JBC Transcription and Nuclear Factor Monoclonals

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Originally published In Press as doi:10.1074/jbc.M413729200 on March 16, 2005

J. Biol. Chem., Vol. 280, Issue 20, 20140-20147, May 20, 2005
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Sequences from the Low Density Lipoprotein Receptor-related Protein (LRP) Cytoplasmic Domain Enhance Amyloid {beta} Protein Production via the {beta}-Secretase Pathway without Altering Amyloid Precursor Protein/LRP Nuclear Signaling*

Il-Sang Yoon{ddagger}, Claus U. Pietrzik{ddagger}§, David E. Kang{ddagger}, and Edward H. Koo{ddagger}

From the {ddagger}Department of Neurosciences, University of California, San Diego, La Jolla, California 92093

Received for publication, December 6, 2004 , and in revised form, March 15, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Increasing evidence suggests that the low density lipoprotein receptor-related protein (LRP) affects the processing of amyloid precursor protein (APP) and amyloid {beta} (A{beta}) protein production as well as mediates the clearance of A{beta} from the brain. Recent studies indicate that the cytoplasmic domain of LRP is critical for this modulation of APP processing requiring perhaps a complex between APP, the adaptor protein FE65, and LRP. In this study, we expressed a small LRP domain consisting of the C-terminal 97 amino acids of the cytoplasmic domain, or LRP-soluble tail (LRP-ST), in CHO cells to test the hypothesis that the APP·LRP complex can be disrupted. We anticipated that LRP-ST would inhibit the normal interaction between LRP and APP and therefore perturb APP processing to resemble a LRP-deficient state. Surprisingly, CHO cells expressing LRP-ST demonstrated an increase in both sAPP secretion and A{beta} production compared with control CHO cells in a manner reminiscent of the cellular effects of the APP "Swedish mutation." The increase in sAPP secretion consisted mainly of sAPP{beta}, consistent with the increase in A{beta} release. Further, this effect is LRP-independent, as the same alterations remained when LRP-ST was expressed in LRP-deficient cells but not when the construct was membrane-anchored. Finally, deletion experiments suggested that the last 50 amino acid residues of LRP-ST contain the important domain for altering APP processing and A{beta} production. These observations indicate that there are cellular pathways that may suppress A{beta} generation but that can be altered to facilitate A{beta} production.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The low density lipoprotein receptor-related protein (LRP)1 is a large type I transmembrane protein that functions as a multifunctional endocytosis receptor for a diverse array of extracellular ligands (1, 2). LRP is synthesized as a 600-kDa precursor protein that is subsequently cleaved in the trans-Golgi compartment by furin to generate a large 515-kDa {alpha}-chain and a smaller 85-kDa membrane-associated {beta}-chain that remain non-covalently linked (3, 4). The cytoplasmic tail contains two NPXY endocytosis motifs and binds a number of cytoplasmic adaptor and scaffold proteins, such as FE65, Mint2, Disabled-1 (Dab1), Shc, and JIP-1 and -2, probably through the second NPXY motif (5–7). In addition, LRP itself undergoes a presenilin-dependent {gamma}-secretase intramembrane proteolysis, releasing a potentially transcriptionally active fragment in a manner similar to APP and Notch (8, 9).

Recent studies have implicated a role for the LRP pathway in Alzheimer disease pathogenesis. LRP and three of its key ligands, apoE, {alpha}2-macroglobulin, and APP, are genetically associated with Alzheimer disease and are found in senile plaques in the brains of Alzheimer disease patients (10–13). LRP also mediates the binding and clearance of amyloid {beta} peptide (A{beta}) complexes bound to apoE or {alpha}2-macroglobulin in cultured cells and in the brain (14–16). In addition, LRP may play a crucial role in brain efflux of A{beta} isoforms at the blood-brain barrier (17, 18). These findings therefore support a model in which LRP plays an important role in A{beta} uptake and removal.

In a dual but opposing effect, it has been proposed that LRP can also promote A{beta} production by altering the trafficking and processing of APP, possibly by APP/LRP interactions via the Kunitz protease inhibitor domain as well as through cytoplasmic adaptor proteins (7, 19–23). Absence of LRP or treatment of receptor-associated protein, an antagonist of all known LRP ligands, substantially reduced A{beta} release, a phenotype that was reversed when full-length or truncated LRP was transfected in LRP-deficient cells (20, 21). The domain in LRP that is responsible for regulating APP processing and A{beta} production was mapped to the cytosolic tail; specifically, a seven-amino acid motif that included the distal NPVY site (20). These results suggested that the cytoplasmic domain of LRP plays a major role in APP processing.

