hFE65L Influences Amyloid Precursor Protein ... - Wiley Online Library

Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry

hFE65L Influences Amyloid Precursor Protein Maturation and Secretion Suzanne Y. Gueńette, Jing Chen, Amber Ferland, Christian Haass, Anja Capell, and Rudolph E. Tanzi Genetics and Aging Unit and Department of Neurology, Massachusetts General Hospital East and Harvard Medical School, Charlestown, Massachusetts, U.S.A.

Abstract: The amyloid precursor protein (APP) is processed in the secretory and endocytic pathways, where both the neuroprotective ␣-secretase-derived secreted APP (APPs␣) and the Alzheimer’s disease-associated ␤-amyloid peptide are generated. All three members of the FE65 protein family bind the cytoplasmic domain of APP, which contains two sorting signals, YTS and YENPTY. We show here that binding of APP to the Cterminal phosphotyrosine interaction domain of hFE65L requires an intact YENPTY clathrin-coated pit internalization sequence. To study the effects of the hFE65L/APP interaction on APP trafficking and processing, we performed pulse/chase experiments and examined APP maturation and secretion in an H4 neuroglioma cell line inducible for expression of the hFE65L protein. Pulse/ chase analysis of endogenous APP in these cells showed that the ratio of mature to total cellular APP increased after the induction of hFE65L. We also observed a threefold increase in the amount of APPs␣ recovered from conditioned media of cells overexpressing hFE65L compared with uninduced controls. The effect of hFE65L on the levels of APPs␣ secreted is due neither to a simple increase in the steady-state levels of APP nor to activation of the protein kinase C-regulated APP secretion pathway. We conclude that the effect of hFE65L on APP processing is due to altered trafficking of APP as it transits through the secretory pathway. Key Words: FE65L protein—Amyloid precursor protein—Binding—Processing—Secretion—Maturation. J. Neurochem. 73, 985–993 (1999).

secretory and endocytic pathways, and it is now well established that events that modulate APP trafficking have an impact on APP processing and ultimately on A␤ production. For example, truncation of the APP carboxy terminus or deletion of the YENPTY sequence attenuates A␤ production and increases APP ectodomain [secreted APP (APPs)] secretion (Haass et al., 1993; Koo and Squazzo, 1994). These effects on APP processing have been attributed to a decrease in the amount of APP reinternalized in the endocytic pathway (Koo et al., 1996). APPs secretion is a constitutive process that can be stimulated by phorbol esters, interleukin-1, cholinergic agonists, and neuronal activity (Buxbaum et al., 1992; Nitsch et al., 1996). APPs enhances cell adhesion (Saitoh et al., 1989; Schubert et al., 1989) and neurite outgrowth (LeBlanc et al., 1992; Koo et al., 1993; Allinquant et al., 1995) and increases the number of presynaptic structures in the rat brain cortex (Roch et al., 1994). APPs also has a protective effect: It decreases neuronal vulnerability to the excitatory amino acid glutamate via K⫹ channel activation (Mattson et al., 1993; Schubert and Behl, 1993). Yet, very little is known about the intracellular molecular components modulating constitutive APPs release and APP trafficking. The FE65 and X11 proteins are good candidate molecules for proteins that modulate APP trafficking because they bind the carboxy-terminal region of APP. Recent studies have shown that overexpression of X11 decreases ␣-secretase-

Alzheimer’s disease (AD), a progressive neurodegenerative disorder of the CNS, is characterized by intracellular neurofibrillary tangles and extracellular deposits of amyloid. Amyloid is largely composed of the 39 – 43amino acid ␤-amyloid peptide, A␤, that is derived from the amyloid precursor protein (APP). To date, it is unclear whether A␤ contributes to AD pathogenesis or is simply a marker for neuronal dysfunction in AD. However, enhanced A␤ production is clearly associated with familial AD mutations in APP and the presenilin genes (Citron et al., 1992; Cai et al., 1993; Suzuki et al., 1994; Scheuner et al., 1996). A␤ generation occurs in both the

Received February 19, 1999; revised manuscript received April 29, 1999; accepted April 30, 1999. Address correspondence and reprint requests to Dr. S. Y. Gueńette at Department of Neurology, Massachusetts General Hospital East and Harvard Medical School, 149 13th Street, Charlestown, MA 02129, U.S.A. Abbreviations used: A␤, ␤-amyloid peptide; AD, Alzheimer’s disease; APLP2, amyloid precursor-like protein 2; APP, amyloid precursor protein; APPs, secreted amyloid precursor protein; APPs␣, ␣-secretase-derived secreted amyloid precursor protein; BFA, brefeldin A; HA, hemagglutinin; HEK, human embryonic kidney; PDBU, phorbol 12,13-dibutyrate; PI, phosphotyrosine interaction; PID, phosphotyrosine interaction domain; PKC, protein kinase C; STS, staurosporine.

