The Low Density Lipoprotein Receptor-related Protein Complexes with Cell Surface Heparan Sulfate Proteoglycans to Regulate Proteoglycan-mediated Lipoprotein Catabolism*

Larissa C. Wilsie and Robert A. OrlandoDagger

From the Department of Biochemistry and Molecular Biology, University of New Mexico, Health Sciences Center, Albuquerque, New Mexico 87131-0001

Received for publication, August 27, 2002, and in revised form, February 19, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been proposed that clearance of cholesterol-enriched very low density lipoprotein (VLDL) particles occurs through a multistep process beginning with their initial binding to cell-surface heparan sulfate proteoglycans (HSPG), followed by their uptake into cells by a receptor-mediated process that utilizes members of the low density lipoprotein receptor (LDLR) family, including the low density lipoprotein receptor-related protein (LRP). We have further explored the relationship between HSPG binding of VLDL and its subsequent internalization by focusing on the LRP pathway using a cell line deficient in LDLR. In this study, we show that LRP and HSPG are part of a co-immunoprecipitable complex at the cell surface demonstrating a novel association for these two cell surface receptors. Cell surface binding assays show that this complex can be disrupted by an LRP-specific ligand binding antagonist, which in turn leads to increased VLDL binding and degradation. The increase in VLDL binding results from an increase in the availability of HSPG sites as treatment with heparinase or competitors of glycosaminoglycan chain addition eliminated the augmented binding. From these results we propose a model whereby LRP regulates the availability of VLDL binding sites at the cell surface by complexing with HSPG. Once HSPG dissociates from LRP, it is then able to bind and internalize VLDL independent of LRP endocytic activity. We conclude that HSPG and LRP together participate in VLDL clearance by means of a synergistic relationship.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Remnant lipoproteins originating from lipolytic processing of dietary chylomicrons and very low density lipoproteins (VLDL)1 synthesized by the liver are primary carriers in lipid transport and facilitate the delivery of cholesterol and triglycerides to hepatic and extrahepatic tissues. VLDL particles consist of apolipoprotein B (apoB) and apolipoproteins CI, CII, and CIII (1) with esterified cholesterol and triglycerides at their core, and an outer association of apolipoprotein E (apoE). ApoCII is thought to activate lipoprotein lipase, necessary for lipolytic processing (2-4). ApoB (5, 6) and apoE (7, 8) are required for receptor-mediated clearance of VLDL particles.

Receptor-mediated clearance of circulating VLDL is a rapid and critical step for maintenance of proper plasma lipid levels. The half-life of injected VLDL is reported to be ~20 min in mice and rabbits (9). Elevated VLDL levels, as seen in type III hyperlipoproteinemia (10, 11), lead to elevated plasma triglyceride levels making VLDL highly atherogenic (5). Moreover, excessive VLDL uptake by macrophages results in cholesterol accumulation and foam cell formation (12, 13) increasing the risk for coronary heart disease and stroke.

Clearance of VLDL is thought to occur through a multistep process. VLDL initially binds to cell surfaces through interactions with heparan sulfate proteoglycans (HSPG) (14), and this binding is mediated by apoE, which contains a high affinity heparan sulfate binding site (15, 16). Accordingly, apoE knockout mice display severe hypercholesterolemia with a marked elevation of circulating remnants (17, 18). Although it is established that HSPGs are the primary binding sites for VLDL at the cell surface, their path of uptake into cells is less clear and can occur through several independent receptor-mediated events (19, 20). Studies have shown that the LDL receptor participates in VLDL clearance in vivo (8, 21), however, other receptors also contribute to its uptake, including the LDL receptor-related protein (LRP) (22, 23) and HSPG (12, 14, 24, 25). LRP is a large cell surface multiligand-binding protein and member of the LDL receptor family (26). LRP is expressed predominantly in the liver but can also be found in many diverse cell types (27). Impairment of LRP function in vivo has been shown to result in an increase in circulating levels of cholesterol and triglycerides (28, 29). Also, aged rats were shown to have decreased LRP expression, which is accompanied by a delayed clearance of VLDL remnants (30). HSPGs are also able to directly internalize bound lipoproteins and mediate their catabolism, although their internalization rates are slow when compared with those of the LDL receptor family members (31, 32). Syndecan-1, which is expressed on the peri-sinusoidal membrane of hepatocytes, is one such example (33). Syndecan-1 is of the transmembrane class of proteoglycans (34, 35) and has been shown to independently mediate the binding, internalization, and lysosomal delivery of lipoproteins with internalization kinetics of t1/2 ~ 1 h (31).

To attempt to clarify this complex mechanism of lipoprotein clearance, a multistep model has been proposed suggesting that remnant lipoprotein catabolism involves three distinct steps (20, 36). This model is based on a number of independent, corroborative studies (25, 37-41). First, circulating lipoproteins are sequestered to the cell surface by binding to HSPG. Second, the "captured" remnants undergo further lipolytic processing by hepatic lipase (42, 43) and lipoprotein lipase (38, 44). And third, internalization of the remnants is mediated through combined activities of the LDL receptor, LRP, and cell surface HSPG. Whether these three receptors interact to facilitate lipoprotein clearance is not known.