We hypothesized that formation of a LRP·APP complex is important for normal APP processing and have shown that indeed, APP and LRP appear to be functionally linked by the adaptor protein FE65 (24). In this study, we tested the idea that blocking the interaction between LRP and APP could reduce A{beta} production in a manner similar to that observed in LRP-deficient cells. We hypothesized that expression of a LRP construct, consisting of only the last 97 amino acid residues of the cytosolic tail of LRP, will block LRP/APP interaction and, in so doing, alter APP processing and A{beta} generation. Surprisingly, expression of the soluble LRP cytosolic fragment did not impair A{beta} production as expected. Instead this fragment altered APP processing by positively regulating the {beta}-secretase pathway of APP cleavage.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
cDNA Constructs—The mutant LRP constructs used in this study are schematically illustrated in Fig. 1. The Myc-tagged last 97 amino acids of the cytoplasmic region of the LRP {beta}-subunit (Myc-LRP-ST) and various LRP derivates were generated by PCR. The fatty acylation (i.e. myristoylation and palmitoylation) signal sequence of Fyn tyrosine kinase (GCVQCKCKDKEATKLTEER) was fused to the N-terminal end of LRP-ST (FA-LRP-ST) (25). All of the constructs were subcloned into pLHCX vector (Clontech, Palo Alto, CA) for retrovirus production. Plasmids encoding the Gal4 DNA-binding domain (pMST), the Gal4 DNA-binding domain engineered into the cytoplasmic tail of APP695 (pMST-APP), the APP-Gal4 fusion protein with a deletion in the NPTY motif of the APP cytoplasmic tail (pMST-APP{Delta}), and a Gal4 reporter plasmid encoding firefly luciferase (pG5E1B-luc) have been described (26) (kindly provided by Dr. T. Sudhof). The pLHCX-FE65 construct was engineered from pCMV-FE65 vector (a generous gift from Dr. T. Sudhof). Plasmid pRL-TK encoding Renilla luciferase was from Promega. All constructs were verified by sequencing.



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  		FIG. 1.
cDNA constructs used in this study. Diagram represents cDNA constructs used in this study. Myc-LRP-ST constructs contain the C-terminal 97 amino acid residues of LRP fused to a Myc tag (abbreviated as LRP-ST for soluble tail). The Myc-LRP-ST{Delta}NPTY and Myc-LRP-ST{Delta}NPVY are engineered from the Myc-LRP-ST construct, in which the first and second NPXY motif of LRP-ST was deleted, respectively. (LRP-ST1–47) contains the first 47 amino acid residues of LRP-ST and has a FLAG tag at its C terminus. (LRP-ST48–97) contains the last 50 amino acid residues of LRP-ST and has a FLAG tag at its N terminus. The FA-LRP-ST is engineered from the LRP-ST by appending the fatty acylation signal peptide of Fyn tyrosine kinase at its N terminus using PCR.

 
Cell Lines—Chinese hamster ovary (CHO) cells and the LRP-deficient CHO cell line (13-5-1) stably expressing wild type APP751 (APP-WT) have been described previously (20). To generate stable cell lines expressing various LRP constructs, VSV-G (glycoprotein of the vesicular stomatitis virus) pseudo-typed retroviruses were packaged and used to infect CHO cells (27). After infection, the cells were selected using hygromycin (250 µg/ml), and antibiotic-resistant cells were expanded without further clonal selection.

Antibodies—The polyclonal antibody 1704 recognizes the cytoplasmic domain of human LRP (20). The polyclonal antibody CT15, which reacts with the C-terminal 15 amino acid residues of APP, and the monoclonal antibodies 1G7/5A3 and 26D6, which recognize the ectodomain of APP, have been described previously (28, 29). Monoclonal antibodies 9E10 against the Myc epitope sequence and Ab-1 against the {beta}-tubulin were obtained from Calbiochem.

Western Blot Analyses—Cell lysates solubilized with 1% Nonidet P-40 or conditioned media were fractionated by SDS-PAGE in 4–12% NuPAGE® Novex BisTris gels (Invitrogen) or 8% Tris-glycine gels to detect low molecular APP C-terminal fragments and A{beta} or sAPP, respectively. In all cases, gel loading was normalized to total protein content in the cell extract or the corresponding cell extracts when medium samples were used. Western blotting was carried out with the indicated antibodies and detected by enhanced chemiluminescence (Pierce). Quantitation of the chemiluminescence signal was done using a charge-coupled device imaging system (GeneGnome, Syngene, Frederick, MD). All experiments were performed at least three times, and each experiment was done in duplicate or in triplicate. Data are presented as the mean ± S.E. Differences were analyzed by Student's t test or one-way analysis of variance, followed by post hoc comparisons of group means using the Tukey test. Statistical differences with two-tailed probability values of p < 0.05 were taken as significant.