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derived APPs (APPs␣) and leads to retention of cellular APP (Borg et al., 1998; Sastre et al., 1998). We reported the identification of hFE65L as a protein that interacts with the carboxy-terminal region of APP and amyloid precursor-like protein 2 (APLP2) (Gueńette et al., 1996). hFE65L is one of three proteins comprising the human FE65 family of proteins: hFE65, hFE65L, and hFE65L2 (Bressler et al., 1996; Gueńette et al., 1996). The heterologous rat FE65 gene was originally cloned as a cDNA that is mainly expressed in brain and was described as having homology to the DNA binding domain of retroviral integrases (Duilio et al., 1991). The subsequent identification of two phosphotyrosine interaction (PI) domains (PIDs) in FE65 (Bork and Margolis, 1995) provided the theoretical means by which the interaction of FE65 with the APP carboxy terminus was plausible. The PID protein–protein interaction domain had been shown to mediate the binding of the mitogenic protein Shc to NPXYp motifs in growth factor receptors (Blaikie et al., 1994). Furthermore, the C-terminal 203 amino acids of hFE65L containing PID2 was shown to coprecipitate endogenous APP and APLP2 (Gueńette et al., 1996). In addition, enrichment of APP from PC12 cell extracts by incubation with beads coated with glutathione S-transferase–FE65PID fusion protein of the heterologous rat FE65 had previously been reported (Fiore et al., 1995). Fusion proteins of the rat and mouse FE65 proteins, including rat FE65L2, also bind the carboxy terminus of APP (Borg et al., 1996; Bressler et al., 1996; Duilio et al., 1998). The 43 carboxy-terminal amino acids of APP extend into the cytoplasmic domain of the cell and contain a clathrin-coated pit internalization sequence, YENPTY, that targets cell surface APP to the endocytic compartment (Lai et al., 1995). It is interesting that the binding of rat FE65 and X11 to APP is abrogated by the Y682G substitution within the 682YENPTY687 sequence of APP (Borg et al., 1996). Furthermore, phosphorylation of tyrosine residues in the YENPTY sequence of APP has not been detected and is not required for binding of FE65 to the APP carboxy terminus (Borg et al., 1996; Zambrano et al., 1997). Taken together, these results suggest that the interaction of hFE65L with the YENPTY sequence in the carboxy terminus of APP may modulate APP processing. In this study, we show that overexpression of the cytosolic protein hFE65L enhances APP maturation and increases APP secretion. Furthermore, we show that the effect of hFE65L on APP secretion does not appear to be via activation of protein kinase C (PKC). MATERIALS AND METHODS Antibodies and cDNA constructs Commercially available polyclonal rabbit antisera (Berkeley Antibody Co.) and monoclonal antibody (Boehringer Mannheim) specific for the hemagglutinin (HA) epitope were used for western blot analysis and immunoprecipitations of the HA– hFE65L fusion protein. The APP antibodies used were monoclonal antibody 22C11 (Weidemann et al., 1989), 6E10 monoclonal antibody raised against A␤1–17 (Kim et al., 1990) J. Neurochem., Vol. 73, No. 3, 1999

(Senetek), 369W antisera directed against APP645– 694 (Buxbaum et al., 1990), and 1G7/5A3 (Koo and Squazzo, 1994). Transferrin receptor immunoprecipitations were obtained with a commercially available monoclonal antibody (Zymed Laboratories). Finally, an anti-␣-tubulin antibody (Sigma Chemical Co.) was used as a control for relative protein loads on total protein extracts obtained from H4 neuroglioma cells. The HA-FE65L plasmid is a derivative of pUHD10-3 (Gossen and Bujard, 1992). An HA epitope tag was introduced into the hFE65L gene by annealing the phosphorylated oligonucleotides 5⬘-TACCCATACGACGTCCCAGACTACGCTAGCCTC-3⬘ and 5⬘-GAGGCTAGCGTAGTCTGGGACGTCGTATGGGTA-3⬘ and ligating the annealed products to either HpaI-digested or PmlI-digested and phosphatase-treated hFE65L plasmid. The resulting plasmids were sequenced to confirm the in frame insertion of YPYDVPDYAM. HA-tagged hFE65L fusion protein was detectable only in cells transfected with the HA tag inserted at the HpaI site.