In the present study, we have begun to examine the relationship between HSPG and LRP at the cell surface to identify if these receptors act independently in lipoprotein clearance or if they coordinate their activities in a synergistic manner. Using a cell line deficient in LDL receptor expression, we have identified that HSPG and LRP are present in a co-immunoprecipitable complex at the cell surface. Moreover, incubation of cells with an antagonist of LRP ligand-binding activity increases the availability of HSPG for VLDL binding resulting in increased VLDL catabolism. These results suggest that HSPG and LRP are associated in a macromolecular complex at the cell surface and upon dissociation increase the availability of free HSPG to augment VLDL binding to cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Human VLDL, purchased from Intracel, Inc. (Frederick, MD), was obtained from a mixed pool of normal, non-fasting donors. Human alpha 2-macroglobulin (alpha 2M), heparin, heparinase I, and 4-methylumbelliferyl-beta -D-xylopyranoside were purchased from Sigma-Aldrich (St. Louis, MO). Protein A-agarose was obtained from Bio-Rad (Hercules, CA) and p-nitrophenyl-beta -D-xylopyranoside was from Calbiochem (La Jolla, CA). [35S]Methionine and 35SO4 were purchased from PerkinElmer Life Sciences (Boston, MA). Precast SDS-PAGE gels were from BioWhittaker (East Rutherford, NJ). RAP-GST fusion protein was purified as previously described (45). Anti-LRP polyclonal antibody was raised against an 18-amino acid peptide from the cytoplasmic tail of human LRP (46). Tissue culture plastics were purchased from Corning (Corning, NY). Buffers, salts, and detergents were obtained from either Sigma-Aldrich or Calbiochem.

Cell Culture-- Mouse embryo fibroblasts deficient in LRP expression (MEF-2) or deficient in LDL receptor expression (MEF-3) were generous gifts from Dr. Joachim Herz, University of Texas Southwestern Medical Center, Dallas, TX. Cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Irvine Scientific, Santa Ana, CA), 100 µg/ml streptomycin sulfate, and 100 units/ml penicillin G. Cells were cultured at 37 °C with 5% CO2 and passaged twice weekly.

Immunoblotting and Immunoprecipitation-- For immunoblotting, cell lysates were prepared in 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 10 mM CHAPS, and cellular proteins were separated by SDS-PAGE and electrotransferred to polyvinylidene difluoride membranes using a wet tank transfer method (Bio-Rad, Hercules, CA). Membranes were blocked with 20 mM Tris, pH 7.4, 150 mM NaCl (TBS), 0.1% Tween-20, 5% calf serum for 15 min at 23 °C and incubated with anti-LRP antisera (1:2000) in blocking buffer for 2 h at 23 °C. Membranes were washed three times (10 min each) with TBS, 0.1% Tween-20, 1% calf serum, and bound antibodies were detected with species-specific HRP-conjugated secondary antibodies followed by chemiluminescence detection according to the manufacturer's instructions (Pierce, Rockford, IL).

For immunoprecipitation, cells were incubated with 125 µCi/ml of either [35S]methionine in complete culture media or 35SO4 in sulfate-free media for 16 h. Cells were rinsed twice with TBS and solubilized with TBS containing 1% Triton X-100, 1% bovine serum albumin (BSA), and protease inhibitor mix (2 µg/ml chymostatin, leupeptin, antipain, and pepstatin) for 30 min at 4 °C. Detergent-insoluble material was removed by centrifugation (12,000 × g, 5 min, 4 °C), and soluble proteins were incubated with anti-LRP antisera (1:200) and protein A-agarose for 16 h at 4 °C. Immunoprecipitates were washed two times with radioimmune precipitation assay buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS) followed by two times with TBS containing 0.1% Tween-20. Antibody-bound proteins were solubilized in Laemmli sample buffer supplemented with 4% beta -mercaptoethanol and heated at 95 °C for 5 min. Proteins were resolved by SDS-PAGE, stained with 0.1% Coomassie Blue-R in water:methanol:acetic acid (50:40:10, v/v), destained, and incubated for 30 min in Amplify (Amersham Biosciences, Piscataway, NJ). Gels were then dried and exposed to Kodak X-AR film at -80 °C.

Activation of alpha 2-Macroglobulin-- alpha 2M was activated for receptor binding by incubating with an equal volume of 0.4 M methylamine in 0.1 M Tris-HCl, pH 8, for 2 h at room temperature. Unbound methylamine was then removed by passage over a desalting column (PD-10, Amersham Biosciences).

Radioiodination-- VLDL and alpha 2M were radioiodinated with Na125I (PerkinElmer Life Sciences) using IODO-BEADs (Pierce) as previously described (47). Specific activities were routinely between 3000 and 4000 cpm/ng of protein. Using the Bligh-Dyer lipid extraction procedure, we determined that ~10% of the 125I label was incorporated into lipid of the VLDL particle while the remaining 90% of label was found coupled to protein.

Cell Surface 4 °C Binding Assay-- Cells were grown on tissue culture plates precoated with 1% gelatin and used when confluent. In some cases, cells were cultured for 72 h in 3 mM 4-methylumbelliferyl-beta -D-xylopyranoside or p-nitrophenyl-beta -D-xylopyranoside or incubated for 4 h at 37 °C with heparinase I (1 Sigma unit/ml) prior to performing the binding assay. Cells were rinsed twice with 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM CaCl2 (buffer A), followed by incubation for 3 h with 125I-VLDL or 125I-alpha 2M (2 µg/ml) at 4 °C in the presence or absence of the indicated potential competitors. 125I-Labeled ligands were diluted into buffer A containing 1% BSA and chilled to 4 °C before adding to cells. Unbound 125I-ligand was removed by rinsing cells three times with cold buffer A after which cells with bound ligand were solubilized with 0.1 N NaOH. Solubilized proteins were added to EcoLume (ICN Biomedicals, Costa Mesa, CA) and subjected to scintillation counting (73% efficiency for iodine 125). Results were normalized to total cellular protein (BCA Protein Assay, Pierce, Rockford, IL). Specificity was determined as the difference between total binding (without competition) and nonspecific binding (non-competable) (48). The actual amount of ligand bound to cells was calculated as cpm divided by the specific activity of 125I-labeled ligand. All data points represent averages of duplicates or triplicates with standard errors of <5%. Experiments were repeated a minimum of three times. The paired t test was used to determine statistical significance.