Extraction of Membrane Fraction—Cells in a 10-cm dish were washed twice with ice-cold phosphate-buffered saline and scraped in 1 ml of phosphate-buffered saline. The scraped cells were collected by low speed centrifugation (500 x g for 1 min). Cells were then resuspended in 1 ml of hypotonic buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA) containing 1x complete protease inhibitor mixture (Roche Applied Science) and homogenized by passing five times through a 25-gauge needle and five times through a 27-gauge needle. To prepare a post-nuclear supernatant, the homogenate was centrifuged at 1000 x g for 15 min at 4 °C. The membrane fraction was isolated from the post-nuclear supernatant by centrifugation at 20,000 x g for 45 min at 4 °C. The supernatant (cytosolic fraction) was saved for further analysis at –80 °C. The pellet (membrane fraction) was resuspended in 100 µlof radioimmune precipitation assay buffer (50 mM Tris, 150 mM NaCl, 0.02% NaN3, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) and cleared by a spin at 20,000 x g for 10 min at 4 °C.

Metabolic Labeling—Confluent cells were incubated in 150 µCi/ml [35S]methionine/cysteine for 10 min or 3 h to determine the turnover of full-length APP or APP C-terminal fragments (CTFs), respectively. Following metabolic labeling, the cells were either lysed immediately (time 0) or chased for 1, 2, or 4 h. The full-length APP and APP CTFs were immunoprecipitated with polyclonal antibody CT15. The immunoprecipitates were fractionated by SDS-PAGE (8% Tris-glycine gels for full-length APP and 4–12% NuPAGE® Novex BisTris gels for APP-CTF) and exposed to a phosphorimaging screen for quantitation.

Internalization Assay—To measure internalization of cell-surface APP, iodinated 1G7 antibody was added to confluent cultures of APP-WT CHO and MycST cells at 37 °C for 30 min exactly as described previously (20). In brief, after incubation, the cells were rapidly chilled on ice, and the reaction was quenched by the addition of ice-cold binding medium. The chilled cells were washed extensively, and the remaining surface antibody was detached by two acid washes. The cells were then lysed and collected for analysis. Acid-labile and acid-resistant radioactivity represents the surface and internalized pools of APP, respectively. Specific binding was determined after subtraction of the radioactivity counts obtained from parallel cultures of untransfected CHO cells. The ratio of acid-resistant to acid-labile counts therefore provided a measure of the internalized versus cell-surface pools of APP. Data are from three separate experiments, and each experiment was performed in triplicate.

Luciferase Reporter Assay—Subconfluent CHO cells were transiently transfected with the following combinations of plasmids (0.25 µg of each): 1) pMST + pG5EIB-luc + pLHCX; 2) pMST-APP + pG5EIB-luc + pLHCX; 3) pMST-APP + pG5EIB-luc + pLHCX-FE65 + pLHCX; 4) pMST-APP{Delta}+ pG5EIB-luc + pLHCX-FE65 + pLHCX; and 5) pMST-APP + pG5EIB-luc + pLHCX-FE65 + pLHCX-MycST. 20 ng of pRL-TK was added to each plasmid mix to control for transfection efficiency. 48 h after transfection, the cells were washed twice with phosphate-buffered saline and lysed using the 1x lysis buffer from the dual luciferase reporter assay system (Promega). The firefly and Renilla luciferase activities were quantified using a dual injector luminometer (EC & G Berthold). The firefly luciferase values were normalized against the Renilla luciferase value after background values from the wells with no cell lysates were subtracted. The values were expressed as fold induction over Gal4 (i.e. value from plasmid combination 1).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of LRP-soluble Tail (LRP-ST) Expression on APP Processing—To examine the effects of LRP-ST expression on APP processing, Myc-tagged LRP-ST (Myc-LRP-ST) was stably expressed in APP-WT CHO cells (herein referred to as MycST cells). Expression of Myc-LRP-ST was examined by immunoblotting with a LRP C-terminal antibody. When whole cell lysates were separated into cytosolic and membrane fractions, full-length LRP (~600 kDa) and LRP {beta}-chain (~85 kDa) were detected in membrane fractions from both parental control and Myc-LRP-ST-transfected cells (Fig. 2A, upper panel). As anticipated, the Myc-LRP-ST lacking the transmembrane domain was detected in the cytosolic fraction of MycST cells only. The recently described ~25-kDa C-terminal fragment of LRP representing the substrate for {gamma}-secretase intramembrane proteolysis (9) was also detected in membrane fractions of both cell lines (Fig. 2A, lower panel, asterisk). Expression of the Myc-LRP-ST fragment was also detected by immunoblotting with an antibody that recognized the Myc epitope tag (Fig. 2B). Expression of Myc-LRP-ST did not alter the expression of APP (Fig. 3, top panel). However, there was a significant ~2-fold increase in the level of APP CTF in MycST cells as compared with control APP-WT CHO cells (Fig. 3, middle panel). Previously, we reported that in LRP-deficient cells, the levels of APP CTFs were substantially reduced (20).