Cell culture and generation of cell lines The carboxy-terminal 203 amino acids of hFE65L encoded by the pCMV4-2A plasmid was expressed in human embryonic kidney (HEK) 293 cell lines by transient transfection using Lipofectamine (BRL) as previously described (Gueńette et al., 1996). The HEK293 cell lines used are stable cell lines overexpressing APP695 (a gift from Dennis Selkoe), APPY653A, APPY682A, or APPY687A (produced by Anja Cappell and Christian Haass). Establishment of the H4 neuroglioma lines for tetracycline-repressible HA– hFE65L expression was previously described (Gueńette et al., 1999). The results shown below are those obtained for clone 32-6, which expresses the highest level of HA– hFE65L. However, we obtained similar results with two additional clones that expressed a lower level of HA– hFE65L, clones 15-2 and 32-9 (data not shown). The brefeldin A (BFA) treatment was performed by culturing H4 neuroglioma 32-6 cells in the presence or absence of tetracycline for 72 h and treating with 10 ␮g/ml BFA in dimethyl sulfoxide for 1 h at 37°C. The phorbol ester phorbol 12,13dibutyrate (PDBU; 1 ␮M) and staurosporine (STS; 10 nM) treatments were performed during the 2-h chase period.

Immunofluorescence staining and crude subcellular fractionation HA– hFE65L staining was performed on cells induced for HA– hFE65L synthesis for 48 h. The primary antibody used is the monoclonal anti-HA antibody 12CA5 (Boehringer Mannheim) and fluorescein isothiocyanate-goat anti-mouse immunoglobulin (BioSource International). A crude fractionation protocol using differential centrifugation (Graham, 1993) was used to determine the subcellular localization of the HA– hFE65L protein.

Metabolic labeling and immunoprecipitations The H4 neuroglioma clones expressing HA– hFE65L were grown to confluency in either the presence or absence of tetracycline on six 100-mm-diameter culture dishes. Cells were trypsinized, resuspended in the respective culture medium, pelleted by centrifugation (1,000 rpm for 10 min), resuspended in 1 ml of methionine-free Dulbecco’s modified Eagle’s medium, and incubated for 20 min at 37°C. Cells were pelleted by brief centrifugation, resuspended in 1 ml of methionine-free Dulbecco’s modified Eagle’s medium containing 1 mCi of [35S]methionine (⬎1,000 Ci/mmol; NEN Research Products), and labeled for 20 min at 37°C. The chase period was initiated using Dulbecco’s modified Eagle’s medium containing a five-

hFE65L OVEREXPRESSION ENHANCES APP PROCESSING fold excess of unlabeled methionine after washing the cells once with methionine-free medium. Portions (1 ml) of each labeling reaction were aliquoted in six microfuge tubes at the beginning of the chase period and incubated for various times at 37°C. Cell extract preparation, immunoprecipitations, and western blot analyses were performed as previously described (Gueńette et al., 1996) with the exception that immune complexes were collected with either protein A or goat anti-mouse IgG magnetic beads (Perseptive Diagnostics). Chase media were collected and treated as described by Caporaso et al. (1992). APP was immunoprecipitated from total cell lysate protein (100 ␮g) using the 369W antisera. APPs was immunoprecipitated using either 6E10 or 1G7/5A3 antibodies from chase medium volumes normalized using total cell lysate protein values estimated by the BCA protein assay (Pierce). Immunoprecipitates were separated on sodium dodecyl sulfatepolyacrylamide gradient gels (4 –20%), and the gels were dried with or without treatment with Autofluor (National Diagnostics). The 35S-APP signal was quantified by phosphorimage analysis using the Bio-Rad model GS-525 Molecular Imager.

Enrichment of APPs by anion exchange chromatography APPs was enriched from conditioned medium obtained from cells uninduced or induced for synthesis of HA– hFE65L for 72 h using MacroPrep High Q chromatography as previously described (Moir et al., 1998). In brief, cell extracts were incubated with MacroPrep High Q resin in 50 mM Tris-HCl (pH 7.4) and 350 mM NaCl, beads were washed three times using the same buffer to remove unbound proteins, and bound proteins were eluted with 50 mM Tris-HCl (pH 7.4) and 1.2 M NaCl. Cell lysates corresponding to each of the conditioned medium samples were also collected. APPs␣ and cellular APP were detected by western blot analysis of the MacroPrep High Q eluates and equal amounts of total protein from cell lysates (50 ␮g of protein) using the 6E10 antibody. The 6E10 signals for APPs␣ and cellular APP were quantitated using SuperSignal Ultra (Pierce) and capture of the chemiluminescent signal using the Bio-Rad Fluor-S MultiImager. The APPs␣/total APP values represent the amount of secreted APP over the sum of secreted and cellular APP. The APPs␣/total APP values were derived from the pixel values obtained for known medium and cell lysate volumes. The pixel value was adjusted for total cell lysate and conditioned medium volumes and normalized to total cellular APP for the 0 chase time point of the uninduced cell lysate.