Solid-phase Assay-- Wells of 96-well plates contained either confluent MEF-3 cells or were pre-coated with RAP-GST or BSA by incubating for 16 h at 4 °C with 20 µg/ml protein diluted into phosphate-buffered saline. Nonspecific sites were blocked by incubating at 4 °C for 2 h with TBS containing 3% BSA. 125I-VLDL was diluted to 2 µg/ml in TBS, 3% BSA and added to wells for 2 h at 4 °C in the presence or absence of unlabeled VLDL (50 µg/ml). Wells were then rinsed with TBS, and proteins were solubilized with 0.1 N NaOH and subjected to scintillation counting as described above.

37 °C Ligand Degradation Assay-- Cells were incubated at 37 °C/5% CO2 with 2 µg/ml 125I-VLDL or 125I-alpha 2M diluted into Dulbecco's modified Eagle's medium containing 1% BSA in the presence or absence of the indicated potential competitor. At the indicated times, media was removed and processed for trichloroacetic acid precipitation (49). Trichloroacetic acid-soluble material was added to EcoLume and subjected to scintillation counting. Degradation was calculated as trichloroacetic acid-soluble cpm divided by the specific activity of the radioiodinated ligand.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Because we have chosen to examine the relationship between HSPG and LRP toward VLDL clearance, we have selected a cell line (mouse embryo fibroblasts, MEF-3) that is devoid of LDL receptor expression to remove its contribution toward VLDL binding and clearance. MEF-3 cells were originally established from LDL receptor knockout mice (50). To confirm LRP expression in these cells, detergent lysates were prepared and subjected to immunoblot analysis using polyclonal antibodies (pAb) specific for the cytoplasmic tail of LRP (46). In MEF-3 cells, anti-LRP antibodies clearly reacted with the light chain of LRP (~95 kDa), which contains the receptor's cytoplasmic tail sequence (Fig. 1, lane 2). MEF-2 cells, which are devoid of LRP expression (51), show no reactivity with anti-LRP demonstrating the specificity of this antibody (Fig. 1, lane 3).


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Fig. 1.   LRP is expressed in LDL receptor-deficient mouse embryo fibroblasts (MEF-3). Cell lysates were prepared using CHAPS detergent and proteins (20 µg/lane) were separated by SDS-PAGE and immunoblotted with anti-LRP pAb. MEF-3 cells demonstrate LRP expression (lane 2). The light chain of LRP (~95 kDa) is the same molecular mass as that seen in normal rat liver (lane 1). MEF-2 cells are LRP-deficient and serve as a negative control for anti-LRP pAb (lane 3).

Because VLDL is known to bind to HSPG on the cell surface prior to its internalization, we examined the relationship between LRP and this primary binding event by first determining if LRP is associated with HSPG at the cell surface. MEF-3 cells were incubated with [35S]methionine to label proteins or 35SO4 to label proteoglycans. Cell lysates were prepared using a mild, non-denaturing detergent (1% Triton X-100) and immunoprecipitated with pAb to LRP. With [35S]methionine labeling, the heavy chain of LRP can be seen at 515 kDa (Fig. 2A). In cells labeled with 35SO4, a very large (>600 kDa) sulfated molecule is found to co-immunoprecipitate with LRP. To determine if this large sulfated molecule is an HSPG, immunoprecipitates were prepared from 35SO4-labeled cells using anti-LRP antibodies, and antibody-bound proteins were treated with or without heparinase. Confirming our results obtained in Fig. 2A, anti-LRP antibodies co-immunoprecipitate a large sulfated molecule from 35SO4-labeled cells (Fig. 2B). When this material is treated with heparinase, little or none of the high molecular weight sulfated molecule remains thus identifying it as an HSPG. These data indicate that LRP is associated with HSPG in a co-immunoprecipitable complex at the cell surface in MEF-3 cells.


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Fig. 2.   HSPG co-immunoprecipitates with LRP. A, cells were metabolically labeled for 16 h with either [35S]methionine or 35SO4. Cell lysates were prepared and immunoprecipitated with anti-LRP polyclonal antibody. LRP is readily detected following [35S]methionine labeling. In cells labeled with 35SO4, a high molecular weight, sulfated molecule is seen to co-immunoprecipitate with LRP. B, immunoprecipitates prepared from cells labeled with 35SO4 using anti-LRP antibodies were treated with or without heparinase I (1 unit/ml). Heparinase I treatment significantly reduced the amount of the large sulfated molecule associated with LRP thus identifying it as an HSPG.

We next examined the binding properties of VLDL on MEF-3 cells. Cells were incubated at 4 °C (to prevent internalization, but permit lateral movement of protein in the plasma membrane) with 125I-VLDL alone or together with unlabeled VLDL, heparin, RAP, or mix of RAP and heparin. Unlabeled VLDL effectively competed for the binding of 125I-VLDL demonstrating the specificity of its binding to the surface of MEF-3 cells (Fig. 3A). Heparin also effectively competed for 125I-VLDL binding confirming that the initial binding of VLDL to cells occurs through HSPG.