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  		FIG. 2.
Stable expression of Myc-LRP-ST (MycST) in CHO cells overexpressing APP751 (APP-WT CHO). Full-length (FL-LRP) and the {beta}-chain of LRP are recovered in the membrane fraction (A, upper panel) and detected using LRP polyclonal antibody 1704. The Myc-LRP-ST fragment in the cytosolic fraction is detected using LRP (A) or Myc (B) antibodies. Asterisk indicates the ~25-kDa C-terminal fragment of LRP representing the substrate for {gamma}-secretase intramembrane proteolysis (9).

 



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  		FIG. 3.
Increased level of C-terminal fragment (CTF) of APP in MycST cells. Expression of the full-length APP level in a duplicate sample of cell extracts is unchanged following stable expression of Myc-LRP-ST (top panel). However, there is an ~2-fold increase in APP CTF levels in MycST cells as compared with APP-WT CHO cells (middle panel). {beta}-tubulin levels were determined with a monoclonal antibody (Ab-1) in the same cell lysate to ensure equal loading (bottom panel).

 
We next assayed for sAPP secretion, because CTF generation and sAPP secretion are frequently correlated with each other. As predicted, there was an ~2-fold increase in total sAPP secretion in conditioned medium from MycST cells as compared with controls (Fig. 4A). To determine whether the {alpha}-or {beta}-secretase-derived sAPP was preferentially affected by LRP-ST, the same conditioned medium was subjected to additional immunoblotting using monoclonal antibody 26D6, which reacts with the first 12 residues of the A{beta} sequence contained in sAPP{alpha} but not in sAPP{beta} (14, 28). Surprisingly, there was only a small non-significant (~15%) increase in sAPP{alpha} in MycST cells (Fig. 4B), suggesting that the observed ~2-fold increase in total sAPP secretion mainly resulted from sAPP{beta}. Consistent with this interpretation, {beta}-secretase-derived APP CTF{beta} was also increased by >50% in Myc-LRP-ST-expressing cells (Fig. 4C) concomitant with an ~2-fold increase in A{beta} secretion (Fig. 4D). The increase in A{beta} level was not because of the Myc tag attached to LRP-ST, because the expression of LRP-ST alone showed similar results (Fig. 4E). These results taken together indicate that LRP-ST expression in CHO cells augments APP processing through a {beta}-secretase-mediated pathway.



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  		FIG. 4.
Increased level of secreted {beta}-cleaved APP (sAPP{beta}), {beta}-secretase-cleaved CTF (CTF{beta}) and A{beta} in MycST cells. A, a 2-fold increase in total sAPP level is detected in the conditioned medium from MycST cells as compared with control cells seen here by immunoblotting sAPP from conditioned medium using APP monoclonal antibodies (5A3 and 1G7). B, only a small increase in {alpha}-secretase-cleaved sAPP (sAPP{alpha}) level was seen following immunoblotting-conditioned medium with APP monoclonal antibody 26D6 that recognizes the C terminus of sAPP (or first 12 residues of A{beta}). C, approximately 50% more CTF{beta} as compared with APP-WT CHO cells was seen in MycST cells using the APP antibody 26D6, which recognizes only CTF{beta} but not CTF{alpha}. D, 2-fold more A{beta} was secreted into the medium of MycST cells as compared with APP-WT CHO cells. Representative immunoblots are shown in the inset. Note that peptide A{beta} 1–40 peptide standard was loaded in the first lane from the left. E, similar effects on sAPP and A{beta} levels were seen in the conditioned media of LRP-ST cells that were transfected with a similar LRP-ST construct but lacking a Myc tag. This shows that the effects are not secondary to the Myc sequences. *, p < 0.05 compared with control APP-WT CHO cells.