RESULTS hFE65L interaction with APP is abolished by Y682A and Y687A mutations in APP695 Immunoprecipitation experiments were performed following transient transfection of an HA-tagged PID fusion of hFE65L [pCMV4-2A (Gueńette et al., 1996)] into HEK293 cells stably overexpressing wild-type APP695 or APP695 mutant alleles bearing Y to A substitutions at amino acid residues 653, 682, and 687 within the cytoplasmic domain of APP. The pCMV4-2A insert represents the hFE65L 203 C-terminal amino acids originally isolated from the interaction trap human fetal brain cDNA library (Gueńette et al., 1996). Because HEK293 cells express APP751, we could distinguish the isoform patterns of endogenous APP from the wild-type

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FIG. 1. Tyrosine mutations in the APP carboxy terminus prevent binding of hFE65L. A: Western blot (W) analysis of immunoprecipitates (IP) obtained with an anti-HA antibody directed against the HA– hFE65L PID2 fusion protein. HEK293 and HEK293 cells stably transfected with APP695 and the Y682A, Y687A, and Y653A mutant alleles of APP695 were transiently transfected with the C-terminal 203 amino acids of hFE65L fused to an HA epitope (pCMV4-2A) or the backbone plasmid (pCDNA3). The APP695 and the endogenous APP751 isoforms were detected with the 22C11 antibody. B: Western blot analysis of total protein using the 22C11 antibody. HEK293 cells predominantly express the Kunitz protease inhibitor isoforms of APP (Kuentzel et al., 1993), which migrate more slowly in sodium dodecyl sulfate– polyacrylamide gel electrophoresis than the corresponding APP695 isoforms found in the HEK293 cells stably transfected with wild-type and mutant alleles of APP695.

and mutant APP695 isoform pattern. Thus, coimmunoprecipitation of the exogenous APP695 could be distinguished from the endogenous APP751 by the APP isoform pattern. Our results showed that Y682A and Y687A mutations abolished the binding of APP to the hFE65L PID2 (Fig. 1A). In contrast, the Y653A mutation had no effect on the binding of the 4-2A hFE65L fusion protein to APP. The HEK293 cell lines expressing APP695 or mutant alleles of APP695 all produced the APP695 isoforms at levels equal to or greater than the endogenous APP751 (Fig. 1B). These results are in agreement with a study showing that FE65, a homologue of hFE65L, also fails to bind APP bearing a Y682G mutation (Borg et al., 1996). hFE65L localizes to cytoplasm of the cell To determine the subcellular localization of HA– hFE65L in H4 neuroglioma cells, we performed immunofluorescence staining of cells induced for 48 h using an antibody directed against the HA epitope. Our results indicated that the hFE65L protein is concentrated either at the periphery of the cell or around the nucleus (Fig. 2A). Cells expressing higher levels of the protein, i.e., at 72 h of induction, showed a more general cytoplasmic staining (data not shown), which suggests that the staining pattern of HA– hFE65L in cells induced for 48 h may be due to higher cytosolic J. Neurochem., Vol. 73, No. 3, 1999

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FIG. 2. Localization of HA– hFE65L in H4 neuroglioma cells. A: Tetracycline-repressible H4 neuroglioma cells stably transfected with an HA– hFE65L construct were cultured in the absence of tetracycline for 48 h. Left panel: Phase image. Right panel: HA– hFE65L was visualized by indirect immunofluorescence using an anti-HA antibody. B: Western blot analysis of the HA– hFE65L protein for fractions obtained from tetracycline-repressible H4 neuroglioma hFE65L cells cultured in the absence of tetracycline for 72 h. Fractionation was performed by differential centrifugation, and 80 ␮g of protein was loaded for each fraction. T, total protein; S1 and P1, 3,000-g supernatant and pellet, respectively; S2 and P2, 27,000-g supernatant and pellet, respectively; S3 and P3, 100,000-g supernatant and pellet, respectively.

volumes in the perinuclear region and the cell periphery. To confirm that hFE65L is a cytosolic protein, we performed a crude fractionation procedure to separate membrane-associated proteins from the cytosol. Crude subcellular fractionation of H4 neuroglioma cells grown in tetracycline-free medium for 72 h was performed using differential centrifugation (Graham, 1993). Western blot analysis using an anti-HA antibody (Fig. 2B) showed that hFE65L is located in the high-speed supernatant (S3), indicative of a cytosolic protein, in agreement with the cytoplasmic staining pattern of the HA– hFE65L protein observed by immunofluorescence staining of induced cells.

