also found that the PTB domain of Dab1 increased secreted apoEr2 and apoEr2 CTF (Fig. 1D). The PTB domain of Dab1 expression also slightly increased cell apoEr2 levels (Fig. 1D). Because Dab1 interacts with apoEr2 through its PTB domain (19), we hypothesized that constructs of Dab1 lacking the PTB domain would not affect apoEr2 processing. We found that PTB deletion construct of Dab1 did not affect sapoEr2 or apoEr2 CTF levels (Fig. 1E). Thus, Dab1 interactions with apoEr2 promote the cleavage of apoEr2, and this effect depends on the Dab1 PTB domain.
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Dab1 Interacts with APP in COS7 Cells and Affects Its Processing— Dab1 was reported to interact with APP by glutathione S-transferase pulldown, yeast two-hybrid, co-immunoprecipitation, and x-ray crystallography (11). We tested whether Dab1 had a similar effect on APP processing as it had for apoEr2. We transfected COS7 cells with APP and the Dab1 PTB domain, immunoprecipitated Dab1 PTB with the anti-FLAG antibody, and probed for APP with the anti-Myc antibody. APP co-precipitated with the PTB domain of Dab1 but did not precipitate in the absence of Dab1 (Fig. 2A, upper panel). We made similar observations with full-length Dab1 (data not shown). Western blot analysis of COS7 cell extracts confirmed similar levels of cell APP and Dab1 across transfections (Fig. 2A, middle and lower panels).
We tested whether the interaction of Dab1 with APP affected processing of APP in COS7 cells. Expression of full-length Dab1 significantly increased secreted APP and APP -CTF levels but significantly decreased APP -CTF levels (Fig. 2B). Thus, Dab1 co-expression promoted the -cleavage of APP and decreased its -cleavage. We also found that the PTB domain lacking the C terminus of Dab1 increased secreted APP and APP CTF (Fig. 2C). The PTB domain lacking the C terminus of Dab1 expression slightly increased cell APP levels (Fig. 2C). We also transfected COS7 cells with APP and the PTB deletion construct of Dab1, and we measured the amount of secreted APP and APP CTF. The PTB deletion construct of Dab1 did not affect levels of any APP proteolytic product as follows: secreted APP, APP -CTF, or APP -CTF (Fig. 2D).
To test the nature of soluble and cell-associated protein bands, we transfected APP and measured the cell-associated APP and secreted APP. We found that the band for cell-associated APP is slightly higher than that for sAPP (Fig. 2F). Finally, because Dab1 affected APP processing, we examined whether it affected A production in COS7 cells. Full-length Dab1 significantly decreased secreted A40 levels (by 68%) and A42 levels (by 44%) (Fig. 2E).
APP-Dab1 Interactions Do Not Alter APP or Dab1 Phosphorylation—Tyrosine phosphorylation of Dab1 is important for its signal-transducing effects in development (20). To test whether the interaction of Dab1 with APP affects Dab1 phosphorylation, we expressed APP with Dab1 in COS7 cells as above. We included a form of Dab1 in which its five tyrosine residues were replaced with phenylalanine (5F) as a negative control. As a positive control, we also included a constitutively active form of the kinase Fyn, which phosphorylates both Dab1 and APP (21). We immunoprecipitated Dab1 and probed for phosphotyrosine. Both full-length and mutant 5F Dab1 did not show anti-phosphotyrosine immunoreactivity (Fig. 3A). However, co-transfection with Fyn significantly increased anti-phosphotyrosine Dab1 immunoreactivity (Fig. 3A, 1st panel). We also performed the reverse experiment, immunoprecipitating with anti-phosphotyrosine antibody and probing with anti-Dab1 antibody (Fig. 3A, 2nd panel). Co-transfection with Fyn significantly increased anti-Dab1 immunoreactivity, but no phospho-Dab1 was observed in the presence of APP alone. To measure the expression levels of Dab1, we immunoprecipitated with an anti-Dab1 antibody, and we probed with a second anti-Dab1 antibody (Fig. 3A, 3rd panel). Strong Dab1 and Dab1 5F immunoreactivity were observed in all the transfected samples. Finally, we measured Fyn and APP levels in transfected cells via immunoblot to demonstrate their expression (Fig. 3A, 4th and 5th panels).