 
Reduction in Cell-surface APP by LRP-ST—To test whether the increased sAPP, CTFs, and A{beta} levels in MycST cells were because of the increased stability of APP, we next examined the turnover of full-length APP by pulse-chase analysis in APP-WT CHO and MycST cells. However, there was no change in the turnover rate of APP in MycST cells as compared with controls (t1/2, ~60 min) (Fig. 5, A and B). Similarly, the increase in CTF levels in MycST cells might also be due to its enhanced stability. As with full-length APP, there was no significant difference in the half-life of APP CTFs (~2 h) in MycST cells as compared with controls (Fig. 5C), suggesting that the increased APP CTF level in MycST cells is secondary to enhanced production.



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  		FIG. 5.
Turnover of full-length APP and APP CTF, APP internalization and surface APP level in MycST cells. There is no change in either APP maturation and turnover or APP CTF turnover in MycST cells as compared with APP-WT CHO cells. Representative autoradiograph from pulse-chase experiments after metabolic labeling with [35S]methionine/cysteine is shown for APP (A) and quantitation of full-length APP and APP CTF turnover from three separate experiments expressed as mean ± S.E. (B and C, respectively). Steady-state internalization of APP from the cell surface (D) and the cell-surface APP levels (E) were measured with iodinated 1G7 antibody at 37 °C and at 4 °C, respectively, as described under "Experimental Procedures." The ratios of internalized to cell-surface APP and the surface APP levels in MycST cells are normalized to the APP-WT CHO cells. The APP internalization was comparable between the two cell lines. However, there was ~30% reduction in the cell-surface APP level in MycST cells. *, p < 0.05; **, p < 0.0001.

 
In CHO cells, a major pathway for the generation and secretion of A{beta} appears to be derived from endocytic processing of APP from the cell surface (30, 31). Accordingly, we next measured the internalization of APP using an iodinated APP antibody (1G7). The internalization of APP was expressed as a ratio between the 125I-1G7 antibody bound to intracellular and cell-surface APP. Although APP internalization was essentially unchanged (7% reduction, Fig. 5D), a 30% reduction in steady-state levels of cell-surface APP was observed in MycST cells as compared with control APP-WT CHO cells (Fig. 5E). These results suggested that LRP-ST altered the processing of APP in compartments prior to trafficking to the cell surface. Such a phenotype resembles that of the APP "Swedish mutation" where decreased cell-surface APP and increased {beta}-secretase cleavage products were documented (31, 32). However, we failed to document any increase in either the protein or in the mRNA level of {beta}-secretase (data not shown).

The alterations in the level of cell-surface APP level as well as the changes in APP/A{beta} processing in MycST cells indicated that there may be perturbed trafficking of APP to different subcellular organelles. Therefore, we examined the subcellular localization of APP by immunolocalization and sucrose density gradient fractionation after sodium carbonate extraction or Triton X-100 solubilization. However, we could not detect any significant changes in APP subcellular localization in MycST cells with either of these two approaches (data not shown).

Effect of MycST Deletion Mutants on APP Processing—A recent study using the yeast two-hybrid assay reported that the cytoplasmic tail of LRP could interact with several functionally distinct proteins, such as JIP1 and -2, PSD-95, and Mint2 (6). In addition, it has been shown that the NPXY domains of both LRP and APP cytoplasmic tails bind to adaptor proteins FE65, mDab, and Shc (5–7, 24, 26, 33). Because FE65, Shc, and Mint proteins have been shown to differentially modulate A{beta} secretion (33–37), it is possible that binding of the cytoplasmic adaptor proteins to the NPXY motifs of the LRP cytoplasmic tail region could account for the increased A{beta} secretion observed in MycST cells. Thus, we first expressed Myc-LRP-ST constructs lacking the first (Myc-LRP-ST {Delta}NPTY) or second (Myc-LRP-ST {Delta}NPVY) NPXY motifs in parental APP-WT CHO cells. Surprisingly, expression of both Myc-LRP-ST deletion constructs still increased the levels of A{beta} as compared with control APP-WT CHO cells (Fig. 6A), indicating that neither NPXY core motif alone in LRP-ST is functional with respect to the increase in A{beta} production. Based on these results, we therefore hypothesized that regions flanking either one or both NPXY motifs must account for the increased A{beta} secretion by LRP-ST. To test this possibility, additional mutant LRP-ST constructs were engineered. Although expression of the last 50 amino acid residues of LRP-ST (LRP-ST48–97) still increased A{beta} secretion, expression of the first 47 amino acid residues of LRP-ST (LRP-ST1–47) had no effect on A{beta} secretion in APP-WT CHO cells (Fig. 6B). These results suggest that amino acid residues in between positions 48 and 97 but not the core NPXY motif of LRP-ST (residues 57NFTNPVYATL66) are critical for the increased production of A{beta} following forced expression of the LRP cytosolic tail.