Steady-state levels of APP are unaffected by overexpression of the APP C-terminal interacting protein hFE65L To study the consequence of the hFE65L/APP interaction on the cellular levels of APP, we induced the 80-kDa HA– hFE65L fusion protein at 24-h intervals over 72 h. We found that the HA– hFE65L fusion protein continues to accumulate over a 72-h period while being absent from cell lysates obtained from uninduced cells (Fig. 3A). Because hFE65L was initially isolated as a protein that interacts with APP, we examined the effect of hFE65L overexpression on the total steady-state amount of full-length APP per unit protein. We found no

FIG. 3. hFE65L overexpression has no effect on APP steady-state levels. A: Crude protein extracts (50 ␮g) obtained from the HA– hFE65-inducible H4 cells (clone 32-6) grown in either the presence or absence of tetracycline for 24, 48, or 72 h were separated on a 4 –20% sodium dodecyl sulfate-polyacrylamide gradient gel, transferred to a polyvinylidene difluoride membrane, and western blotted (W) with rabbit anti-HA antibody (top panel), 369W (middle panel), and anti-␣-tubulin antibody (lower panel). B: Western blot analyses using the 6E10 antibody of two sets of crude protein extracts (50 ␮g) obtained from the HA– hFE65L-inducible H4 cells (clone 32-6) either uninduced or induced for HA– hFE65L for 72 h. The signal was detected with SuperSignal Ultra (Pierce) and reprobed with the anti-HA antibody to show induction of HA– hFE65L. C: Levels of APP isoforms in cellular extracts of H4 cells uninduced (Unind.) and induced for hFE65L synthesis over a 72-h period. imm., immature; mat., mature. Pixel values were obtained by capturing the chemiluminescent signal with a Bio-Rad Fluor-S MultiImager. Data are average ⫾ SD (bars) values of 10 independent experiments with p values obtained from t test analyses. SDs are for total cellular APP.

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FIG. 4. The 80-kDa HA– hFE65L fusion protein coimmunoprecipitates with mature and immature forms of APP. A: Coimmunoprecipitation of HA– hFE65L and APP were obtained from H4 cells (clone 32-6) grown in the presence or absence of tetracycline for 72 h and either untreated (C) or treated with dimethyl sulfoxide (DMSO) or BFA (10 ␮g/ml) for 1 h. Immune complexes were obtained from crude protein extracts (500 ␮g) with the rabbit-HA antibody and detected by western blot (W) analysis using the APP 22C11 antibody. IP, immunoprecipitation. B: Crude protein extracts (50 ␮g) obtained in A were analyzed by western blot analysis using the APP 22C11 antibody.

difference in the steady-state level of APP when comparing uninduced cells with cells induced for synthesis of HA– hFE65L (Fig. 3A). However, a subtle shift toward increased mature APP was noted for cells overexpressing hFE65L (see Fig. 3A and B). To confirm this result, we measured the steady-state APP isoform pattern in cells overexpressing hFE65L by quantitation of the 6E10 signal. We found that the ratio of the steady-state levels of mature to immature APP was increased, owing to a decrease in the amount of immature APP in H4 cells overexpressing hFE65L. Furthermore, as with the 369W antibody, the steady-state level of APP in cells uninduced or induced for hFE65L overexpression was not significantly different (Fig. 3C).

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APP could be recovered in anti-HA immunoprecipitates from H4 32-6 induced for HA– hFE65L synthesis (Fig. 4A). APP was also recovered in HA– hFE65L immunoprecipitates obtained from the H4 32-6 cells treated with BFA. The intermediate mobility of APP observed from these immunoprecipitates (Fig. 4A) is characteristic of the partially glycosylated APP obtained from BFA-treated cells (Haass et al., 1993). Coimmunoprecipitation of immature and partially glycosylated APP with HA– hFE65L indicates that there is no limitation on the interaction of hFE65L with all full-length forms of APP. Because hFE65L binds both mature and immature APP (Fig. 4) and because we noted a subtle shift in the proportion of the APP pattern (Fig. 3), we postulated that hFE65L could modulate APP maturation, most likely by modulating the trafficking of APP as it transits from the endoplasmic reticulum and Golgi en route to the cell surface. hFE65L increases the rate of mature/immature APP To examine the effect of hFE65L on the rate of APP maturation, we performed [35S]methionine pulse/chase labeling experiments comparing APP maturation in H4 neuroglioma cells induced or uninduced for expression of HA– hFE65L. Cells were labeled for 20 min with [35S]methionine and chased in medium containing a fivefold excess of unlabeled methionine for 0, 15, 30, 60, 120, and 180 min (Fig. 5A). The cell lysates were immunoprecipitated with the 369W anti-APP C-terminal antibody, and the immunoprecipitates were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by autoradiography. As previously reported for APP isolated from H4 neuroglioma cells (Kuentzel et al., 1993), label was initially incorporated into a 120-kDa immature precursor protein, which was