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Fig. 3.   RAP increases the availability of binding sites for VLDL. Cells were incubated at 4 °C with 125I-VLDL (2 µg/ml) (A) or 125I-alpha 2M (2 µg/ml) (B) in the presence or absence of the indicated unlabeled ligands (VLDL, 50 µg/ml; heparin, 500 µg/ml; RAP, 50 µg/ml; alpha 2M, 40 µg/ml). A, incubation of cells with RAP significantly increased the binding of 125I-VLDL. Competition for this binding by heparin, VLDL, or a mix of RAP and heparin suggests that the increased 125I-VLDL binding due to RAP incubation is a result of increased availability of cell surface HSPG. B, RAP, alpha 2M, or a combination of RAP and heparin compete for the binding of 125I-alpha 2M to cells demonstrating that our preparation of RAP is an effective ligand-binding antagonist for LRP. As expected, heparin alone has no effect on 125I-alpha 2M binding to cells. The asterisk indicates a statistically significant difference (p < 0.05).

To determine if, in addition to HSPG, 125I-VLDL also bound directly to LRP, we challenged its binding with the receptor-associated protein (RAP), because RAP is known to compete for the binding of all ligands to LRP (52, 53), including VLDL (54, 55). Surprisingly, co-incubation of 125I-VLDL with RAP resulted in a significant increase in VLDL binding (Fig. 3A). Incubation of 125I-VLDL together with RAP and heparin reduced its binding to background levels suggesting that the increase in VLDL binding by co-incubation with RAP is due to increased availability of HSPG binding sites. To confirm that our preparation of RAP is active as a ligand binding antagonist for LRP, we incubated MEF-3 cells with 125I-alpha 2M in the presence or absence of RAP. alpha 2M is known to be a specific ligand for LRP (56). Our preparation of RAP proved to be an effective competitor for 125I-alpha 2M binding to LRP (Fig. 3B). Heparin, as expected, had no effect on 125I-alpha 2M binding to LRP.

To determine if the effect of RAP on VLDL binding to MEF-3 cells is concentration-dependent, cells were incubated at 4 °C with 125I-VLDL and varying concentrations of RAP. As shown in Fig. 4, increasing amounts of RAP do increase the availability of specific VLDL binding sites in a concentration-dependent manner.


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Fig. 4.   The effect of RAP on VLDL binding to MEF-3 cells is concentration-dependent. MEF-3 cells were incubated at 4 °C with 125I-VLDL (2 µg/ml) and the indicated concentrations of RAP. Cells were also incubated in parallel with RAP and heparin (500 µg/ml) together, and these values were subtracted from total binding to obtain specific binding of 125I-VLDL to cells (shown). Increasing amounts of RAP increased the availability of specific binding sites for VLDL on MEF-3 cells.

The fact that heparin effectively competes for 125I-VLDL binding to the increased sites made available by incubation with RAP suggests that these new sites are HSPG. To explore this possibility, we treated MEF-3 cells with 4-methylumbelliferyl-beta -D-xylopyranoside or p-nitrophenyl-beta -D-xylopyranoside, both of which are known to be potent competitors of heparan sulfate polysaccharide chain addition to proteoglycan core proteins (57, 58). Cells treated with these reagents have reduced amounts of glycosaminoglycan associated at the cell surface and have been shown to bind reduced amounts of VLDL (59, 60). We incubated MEF-3 cells with 4-methylumbelliferyl-beta -D-xylopyranoside (Fig. 5A) or p-nitrophenyl-beta -D-xylopyranoside (Fig. 5B) for 72 h, followed by incubation at 4 °C with 125I-VLDL in the presence or absence of RAP. As expected, both 4-methylumbelliferyl-beta -D-xylopyranoside and p-nitrophenyl-beta -D-xylopyranoside reduced overall binding of 125I-VLDL to cells. In addition, both reagents also significantly reduced VLDL binding to the additional sites made available by RAP incubation indicating that these sites are HSPG. To further confirm this observation, 125I-VLDL binding study was performed on MEF-3 cells treated with heparinase (Fig. 6). Following heparinase treatment, the binding of 125I-VLDL to cells was markedly reduced as expected. Moreover, the RAP-inducible binding sites were also significantly reduced due to heparinase treatment. Together, these data indicate that the additional VLDL binding sites made available by incubation with RAP are HSPG.


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Fig. 5.   The RAP-inducible VLDL binding sites are HSPG. MEF-3 cells were grown in the absence or presence of 3 mM 4-methylumbelliferyl-beta -D-xylopyranoside (A) or 3 mM p-nitrophenyl-beta -D-xylopyranoside (B). After 72 h cells were incubated at 4 °C with 125I-VLDL (2 µg/ml) in the presence or absence of RAP (50 µg/ml). Cells were also incubated in parallel with RAP and heparin (500 µg/ml) together to determine nonspecific binding of 125I-VLDL; this value has been subtracted from the reported values. Treatment of MEF-3 cells with either 4-methylumbelliferyl-beta -D-xylopyranoside or p-nitrophenyl-beta -D-xylopyranoside significantly reduced 125I-VLDL binding. Moreover, these treatments also reduced 125I-VLDL binding to cells incubated with RAP indicating that the RAP-inducible binding sites are HSPG. The asterisk indicates a statistically significant difference (p < 0.05).