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  		FIG. 6.
Reduced A{beta} level in conditioned media from APP-WT CHO cells stably expressing N-terminal 47 amino acids of LRP-soluble tail (LRP-ST1–47). Deletion of the first (Myc-LRP-ST{Delta}NPTY) or second (Myc-LRP-ST{Delta}NPVY) NPXY domain of LRP-ST had no effect on the elevation of A{beta} levels induced by LRP-ST construct as compared with APP-WT CHO cell level (A). When APP-WT CHO cells were stably transfected with the construct in which the second half of LRP-ST was deleted (LRP-ST1–47), but not the proximal half (LRP-ST48–97), A{beta} levels in conditioned media were restored to control levels (B). When the fatty acylation signal peptide of Fyn tyrosine kinase was appended to the N terminus of LRP-ST (FA-LRP-ST), most of the expressed FA-LRP-ST was found in the membrane fraction (E, middle panel). Prolonged exposure of the immunoblot revealed low levels of FA-LRP-ST fragment in the cytosolic fraction (E, bottom panel). The translocation of the LRP-ST fragment from cytosolic to membrane fraction caused a reduction in both secreted sAPP (C) and A{beta} levels (D). *, p < 0.001; {dagger}, p < 0.05 compared with control APP-WT cells.

 
Effect of Translocation of the LRP-ST to the Membrane Fraction on APP Processing—Because LRP-ST is soluble, we infer that it modulates APP processing in the cytoplasm. This notion predicts that translocation of LRP-ST to the membrane should attenuate or even abolish its effect on APP processing. To test this hypothesis, we engineered a mutant LRP-ST construct (FA-LRP-ST), which contains the fatty acylation (i.e. myristoylation and palmitoylation) signal sequences of Fyn tyrosine kinase (25) in its N-terminal end (Fig. 1). When these signal sequences were added to the N terminus of green fluorescent protein, the resultant green fluorescent protein chimera became primarily membrane-bound, whereas green fluorescent protein alone was found essentially only in the soluble fraction (>90%) (25). As expected, the majority of FA-LRP-ST protein was membrane-associated when expressed in CHO cells with only a small amount recovered in the cytosolic fraction (<5%) (Fig. 6E, middle panel). After prolonged exposure, the FA-LRP-ST could be observed in the cytoplasmic fraction (Fig. 6E, bottom panel). Next, we measured total sAPP and A{beta} levels in conditioned media from FA-LRP-ST-expressing cells. Interestingly, FA-LRP-ST-expressing cells secreted a significantly reduced level of total sAPP (Fig. 6C) and A{beta} (Fig. 6D) as compared with MycST cells. Levels of sAPP and A{beta} were intermediate between MycST and control cells, a finding that we attribute to the residual amount of FA-LRP-ST present in cytoplasmic fraction (Fig. 6E, bottom panel). Nevertheless, these results indicate that the subcellular localization of LRP-ST is important for its role in APP processing.

Effects of LRP-ST on APP Processing Do Not Require Endogenous LRP Expression—Although we initially predicted that LRP-ST would block the APP/LRP interaction to lower A{beta} production, this outcome was not seen in the studies. This indicated that LRP-ST may not be blocking the APP·LRP complex. Accordingly, to test whether the perturbations in APP processing we observed are independent of LRP, the LRP-ST construct was expressed in the CHO 13-5-1 cell line that is deficient in LRP. Surprisingly, both sAPP and A{beta} levels were indeed increased in 13-5-1 cells expressing Myc-LRP-ST, much like that seen in LRP-expressing APP-WT CHO cells (Fig. 7). Thus, these results are consistent with the hypothesis that the LRP-ST effect on APP processing is independent of endogenous LRP and therefore did not inhibit APP/LRP interaction as predicted.



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  		FIG. 7.
Effect of Myc-LRP-ST is independent of endogenous LRP expression. Transfection of Myc-LRP-ST in LRP-deficient 13-5-1 CHO cells increased sAPP and A{beta} secretion similar to that seen in APP-WT CHO cells. Comparable levels of full-length APP (FL-APP) are seen in the two 13-5-1 cell lines. Immunoblots for FL-APP, sAPP, and A{beta} were performed using antibodies CT15, 1G7/5A3, and 26D6, respectively.