FIG. 5. Maturation of APP is accelerated by overexpression of hFE65L. A: APP species were immunoprecipitated with 369W antisera from cell lysates (100 ␮g) of [35S]methionine pulse-labeled H4 cells (clone 32-6). IP, immunoprecipitation. Cells were labeled for 20 min and chased for the indicated times (top panel). HA– hFE65L induction was achieved by culturing cells in the absence of tetracycline for 72 h. Transferrin receptor (TfR) species were precipitated from identical cell lysates (500 ␮g) with anti-transferrin antibody (bottom panel). B: Immunoprecipitations of APP using the 369W antisera of pulselabeled cells as in A but with chase intervals of 10 min. C: Ratio of mature/immature APP from three independent pulse/chase experiments shown in B. Values were normalized so that APP signals were equivalent at time 0. Quantitation of the 35S-APP signal was obtained by phosphor capture using the Bio-Rad Molecular Imager.


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chased into a 145–150-kDa band representing mature APP. We found that by 60 min of chase time the proportion of labeled APP chased from the immature form to the mature form(s) was increased in cells overexpressing hFE65L. The increase in the ratio of mature to immature APP when hFE65L synthesis was induced was maintained for the additional 120- and 180-min chase time points (Fig. 5A). In contrast, the rate of transferrin receptor maturation was unaffected by hFE65L overexpression (Fig. 5A). To examine the effect of hFE65L on APP processing more closely, we repeated the experiment using chase intervals of 10 min (Fig. 5B) for a 60-min period. We chose 60 min as the final time point because APPs␣ could be detected in conditioned medium at 60 min of chase, indicating that secretion of the newly synthesized APP is underway (see Fig. 6A). A comparison of the maturation profiles of APP in the presence or absence of HA– hFE65L showed no shift in the time at which APP starts to mature. However, the amount of mature APP represented a larger fraction of total APP in cells overexpressing hFE65L (Fig. 5C). This difference was statistically significant ( p ⬍ 0.01 by t test) for chase time points of 30 – 60 min (Fig. 5C) and is due to a decrease in the level of immature APP in cells overexpressing HA– hFE65L ( p ⱕ 0.03 for 10 – 60 min). Results of immunoprecipitation of APP with the 6E10 antibody on the cell extracts used in Fig. 5B showed a similar trend (data not shown). However, in the 6E10 immunoprecipitates the difference in APPs␣/total APP was not statistically significant ( p ⬍ 0.08 by t test) for chase time points between 30 and 60 min, nor was there a statistical difference in the levels of immature APP ( p ⬍ 0.08 by t test). Failure to detect lower levels of immature APP in the 6E10 immunoprecipitates was not consistent with the detection of lower steady-state levels of immature APP in cells overexpressing HA– hFE65L by western blot analysis with 6E10. Because we observed the same trend for lower levels of immature APP in immunoprecipitations with both antibodies, we believe the inconsistency between the 6E10 western blot data and the 6E10 immunoprecipitation data is likely due to variable affinity of 6E10 for full-length APP species under different conditions, i.e., denatured APP on western blot versus native APP in cell lysates. APPs␣ secretion is enhanced by hFE65L overexpression An increase in the rate of maturation of APP may also be reflected in the amount of APPs released from the cell if its secretion from the cell is not rate-limiting. We investigated the effects of hFE65L overexpression on APP secretion by recovering APPs from H4 neuroglioma cells cultured for 72 h in the presence or absence of tetracycline. APPs comparisons were made by immunoprecipitating [35S]methionine-labeled APPs from chase media using the different anti-APP antibodies 1G7/5A3 (Koo and Squazzo, 1994) and 6E10 (Kim et al., 1990). Representative results of APPs immunoprecipitated from J. Neurochem., Vol. 73, No. 3, 1999