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Fig. 6.   Heparinase treatment of MEF-3 cells reduced the number of RAP-induced binding sites for VLDL. MEF-3 cells were incubated with or without heparinase (1 unit/ml) for 4 h at 37 °C, followed by incubation at 4 °C with 125I-VLDL (2 µg/ml) in the presence or absence of unlabeled VLDL (50 µg/ml), RAP (50 µg/ml), heparin (500 µg/ml), or combination of RAP and heparin. Heparinase treatment of cells reduced VLDL binding to levels comparable to those obtained by competition with unlabeled VLDL and heparin. Similarly, heparinase also reduced 125I-VLDL binding to RAP-inducible sites to levels comparable to those obtained by competition with a combination of RAP and heparin. The asterisk indicates a statistically significant difference (p < 0.05).

Previous studies have shown that lipoprotein lipase (LPL) is able to increase the binding of VLDL to cells (25, 36, 38, 40, 41) by binding directly to both VLDL particles and HSPG and bridge their interaction. To determine if RAP acts in a similar manner as LPL, we assessed if RAP is able to directly interact with VLDL. To this end, 125I-VLDL was incubated in wells coated with RAP or BSA or containing MEF-3 cells in the presence or absence of unlabeled VLDL. Specific binding of 125I-VLDL to MEF-3 cells was seen as expected, however, no specific binding of the ligand was measured to RAP or BSA (Fig. 7) demonstrating that RAP is unable to bind to VLDL and mediate its binding to cells.


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Fig. 7.   RAP does not bind to VLDL. 125I-VLDL (2 µg/ml) was incubated with RAP or BSA immobilized in 96-well plates or with MEF-3 cells at 4 °C for 2 h in the presence or absence of unlabeled VLDL (50 µg/ml). Binding of 125I-VLDL to MEF-3 cells was competed by unlabeled VLDL confirming specific binding of the ligand. No measurable specific binding of 125I-VLDL to RAP or BSA was detected. Differences in binding of 125I-VLDL to RAP and BSA are not statistically significant (p < 0.05).

After establishing that RAP is able to increase the availability of HSPG for VLDL binding at the cell surface, we next determined the effect of RAP incubation on VLDL internalization and degradation. To this end, cells were incubated at 37 °C with 125I-VLDL in the presence or absence of unlabeled VLDL, RAP, heparin, or RAP together with heparin. After 16 h, culture media was subjected to trichloroacetic acid precipitation and radioactivity in the trichloroacetic acid-soluble fraction, representing degraded ligand, was quantitated. 125I-VLDL uptake and degradation was inhibited by both unlabeled VLDL and heparin demonstrating that specific receptor-mediated internalization is dependent on cell surface HSPG (Fig. 8A). Interestingly, RAP increased the intracellular degradation of VLDL, which is consistent with its ability to increase the number of VLDL binding sites as shown in Figs. 3-6. The RAP-induced increase in 125I-VLDL degradation is competable by heparin suggesting that degradation occurs through an HSPG-mediated internalization pathway. To confirm that our preparation of RAP maintains its inhibitory properties for ligand binding to and uptake by LRP, we incubated MEF-3 cells with 125I-alpha 2M in the presence or absence of RAP (Fig. 8B). RAP effectively blocked uptake and intracellular degradation of 125I-alpha 2M verifying its strong ligand-binding inhibitory property toward LRP. Heparin showed no competition for 125I-alpha 2M binding as expected.


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Fig. 8.   RAP increases 125I-VLDL degradation by MEF-3 cells. MEF-3 cells were incubated at 37 °C with 125I-VLDL (2 µg/ml) (A) or 125I-alpha 2M (2 µg/ml) (B) in the presence or absence of unlabeled VLDL (100 µg/ml), unlabeled alpha 2M (40 µg/ml), RAP (50 µg/ml), heparin (500 µg/ml), or a combination of RAP and heparin. After 16 h, culture media were subjected to trichloroacetic acid precipitation, and soluble radioactivity, representing degraded ligand, was quantitated. A, unlabeled VLDL and heparin inhibited the degradation of 125I-VLDL. RAP increased 125I-VLDL degradation, however, this increase was prevented by a combination of RAP and heparin. B, unlabeled alpha 2M and RAP blocked the degradation of 125I-alpha 2M confirming the inhibitory properties of RAP toward ligand uptake by LRP. Heparin showed little or no effect on 125I-alpha 2M degradation. C, MEF-3 cells were incubated with 125I-VLDL in the presence or absence of RAP at 37 °C for 5.5 or 29 h. Trichloroacetic acid-soluble radioactivity in the media was quantitated. RAP competed for 125I-VLDL degradation during shorter incubations (5.5 h), however, increased 125I-VLDL degradation during longer incubations (29 h). The asterisk indicates a statistically significant difference (p < 0.05).