 
Myc-LRP-ST Expression Does Not Impair AICD/FE65-mediated Transcription—Recently, it was reported that the expression of the cytoplasmic domain of LRP abolished the transcriptional activation mediated by the APP intracellular domain released after {gamma}-secretase cleavage, AICD, and FE65 (8). Therefore, we examined whether abnormal APP processing by Myc-LRP-ST expression is associated with changes in AICD/FE65-mediated transcription. As expected, co-transfection of FE65 together with APP-Gal4 substantially increased the transactivation of the reporter, whereas FE65 was unable to augment the transactivation of the mutant APP lacking the NPXY domain (APP{Delta}-Gal4) (Fig. 8). However, co-transfection of Myc-LRP-ST had no effect on the transcriptional activation mediated by AICD/FE65 (Fig. 8). Thus, these data indicate that transcriptional consequences associated with AICD/FE65 do not underlie the changes in abnormal APP processing seen by LRP-ST expression.



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  		FIG. 8.
Myc-LRP-ST does not inhibit AICD-mediated transcription in a heterologous reporter assay system. Co-expression of an APP-Gal4 fusion protein induced only minimal transactivation of the gene (luciferase). Co-expression of FE65 and APP-Gal4 fusion protein strongly stimulated transcription of the Gal4-dependent reporter that was not attenuated by Myc-LRP-ST. No induction of reporter gene activity was seen by co-expression of FE65 with a mutant APP-Gal4 construct, where the NPTY motif was deleted (APP{Delta}-Gal4). The results are expressed as fold induction in transcription over control cells expressing the Gal4 DNA-binding domain alone.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Increasing evidence suggests that LRP has opposing effects on APP processing and A{beta} metabolism. On one hand, LRP mediates the clearance of A{beta} either by itself or following complex formation of A{beta} with various LRP ligands (14–18). On the other, LRP also influences multiple steps in APP processing to modulate A{beta} formation (20–22). The latter appears to involve an interaction between LRP and APP that is mediated by FE65 as an adaptor molecule linking the respective cytosolic domains to form a functional tripartite complex (24). To further investigate the mechanism by which LRP modulates A{beta} generation, we hypothesized that expression of the LRP cytosolic domain, LRP-ST, should interfere with the APP·LRP complex and in so doing, perturb normal APP processing to inhibit A{beta} generation. To our surprise, expression of LRP-ST in CHO cells not only did not impair A{beta} generation, we found that A{beta} generation was paradoxically increased. Like LRP-deficient cells, sAPP secretion was enhanced by LRP-ST expression, but unlike the LRP deficiency state, this change was due primarily to a preferential increase in sAPP{beta} secretion (21). Although endocytic APP is often correlated with A{beta} generation in non-neural cells, LRP-ST expression did not alter APP internalization (20). These results taken together suggested that cytosolic LRP-ST facilitates the trafficking and processing of APP through a pathway resembling that of the Swedish APP mutation, where A{beta}-, sAPP{beta}-, and {beta}-cleaved APP C-terminal fragment levels are selectively augmented. Further, because increased BACE-mediated cleavage of APP takes place in the trans-Golgi network, there is reduced full-length APP at the cell surface (31, 32), a finding common to both cells expressing APP Swedish mutation and the LRP-ST.

The mechanism by which LRP-ST expression increased APP processing through the {beta}-secretase (BACE) pathway is unclear. The fact that similar results were seen when LRP-ST was expressed in LRP-expressing and LRP-deficient CHO cells indicated that this effect is independent of LRP. In other words, LRP-ST likely did not interfere with the LRP·APP complex. However, in addition to FE65 and LRP, a number of other cytosolic proteins are known to interact with APP including X11/Mint (34–36, 38, 39) and its isoforms (X11L and X11L2) (34, 40), JNK-interacting proteins (JIP1b and JIP2) (41) and mDab (7, 42, 43). These studies suggested that interactions between the GYENPTY motif of APP and the phosphotyrosine-binding/protein interaction (PTB/PI) domains of some of these adaptor proteins can modulate A{beta} secretion (34, 36, 41). Because LRP also binds to JIP-1, mDab, and FE65 probably through its distal NPXY motif (6, 7, 24), it is possible that LRP-ST might effectively compete with APP for binding to adaptor proteins to influence A{beta} secretion. In our study, expression of the distal half of LRP-ST (LRP-ST48–97 fragment) containing the distal NPXY domain, but not the proximal half (LRP-ST1–47 fragment), increased A{beta} secretion (Fig. 6). This suggests that the sequestration of adaptor proteins (e.g. JIP-1b, mDab, and FE65) by LRP-ST might account for this phenotype. However, removal of the second NPVY motif alone (Myc-LRP-ST {Delta}NPVY) was not sufficient to abrogate the effects of LRP-ST on A{beta} secretion, indicating that LRP-ST was interfering with other unknown target molecules that interact with additional sequences beyond the NPVY motif. These findings led us to postulate that LRP-ST may either facilitate the trafficking of APP to compartments containing BACE or enhance BACE-mediated cleavage of APP. The latter scenario is consistent with the recent observations that phosphorylation of APP on Thr-668 changes the conformation of APP cytoplasmic tail to influence the interaction with FE65 as well as to alter A{beta} secretion (33).