FIG. 6. APPs secretion is enhanced by hFE65L overexpression. A: APPs was immunoprecipitated from chase medium normalized for equal amounts of cell lysate total protein with either the 6E10 or 1G7/5A3 antibodies. IP, immunoprecipitation. The chase medium was obtained from HA– hFE65L-inducible H4 cells cultured in the presence or absence of tetracycline for 72 h and pulse-labeled for a 20-min period using [35S]methionine. The nature of the 6E10 cross-reactive band that migrates slightly faster than APPs is unknown, and the arrow indicates APPs. B: Western blot (W) analysis of APPs␣ detected with 6E10 antibody after being enriched from medium conditioned for the indicated times. APPs enrichment from the medium was achieved by anion exchange chromatography using MacroQ resin. C: Comparison of the amount of APPs␣ measured from three independent experiments shown in B. Quantitation of the 6E10 chemiluminescent signal was obtained by capture using the Bio-Rad Fluor-S Imager.

chase media using either the 6E10 or the 1G7/5A3 antibodies are shown in Fig. 6A. We obtained similar results with the P2-1 antibody (Van Nostrand et al., 1989) but chose to show the 6E10 and 1G7/5A3 results because the latter two antibodies do not recognize the secreted form of APLP2. It is clear from these data that APPs secretion was enhanced by hFE65L overexpression within 60 min following de novo synthesis of APP (see 1G7/5A3 results). To quantitate the amount of APPs␣ recovered in medium over a 6-h period, we enriched APPs from conditioned medium using an anion exchange chromatography method previously described by Moir et al. (1998). APPs␣ and the respective cellular APP levels were quantitated by western blot analysis using the 6E10 antibody (Fig. 6B) and capture of the chemiluminescent signal with the Bio-Rad Fluor-S MultiImager. Secreted APPs␣ was 3.0 ⫾ 0.5-fold higher in cells overexpressing HA-hFE65L (Fig. 6C). hFE65L enhancement of APPs␣ secretion occurs in the presence of a PKC inhibitor PKC stimulation with phorbol esters has been shown to increase APP secretion. We examined the effect of

hFE65L OVEREXPRESSION ENHANCES APP PROCESSING

FIG. 7. APP secretion mediated by hFE65L is PKC-independent. Top: Immunoprecipitation of APPs using the 6E10 antibody from H4 neuroglioma cells uninduced and induced for HA– hFE65L expression and labeled with [35S]methionine for 20 min. Cells were either untreated (C) or treated with PDBU (1 ␮M) or STS (10 nM) for 2 h during the chase period. Bottom: Western blot analysis of HA– hFE65L using an anti-HA antibody on total protein (50 ␮g) from cell treatments in the top panel.

increased levels of hFE65L protein on APP secretion in the presence of phorbol esters and in the presence of STS, which is known to inhibit PKC-stimulated secretion of APP (Friedman et al., 1997). We found that APP secretion was highest when both hFE65L was overexpressed and PKC was activated by addition of the phorbol ester PDBu. Furthermore, STS treatment of the cells did not abrogate the effect of hFE65L overexpression on APPs secretion (Fig. 7) but is known to block phorbol ester stimulation of APP secretion. This suggests that hFE65L influences the constitutive secretion of APP, independently of PKC. DISCUSSION Our results show that overexpression of hFE65L increases APPs␣ secretion and decreases the levels of immature APP in cell lysates, presumably as a result of faster maturation and secretion of APP. These data provide physiological evidence that members of the family of FE65 proteins modulate APP processing. In addition to the FE65 proteins, there are two other proteins that affect APP secretion when overexpressed. The dominant-negative Rab6 mutant, Rab6N126I, increases APPs␣ secretion in HEK293 cells (McConlogue et al., 1996). The Rab6 protein localizes to the late Golgi compartment, where it mediates intra-Golgi vesicular trafficking and vesicle budding from the trans-Golgi network and there is no physical interaction of Rab6 with APP. In contrast, X11, like the FE65 proteins, binds the carboxy terminus of APP via a PID (Borg et al., 1996). However, X11 overexpression decreases APPs secretion (Borg et al., 1998; Sastre et al., 1998). The opposite effects of the X11 and FE65 proteins on APP secretion suggest that proteins that bind the YENPTY motif of APP play a role in APP trafficking. This hypothesis is supported by recent studies showing that the X11 proteins are part of an evolutionarily conserved protein complex that mediates protein trafficking in neurons and epithelial cells (Butz et al., 1998; Kaech et al., 1998; Rongo et al., 1998). We believe that the effect of hFE65L on APP secretion is not simply an ectopic gain of function for two reasons. First, APPs secretion can be up-regulated (Mills and Reiner,