Other investigators have reported that RAP inhibits VLDL degradation by cultured cells. In these studies, degradation assays were performed for 2-5 h (36, 40, 41, 61) as compared with 16 h in the current study. To directly compare these studies to our current results, we incubated MEF-3 cells with 125I-VLDL at 37 °C for 5.5 or 29 h, followed by trichloroacetic acid precipitation analysis of the culture media. Consistent with prior studies, RAP effectively competed for the degradation of 125I-VLDL after 5.5 h of incubation. However, after 29 h of incubation RAP increased 125I-VLDL degradation by greater than 2-fold over cells treated without RAP. These data indicate that 125I-VLDL degradation is mediated by a RAP-sensitive, LRP-dependent pathway during shorter incubations and an LRP-independent pathway during longer incubations.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study we have examined the relationship between LRP and HSPG at the cell surface and the functional consequences of this relationship toward lipoprotein binding and internalization. First, we have demonstrated that LRP and HSPG form a co-immunoprecipitable complex at the cell surface. Second, we have shown that incubation of cells with the universal ligand-binding antagonist for LRP, namely RAP, increases the number of VLDL binding sites at the cell surface. Third, we have demonstrated that these newly available VLDL binding sites are HSPG using two different competitors of glycosaminoglycan chain addition and heparinase treatment. Fourth, we have shown that by incubating cells with RAP we are able to increase the uptake and degradation of VLDL through this LRP-independent, HSPG pathway. Based on these data, we propose the following model for LRP-HSPG synergistic activity at the cell surface (Fig. 9). LRP and HSPG are associated at the cell surface in a co-immunoprecipitable complex, and the complexed HSPG is unavailable for VLDL binding (Fig. 9A). Through incubation with RAP, the LRP·HSPG complex dissociates, which increases the availability of VLDL binding sites at the surface (Fig. 9B). This activation of additional VLDL binding sites serves to augment the sequestration of VLDL particles to the cell surface, ultimately increasing the efficiency of particle clearance by cells. The measurable increase in intracellular degradation of VLDL due to RAP incubation is consistent with an increased clearance efficiency. Moreover, the observed increase in VLDL degradation during longer incubations with RAP also suggests that uptake occurs through a HSPG internalization pathway, because RAP is known to block direct binding of lipoproteins to LRP (41). Collectively, our data describe a synergistic activity between LRP and HSPG for VLDL binding and clearance and describe a novel function for LRP in regulating the presentation of HSPG at the cell surface.


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Fig. 9.   RAP-induced dissociation of LRP from HSPG increases the availability of VLDL cell surface binding sites. A, LRP and HSPG associate at the cell surface to form a co-immunoprecipitable complex. HSPG, when complexed with LRP, is unable to bind VLDL particles. B, when LRP is activated to release HSPG, as we have shown by RAP incubation, increased VLDL binding sites become available. As our data shows (Fig. 8), this in turn translates into greater efficiency of VLDL uptake and degradation.

The data presented here contrasts with previous studies identifying the role of LRP and HSPG in lipoprotein clearance (22, 62). The sequestration-internalization hypothesis (20, 36), based on a number of comprehensive studies on lipoprotein clearance (25, 37-41), proposes that circulating lipoproteins bind first to cell surface HSPG (sequestration step), followed by receptor-mediated internalization either by the LDL receptor or LRP. In this model, HSPGs serve a passive role in lipoprotein clearance by simply mediating the localization of lipoprotein particles to the cell surface. Particle uptake is proposed to occur following ligand transfer from HSPG to the classic endocytic receptors, the LDL receptor, and LRP. By contrast, our data describe a mechanism whereby LRP regulates the availability of lipoprotein binding sites by associating with HSPG. Because LRP dissociates from HSPG to uncover additional lipoprotein binding sites, the increased availability of HSPG at the cell surface in turn serves to increase VLDL clearance. Thus, our data propose a more active role for HSPG in lipoprotein clearance, because we show they are able to mediate both the binding and internalization of VLDL. Others have also demonstrated that HSPGs are able to directly bind and internalize lipoprotein particles, however, many of these studies have remained in the shadow of those reporting particle clearance by the more classic endocytic receptors. Our data strongly support an active, independent role for HSPG in lipoprotein clearance and describe a novel role for LRP in regulating the presentation of HSPG at the cell surface.

We also observed a shift in receptor utilization when measuring VLDL internalization and degradation. Our data shows that shorter incubation times with RAP (<5.5 h) reduced VLDL metabolism, as seen in previous studies (36, 40, 41), suggesting that LRP may be responsible for rapid, immediate VLDL internalization. By contrast, longer incubation times with RAP (>16 h) increased VLDL degradation indicating that an LRP-independent HSPG pathway contributes significantly to lipoprotein clearance over more extended time periods.

RAP is a well-characterized chaperone for members of the LDL receptor gene family (52, 53) and is primarily found in the endoplasmic reticulum (63, 64), retained there by its C-terminal His-Asn-Glu-Leu sequence (65). As a chaperone, RAP has been shown to assist in the folding and exocytic trafficking of LRP (66). RAP also has the unique property of blocking the binding of all known ligands to LRP (52, 53), which has proven invaluable for functional studies of LRP. Because of its endoplasmic reticulum retention properties, little or no RAP can be found at the cell surface. Therefore, we believe that RAP is mimicking the effect of an LRP-specific extracellular ligand in dissociating LRP from HSPG to make additional sites available for lipoprotein binding. Recent studies have shown that addition of lipoprotein lipase increases lipoprotein particle binding to cell surface HSPG (25, 36, 38, 41, 67) as well as increase their LRP-mediated clearance (41, 61). LPL is thought to increase lipoprotein metabolism by bridging VLDL particle binding to HSPG and LRP, because it is able to bind both cell surface receptors (36, 38, 39). However, activation of LRP to release HSPG sites likely occurs by a different mechanism than ligand bridging, because we show here that RAP is unable to bind VLDL and thus unable to bridge VLDL binding to both HSPG and LRP. Alternatively, RAP may bind HSPG and simply displace a protein already bound to cell surface HSPG thereby making HPSG available to bind other soluble ligands. However, we have only observed the binding of RAP to heparin following its denaturation (45). We have interpreted this to indicate that the heparin binding domain in RAP is cryptic in the native protein. Other studies have confirmed this showing that radiolabeled RAP is unable to bind to cell surface HSPG in fibroblasts (68).