Another potential explanation for our findings is that the effect is mediated at the level of nuclear transcription. LRP was recently shown to be a {gamma}-secretase substrate, and this cleavage event (S3) releases a C-terminal fragment (LRP-ICD) analogous to the Notch intracellular domain NICD. Furthermore, Kinoshita et al. (8) reported that the last 105 amino acids of LRP {beta}-chain (LRP105) inhibited transcriptional activity of the AICD, FE65, and Tip60 complex in a heterologous signaling system, presumably by interfering with the interaction between AICD and Tip60. However, the latter activity of LRP105 does not explain how LRP-ST affects APP processing, because co-transfection of Myc-LRP-ST had no effect on the transcriptional activation mediated by AICD/FE65 (Fig. 8). The lack of effect in AICD-mediated transactivation by Myc-LRP-ST was not unexpected, because, unlike LRP105, LRP-ST did not show any nuclear localization (data not shown). The addition of a signal peptide sequence to LRP105 displaced LRP105 out of the nucleus with the resultant loss of its inhibitory effect on AICD-mediated transactivation (8). This suggested that nuclear localization of LRP intracellular domain (LRP-ICD) is required to influence AICD-mediated gene transcription activity. In addition, because LRP105 is 8 amino acid residues (VVFWYKRR) longer and exhibits different subcellular localization than LRP-ST, it is likely that LRP105 and LRP-ST are not functionally equivalent. Although both LRP105 and LRP-ST are engineered based on the earlier study which showed a release of the LRP-ICD by the presenilin-dependent mechanism (9), the exact nature of LRP-ICD remains to be clarified. Of note, the cellular localization LRP-ST appears to play an important role in APP processing. When the FA signal peptide was added to LRP-ST to translocate LRP-ST to the membrane, much of the effect of LRP-ST on APP processing was lost (Fig. 6, C and D).

In summary, our study provided additional evidence for the role of LRP in APP processing and specifically demonstrated that the soluble form of LRP cytoplasmic tail (LRP-ST) alone can modulate the processing of APP and secretion of A{beta}. These effects associated with LRP-ST may provide an additional tool for elucidating a mechanism by which APP is guided to the amyloidogenic instead of non-amyloidogenic pathway.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant AG12376 (to E. H. K.). Back

§ Present address: Dept. of Molecular Neurodegeneration, Institute for Physiological Chemistry and Pathobiochemistry, University of Mainz, 55099 Mainz, Germany. Back

To whom correspondence should be addressed: Dept. of Neurosciences, University of California, San Diego, 9500 Gilman Dr., Mail Code 0691, La Jolla, CA 92093-0691. Tel.: 858-822-1024; Fax: 858-822-1021; E-mail: edkoo{at}ucsd.edu.

1 The abbreviations used are: LRP, low density lipoprotein receptor-related protein; APP, amyloid precursor protein; A{beta}, amyloid {beta}; sAPP, soluble form of APP; CTF, C-terminal fragment; ICD, intracellular domain; AICD, APP intracellular domain; ST, soluble tail; FA, fatty acylated; CHO cell, Chinese hamster ovary; WT, wild type; apoE, apolipoprotein E; BACE, {beta}-site APP-cleaving enzyme (or {beta}-secretase); JNK, c-Jun N-terminal kinase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Thomas Sudhof for the gift of the AICD reporter plasmids, Dr. Mark Lawson for assistance with luciferase assays, Drs. Peter Sims, Sascha Weggen, Markus Kummer, and Sarah Sagi for helpful discussions, and Tracy Busse and Hiroko Maruyama for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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