We similarly asked whether the interaction of Dab1 with APP affected APP phosphorylation, using the same cells. We immunoprecipitated APP with an anti-c1/6.1 antibody, and probed for phosphotyrosine. COS7 cells transfected with Fyn had measurable levels of phospho-APP (Fig. 3B, 5th lane, 1st panel), but expression of full-length Dab1 or Dab1 5F constructs did not result in phospho-APP. Levels of full-length Dab1 and Dab1 5F were similar and levels of APP were consistent across transfections (Fig. 3B, lower panels). These results suggest that unphosphorylated Dab1 is capable of binding APP and altering its processing without affecting its tyrosine phosphorylation.
Dab1 Affects APP and ApoEr2 Processing via Their NPXY Motifs—To test whether the NPTY motif of APP was important in the effect of Dab1 on processing of APP (as expected), we used a construct of APP where the NPTY sequence was changed to APTY. We transfected COS7 cells with full-length Dab1 and wild-type or mutant APP, and we measured the amount of APP CTF and secreted APP. Levels of cellular APP, APP CTF, and sAPP were similar in cells transfected with wild-type APP versus mutant APP (Fig. 4A, 1st and 3rd lanes). Dab1 co-expression increased APP CTF and sAPP from wild-type APP (Fig. 4A, 2nd lane). However, Dab1 co-expression did not alter levels of mutant APP CTF or sAPP (Fig. 4A, 4th lane). Levels of full-length cell-associated APP were not altered by Dab1 co-expression (Fig. 4A, middle panel).
To similarly test whether the NPVY motif of apoEr2 was important in the effect of Dab1 on processing of apoEr2, we generated a construct of apoEr2 where the NPVY sequence was changed to APVY. We transfected COS7 cells with full-length Dab1 and wild-type or mutant apoEr2, and we measured the amount of apoEr2 CTF and secreted apoEr2. Levels of cellular apoEr2, apoEr2 CTF, and sapoEr2 were similar in cells transfected with wild-type apoEr2 versus mutant apoEr2 (Fig. 4B, 1st and 3rd lanes). Dab1 co-expression increased apoEr2 CTF and sapoEr2 from wild-type apoEr2 (Fig. 4B, 2nd lane), but Dab1 co-expression did not alter the levels of mutant apoEr2 CTF or sapoEr2 (Fig. 4B, 4th lane). Levels of total cell-associated apoEr2 were not altered by Dab1 co-expression. Thus, the NPXY sequences were important to the effects of Dab1 on both APP and apoEr2 processing.
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As another measure of cell surface levels, we immunostained live cells. HEK293 cells were transfected with APP and either the PTB domain of Dab1, the PTB deletion construct of Dab1, or the full-length Dab1. We found that the PTB domain and full-length Dab1, which interact with APP, increased cell surface expression of APP compared with the PTB truncated Dab1, which does not interact with APP (Fig. 5C). Thus, both assays demonstrated that Dab1 constructs containing the PTB domain increased surface levels of APP.
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-secretase cleavage of APP CTF, the APP intracellular domain (AICD). We used an Fe65-dependent APP luciferase transactivation assay to assess AICD in the presence of a Dab1 deletion mutant or full-length Dab1. HEK293 cells were co-transfected with APP-Gal4, Fe65, a luciferase reporter construct (G5E1B-Luc), and Dab1 constructs. Full-length Dab1 resulted in a 75% reduction in Fe65-dependent transactivation. However, the deletion mutant of Dab1 without the PTB domain did not affect the production of AICD (Fig. 6). Levels of Fe65 were similar among the transfected cells (Fig. 6, lower panel).
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We similarly asked whether the APP and Dab1 interaction was increased by Reelin. We co-expressed full-length APP with Dab1 and treated cells with control or Reelin-containing media for 24 h. As with apoEr2, Reelin significantly increased co-precipitation of Dab1 with APP (Fig. 7C). The levels of APP and Dab1 did not vary across conditions (Fig. 7C). Quantification of Western blots demonstrated that APP/Dab1 co-precipitation was increased by 124% after Reelin treatment. It is unknown how the extracellular Reelin protein affected the intracellular interactions between Dab1 and apoEr2 or APP.
We tested whether Reelin affected the interaction between endogenous APP and Dab1 in primary neurons. We immunoprecipitated APP with antibody 22C11 and probed for Dab1 with an anti-Dab1 antibody. As expected, endogenous Dab1 co-precipitated with APP (Fig. 7D). Reelin treatment increased this co-precipitation but did not affect total levels of APP or Dab1 (Fig. 7D). We also immunoprecipitated Dab1 with an anti-Dab1 antibody and probed for APP with 22C11. Endogenous APP consistently co-precipitated with Dab1, and Reelin treatment increased this co-precipitation (but did not affect total levels of APP or Dab1) (Fig. 7D). Quantification of Western blots demonstrated that Reelin treatment increased the APP/Dab1 interaction by 97%.