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1999). Second, we observe this effect in a cell line that shows endogenous expression of the hFE65L message (Gueńette et al., 1999). APP-specific molecular interactions that modulate APP ectodomain secretion may be important for the regulation of the trophic and protective activities reported for APPs (Koo et al., 1993; Mattson et al., 1993; Schubert and Behl, 1993; Roch et al., 1994). Because mutations in the YENPTY reinternalization sequence decrease binding of the FE65 proteins, both FE65 (Borg et al., 1996) and hFE65L (Fig. 1), to APP, it is possible that overexpression of hFE65L may block access of the APP YENPTY sequence to the reinternalization machinery. Modulation of endocytosis by protein–protein interaction has been reported for the type I transmembrane glycoprotein CD4. Entry of CD4 into the endocytic pathway is modulated by its interaction with the cytoplasmic protein p56lck, most likely as a result of masking of its clathrin-coated pit internalization domain (Pelchen-Matthews et al., 1992). Decreasing reinternalization of cell surface-associated APP could potentially increase APPs secretion. This possibility is supported by the finding that mutations in the YENPTY clathrincoated pit internalization sequence increase APPs release (Haass et al., 1993; Koo and Squazzo, 1994) in cells with high levels of surface-associated APP. However, hFE65L overexpression increases both maturation and secretion of APP in H4 neuroglioma cells. Thus, it is difficult to envisage how modulation of cell surface APP internalization would affect the maturation rate of APP. An alternative explanation is that the cell might achieve a rapid increase in relative levels of mature APP and APPs release via decreased exposure of APP to proteolytic enzymes. This hypothesis is based on the observation that processing of APP in H4 neuroglioma cells is similar to that observed in other cell types: ⬃20 –30% of the newly synthesized APP is transported to the Golgi apparatus, whereas the remaining 70% is presumably degraded in the endoplasmic reticulum (Kuentzel et al., 1993). Our data do not support this hypothesis because there is no difference in the steadystate levels of total cellular APP. Rather, we noted a decrease in the levels of immature APP in cells overexpressing hFE65L. Although it is highly likely that APP trafficking is modulated in part by the direct physical interaction of hFE65L with APP, overexpression of hFE65L could activate a signal transduction pathway that stimulates APPs release from cells. It has previously been shown that APPs secretion is modulated by extracellular signals targeting both G protein- and tyrosine kinase-coupled receptors (Nitsch et al., 1996). A possible role for the FE65 proteins in a signal transduction cascade is also supported by the presence of WW domains and PIDs in these proteins. These domains are protein–protein interaction motifs found in several proteins involved in signal transduction pathways. For example, the cytoplasmic adaptor protein, Shc, mediates the assembly of tyrosinephosphorylated signaling complexes via its PID and SH2 domain (Pelicci et al., 1992; Rozakis-Adcock et al., J. Neurochem., Vol. 73, No. 3, 1999

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1992; Blaikie et al., 1994), and the WW domain has been implicated in a signaling mechanism that underlies the malfunctioning of the epithelial sodium channel in Liddle’s syndrome (Hansson et al., 1995; Staub et al., 1996). Our results suggest that hFE65L is not acting on PKC to regulate secretion of APPs because blocking PKC activation with STS did not interfere with hFE65L-mediated enhancement of APPs secretion. Finally, increases in APPs secretion have been reported to correlate with decreases in A␤ secretion (Buxbaum et al., 1993, 1994; Felsenstein et al., 1994; Jacobsen et al., 1994). It remains to be determined whether hFE65L overexpression has an impact on A␤ generation. In the current study we specifically set out to assess the effects of hFE65L on the processing of endogenous APP. Endogenous levels of secreted A␤ were too low for reliable quantitation by ELISA (Xia et al., 1997). Thus, the data presented here pertain solely to the effects of hFE65L overexpression on endogenous APP processing and not to those that might be observed under conditions involving excess accumulation of APP, which would otherwise have allowed for the quantitation of A␤. The exact cellular site of the hFE65L–APP interaction accounting for the observed effect on endogenous APP maturation and consequences for A␤ production will be important to determine in future studies. Note added in proof: During the review of the manuscript Sabo et al. (1999) reported that FE65 overexpression increases both APPs␣ and A␤ secretion in Madin–Darby canine kidney cells stably transfected with FE65 and APP695. Acknowledgment: We thank Tae-Wan Kim for providing the H4 32neo founder cell line for tetracycline repression, Colin Masters for the 22C11 antibody, Sam Gandy for the 369W antiserum, Eddie Koo for the 1G7/5A3 antibodies, Steve Wagner for the P2-1 antibody, and Dennis Selkoe for the HEK293 cells overproducing APP695. We also thank Wei-Ming Xia and Dennis Selkoe for A␤ ELISA measurements. This research was supported by grants from the American Health Assistance Foundation and the National Institutes of Health.

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