Reelin Affects APP and ApoEr2 Processing—The data above demonstrate that Reelin increased the interaction between apoEr2 and Dab1 and between APP and Dab1. Therefore, we hypothesized that Reelin might also increase processing of apoEr2 and APP, similar to our findings with Dab1 (Figs. 1 and 2). To address this hypothesis, we treated COS7 cells transfected with apoEr2 and Dab1 with Reelin-containing medium. Compared with control medium, Reelin treatment increased the levels of secreted apoEr2 and apoEr2 CTF, without affecting total levels of apoEr2 (Fig. 8A).
In order to assess whether Reelin affected APP processing in the presence of Dab1, we treated COS7 cells transfected with APP and Dab1 with Reelin-containing medium. We found that Reelin also increased the levels of secreted APP and APP CTF, without affecting total levels of APP (Fig. 8B). We also treated COS7 cells transfected with APP and the PTB domain of Dab1 with Reelin-containing medium. We found that Reelin increased the levels of secreted APP and APP CTF (Fig. 8C). Levels of full-length APP also appeared higher in the Dab1 PTB-containing cells.
Because Reelin affected APP processing, we examined whether it affected A production. COS7 cells transfected with APP and Dab1 were treated with Reelin-containing medium. We observed that Reelin significantly decreased secreted levels of both A40 (by 72%) and A42 (by 60%) (Fig. 9A). We also tested whether the PTB domain of Dab1 affected A production. COS 7 cells transfected with APP and the PTB domain of Dab1 were treated with Reelin-containing medium. We observed that Dab1 PTB decreased A40 (by 61%), but Reelin treatment did not lead to a further significant reduction in A (data not shown).
In addition, we examined whether Reelin affected the processing of APP in neurons from APPsw-transgenic mice. Primary cortical neurons were isolated from APPsw-tg and wild-type littermates. After 18 h of Reelin treatment, the levels of secreted A40 collected from the culture media of APPsw-tg neurons were significantly lowered (by 32%) compared with littermate controls (Fig. 9B).
Finally, we assessed the levels of endogenous mouse A in primary neurons from wild-type, DAB+/-, or DAB-/- embryos (Fig. 9, C and D). The DAB1 knock-out phenotype was confirmed by PCR (Fig. 9C). Primary neurons from DAB-/- mice had significantly higher A40 and A42 levels compared with neurons from littermate controls, supporting the hypothesis that Dab1 is important for reducing A production (Fig. 9, C and D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cleavage of APP and decreased -cleavage and a decrease in the levels of A40 and A42 (Fig. 2). No changes in total APP or apoEr2 were observed, suggesting that only a small percentage of APP is cleaved under these conditions. Knock-out of endogenous Dab1 in primary neurons resulted in increased levels of A40 and A42 (Fig. 8).
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-cleavage. However, in contrast to our results, they observed increased APP -CTF and A levels (27) in the presence of Dab1. Cell-line specific differences have been observed for the effects of FE65 on APP processing (3, 5, 28), an observation we have confirmed in COS7 and Chinese hamster ovary cells (29). Cytoplasmic adaptor proteins will affect trafficking and processing of full-length APP as well as APP CTFs, and thus there may be differences in cell lines dependent on which adaptor proteins are endogenously present. In support of this model, we have found that Dab1 competes with Fe65 for interaction with AICD (Fig. 6). Our data in primary neurons and mouse brains suggest that the effect of Dab1 in neurons is similar to what we observed in transfected COS7 cells. Adaptor proteins that interact with cytoplasmic NPXY sequences differ in their preference for binding sequences that have or lack phosphorylation of the tyrosine residue. Disruption of the NPXY motif in APP or apoEr2 prevented the effects of Dab1 on trafficking and processing (Fig. 4). Immunoprecipitation of Dab1 did not co-precipitate a phosphorylated version of APP (Fig. 3), consistent with findings that Dab1 binds preferentially to nonphosphorylated NPXY sequences (13, 20). Thus, phosphorylation of APP or apoEr2 may regulate whether these molecules interact with Dab1 or with other adaptor proteins.
APP and apoEr2 are linked extracellularly by binding different domains of F-spondin (30); the presence of F-spondin increases the interaction between APP and apoEr2 (31). As extracellular interactions cluster the proteins together in the membrane, the intracellular domains interact, and the adaptor proteins bound to the intracellular domains interact. Because Dab1 interacts with both APP and apoEr2 through its PTB domain, these proteins may compete for binding a Dab1 molecule. In contrast, the adaptor protein FE65 binds APP and apoEr2 through two separate PTB domains, forming a tripartite complex (29), similar to a complex demonstrated for APP, FE65, and another apoE receptor, low density lipoprotein receptor-related protein (32). Dab1 and FE65 both bind the NPXY sequences of APP and apoEr2, and thus Dab1 could prevent the FE65 intracellular link between APP and apoEr2.
It is unknown how Dab1 transduces the signal from extracellular ligands binding to receptors such as apoEr2. Dab1 is phosphorylated when Reelin binds to apoEr2, potentially affecting downstream functions. In this study, we did not observe any changes in Dab1 phosphorylation in the presence of APP or apoEr2 (Fig. 3) or after treatment of COS7 cells with Reelin (data not shown). We did observe that Reelin increased the co-immunoprecipitation of Dab1 with apoEr2 or APP (Fig. 7) and affected their trafficking and processing (Fig. 8). Thus, we hypothesize that binding of proteins to the extracellular domains of APP or apoEr2 may alter the trafficking of these transmembrane proteins and favor the binding of Dab1 to their cytoplasmic domains. Reelin alone reduced A levels in the presence of Dab1 (Fig. 9) but did not affect A levels in the presence of only the PTB1 domain of Dab1, supporting this hypothesis. We are currently examining whether binding of other ligands of APP or apoEr2 (such as F-spondin or apoE isoforms) affects the interaction of Dab1 with these cell surface proteins.
Many of the interacting molecules examined in this study are important for migration of neurons in the cortex. Reelin and Dab1 knock-out mice show neuronal migration defects (33), similar to deficits seen in apoEr2 and VLDLr knock-out mice (12). Disrupting APP and its two mammalian homologues, APLP1 and APLP2, is generally lethal, but the rare surviving embryos show cortical dysplasias (34), suggesting that the APP protein family members may also have important functions in neuronal migration. Finally, an Fe65/Fe65L1 double knock-out mouse model also demonstrated cortical dysplasias and axonal projection deficiencies (35). Further research into this complex of proteins may provide insight into the mechanisms necessary for neuronal migration.
The data described in this study demonstrate an interaction between Dab1 and APP and apoEr2, altering processing of both of these transmembrane proteins. These effects are promoted by the presence of Reelin, suggesting that extracellular factors alter intracellular interactions important for protein trafficking and processing. These findings underscore a potentially important role for APP and associated proteins in neuronal migration.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Dept. of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, D. C. 20057-1464. Tel.: 202-687-1534; Fax: 202-687-0617; E-mail: gwr2{at}georgetown.edu.
2 The abbreviations used are: APP, amyloid precursor protein; apoE, apolipoprotein E; apoEr2, apoE receptor 2; PTB, phosphotyrosine binding; VLDLr, very low density lipoprotein receptor; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; PBS, phosphate-buffered saline; HBSS, Hanks' balanced saline solution; AICD, APP intracellular domain; sapo, secreted apo; sAPP, secreted APP; CTF, C-terminal fragment; ECS, extracellular solution.
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ACKNOWLEDGMENTS |
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REFERENCES |
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PTB); 1-555, full-length Dab1(FL). B, COS7 cells were transiently transfected with apoEr2 (HA-tagged) and Dab1 PTB domain. Cell lysates (200 µg) were immunoprecipitated (IP) with anti-FLAG, and the precipitate was probed with anti-HA. ApoEr2 co-precipitated with constructs containing PTB domain of Dab1. Lysates from co-transfected cells (20 µg) were probed for apoEr2 or Dab1 to demonstrate expression levels (lower panel). Lane v, vector alone. C, COS7 cells were transiently transfected with apoEr2 with constructs of full-length Dab1. Secreted apoEr2 was measured in conditioned media with antibody 5810, and apoEr2 CTFs were detected from cell lysates with anti-HA antibody. Dab1 increased secreted apoEr2 and apoEr2 CTF. D, COS7 cells were transiently transfected with apoEr2 (HA-tagged) and Dab1 PTB. The PTB domain of Dab1 increased secreted apoEr2 and apoEr2 CTF. E, COS7 cells were transiently transfected with apoEr2 and Dab1 