The nature of the association between HSPG and LRP is currently unknown. No member of the LDL receptor family has been reported to bind heparin or heparan sulfate moieties, so the interaction likely does not involve direct glycosaminoglycan interactions with LRP. Recently, CASK/LIN-2 has been identified to interact with the cytoplasmic tail of all four members of the syndecan HSPG family (69, 70) and is thought to mediate syndecan coupling to the actin cytoskeleton through interactions with protein 4.1 (70). LRP has also been reported to bind cytosolic adaptors, such as FE65 and Disabled, to its cytoplasmic tail (71). Therefore, it is possible that a bridging protein may bring together HSPG and LRP through cytosolic interactions.

LRP was originally thought to be an independent endocytic receptor for many soluble extracellular ligands. After binding these ligands at the cell surface, LRP rapidly internalizes to deliver ligand to lysosomes for degradation and recycles to the surface for additional rounds of binding and internalization. However, we and others have recently identified a greater complexity in LRP function. LRP is now known to interact with other cell surface receptors and affect their activities. For example, LRP associates with the amyloid precursor protein (72, 73) and through this interaction increases proteolysis of the amyloid precursor protein to generate Abeta peptide (74), the amyloidogenic factor responsible for neurologic plaques in Alzheimer disease. LRP is also known to form a co-immunoprecipitable complex with urokinase receptor on the surface of human fibrosarcoma cells, and through this interaction establishes a functional relationship with the plasminogen activation system that affects the invasive phenotype of this cancer line (75). These studies suggest that LRP activity is more complex than originally thought and affects other cell surface receptor systems in addition to its function in soluble ligand clearance. Our studies here extend the functions of LRP to regulating HSPG availability at the cell surface. Moreover, because integral HSPGs are known to shed from the cell surface through proteolytic cleavage (76), LRP may also protect them from proteolysis and prevent a loss of HSPGs that are critical to cell function. In addition to lipoproteins, HSPGs are also known to bind cytokines such as basic fibroblast growth factor (77) and hepatocyte growth factor (78). Binding of these factors by HSPG affects their binding to receptors and downstream signaling activities (77, 79). Although it remains to be determined if LRP regulates the availability of HSPG sites for these growth factors, such a regulation may suggest that the functional properties of LRP extend to a role in modulating diverse cell signaling events.

    ACKNOWLEDGEMENT

We are grateful for the generosity of Dr. Joachim Herz from the University of Texas Southwestern Medical Center, Dallas, TX, for supplying us with MEF-2 and MEF-3 cells.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL63291 (to R. A. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: MSC08-4670, 1 University of New Mexico, Albuquerque, NM 87131. Tel.: 505-272-5593; Fax: 505-272-3518; E-mail: rorlando@salud.unm.edu.

Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M208786200

    ABBREVIATIONS

The abbreviations used are: VLDL, very low density lipoprotein; HSPG, heparan sulfate proteoglycan; LDL, low density lipoprotein; LRP, lipoprotein receptor-related protein; alpha 2M, alpha 2-macroglobulin; GST, glutathione S-transferase; MEF, mouse embryo fibroblast; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BSA, bovine serum albumin; pAb, polyclonal antibody; RAP, receptor-associated protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. McKeone, B. J., Massey, J. B., Knapp, R. D., and Pownall, H. J. (1988) Biochemistry 27, 4500-4505[Medline] [Order article via Infotrieve]
2. Huang, Y., Liu, X. Q., Rall, S. C., Jr., and Mahley, R. W. (1998) J. Biol. Chem. 273, 17483-17490[Abstract/Free Full Text]
3. MacPhee, C. E., Hatters, D. M., Sawyer, W. H., and Howlett, G. J. (2000) Biochemistry 39, 3433-3440[CrossRef][Medline] [Order article via Infotrieve]
4. Olivecrona, G., and Beisiegel, U. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 1545-1549[Abstract/Free Full Text]
5. Karpe, F. (1999) J. Intern. Med. 246, 341-355[CrossRef][Medline] [Order article via Infotrieve]
6. Breslow, J. L. (1996) Science 272, 685-688[Abstract]
7. Knouff, C., Hinsdale, M. E., Mezdour, H., Altenburg, M. K., Watanabe, M., Quarfordt, S. H., Sullivan, P. M., and Maeda, N. (1999) J. Clin. Invest. 103, 1579-1586[Abstract/Free Full Text]
8. Mahley, R. W. (1988) Science 240, 622-630[Medline] [Order article via Infotrieve]
9. Yu, K. C., and Cooper, A. D. (2001) Front. Biosci. 6, D332-D354[Medline] [Order article via Infotrieve]
10. Havel, R. J., Yamada, N., and Shames, D. M. (1989) Arteriosclerosis 9, I33-I38
11. Havel, R. J. (1994) Curr. Opin. Lipidol. 5, 102-109[Medline] [Order article via Infotrieve]
12. Seo, T., and St Clair, R. W. (1997) J. Lipid Res. 38, 765-779[Abstract]
13. Jaakkola, O., Yla-Herttuala, S., Sarkioja, T., and Nikkari, T. (1989) Atherosclerosis 79, 173-182[Medline] [Order article via Infotrieve]
14. Ji, Z. S., Brecht, W. J., Miranda, R. D., Hussain, M. M., Innerarity, T. L., and Mahley, R. W. (1993) J. Biol. Chem. 268, 10160-10167[Abstract/Free Full Text]
15. Cardin, A. D., Hirose, N., Blankenship, D. T., Jackson, R. L., Harmony, J. A., Sparrow, D. A., and Sparrow, J. T. (1986) Biochem. Biophys. Res. Commun. 134, 783-789[Medline] [Order article via Infotrieve]
16. Libeu, C. P., Lund-Katz, S., Phillips, M. C., Wehrli, S., Hernaiz, M. J., Capila, I., Linhardt, R. J., Raffai, R. L., Newhouse, Y. M., Zhou, F., and Weisgraber, K. H. (2001) J. Biol. Chem. 276, 39138-39144[Abstract/Free Full Text]
17. Plump, A. S., Smith, J. D., Hayek, T., Aalto-Setala, K., Walsh, A., Verstuyft, J. G., Rubin, E. M., and Breslow, J. L. (1992) Cell 71, 343-353[Medline] [Order article via Infotrieve]
18. Zhang, S. H., Reddick, R. L., Piedrahita, J. A., and Maeda, N. (1992) Science 258, 468-471[Medline] [Order article via Infotrieve]
19. Krieger, M., and Herz, J. (1994) Ann. Rev. Biochem. 63, 601-637[CrossRef][Medline] [Order article via Infotrieve]
20. Mahley, R. W., and Ji, Z. S. (1999) J. Lipid Res. 40, 1-16[Abstract/Free Full Text]
21. Chappell, D. A., Fry, G. L., Waknitz, M. A., Muhonen, L. E., and Pladet, M. W. (1993) J. Biol. Chem. 268, 25487-25493[Abstract/Free Full Text]
22. Mahley, R. W., Ji, Z. S., Brecht, W. J., Miranda, R. D., and He, D. (1994) Ann. N. Y. Acad. Sci. 737, 39-52[Medline] [Order article via Infotrieve]
23. Herz, J., and Willnow, T. E. (1995) Curr. Opin. Lipidol. 6, 97-103[Medline] [Order article via Infotrieve]
24. Ji, Z. S., Sanan, D. A., and Mahley, R. W. (1995) J. Lipid Res. 36, 583-592[Abstract]
25. Williams, K. J., Fless, G. M., Petrie, K. A., Snyder, M. L., Brocia, R. W., and Swenson, T. L. (1992) J. Biol. Chem. 267, 13284-13292[Abstract/Free Full Text]
26. Hussain, M. M., Strickland, D. K., and Bakillah, A. (1999) Annu. Rev. Nutr. 19, 141-172[CrossRef][Medline] [Order article via Infotrieve]
27. Moestrup, S. K., Gliemann, J., and Pallesen, G. (1992) Cell Tissue Res. 269, 375-382[Medline] [Order article via Infotrieve]
28. Rohlmann, A., Gotthardt, M., Hammer, R. E., and Herz, J. (1998) J. Clin. Invest. 101, 689-695[Abstract/Free Full Text]
29. Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J. (1994) Science 264, 1471-1474[Medline] [Order article via Infotrieve]
30. Field, P. A., and Gibbons, G. F. (2000) Metab. Clin. Exp. 49, 492-498[Medline] [Order article via Infotrieve]
31. Fuki, I. V., Kuhn, K. M., Lomazov, I. R., Rothman, V. L., Tuszynski, G. P., Iozzo, R. V., Swenson, T. L., Fisher, E. A., and Williams, K. J. (1997) J. Clin. Invest. 100, 1611-1622[Abstract/Free Full Text]
32. Fuki, I. V., Iozzo, R. V., and Williams, K. J. (2000) J. Biol. Chem. 275, 25742-25750[Abstract/Free Full Text]
33. Roskams, T., Moshage, H., De Vos, R., Guido, D., Yap, P., and Desmet, V. (1995) Hepatology 21, 950-958[Medline] [Order article via Infotrieve]
34. Bernfield, M., Kokenyesi, R., Kato, M., Hinkes, M. T., Spring, J., Gallo, R. L., and Lose, E. J. (1992) Annu. Rev. Cell Biol. 8, 365-393[CrossRef][Medline] [Order article via Infotrieve]
35. Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J., and Zako, M. (1999) Ann. Rev. Biochem. 68, 729-777[CrossRef][Medline] [Order article via Infotrieve]
36. Nykjaer, A., Bengtsson-Olivecrona, G., Lookene, A., Moestrup, S. K., Petersen, C. M., Weber, W., Beisiegel, U., and Gliemann, J. (1993) J. Biol. Chem. 268, 15048-15055[Abstract/Free Full Text]
37. Aggerbeck, L. P., Angelin, B., Armstrong, V., Franceschini, G., Humphries, S., Rosseneu, M., Soutar, A., and Zechner, R. (1993) Arterioscler. Thromb. 13, 618-627[Medline] [Order article via Infotrieve]
38. Beisiegel, U., Weber, W., and Bengtsson-Olivecrona, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8342-8346[Abstract]
39. Chappell, D. A., Fry, G. L., Waknitz, M. A., Iverius, P. H., Williams, S. E., and Strickland, D. K. (1992) J. Biol. Chem. 267, 25764-25767[Abstract/Free Full Text]
40. Chappell, D. A., Fry, G. L., Waknitz, M. A., Muhonen, L. E., Pladet, M. W., Iverius, P. H., and Strickland, D. K. (1993) J. Biol. Chem. 268, 14168-14175[Abstract/Free Full Text]
41. Weaver, A. M., Lysiak, J. J., and Gonias, S. L. (1997) J. Lipid Res. 38, 1841-1850[Abstract]
42. Ji, Z. S., Lauer, S. J., Fazio, S., Bensadoun, A., Taylor, J. M., and Mahley, R. W. (1994) J. Biol. Chem. 269, 13429-13436[Abstract/Free Full Text]
43. Shafi, S., Brady, S. E., Bensadoun, A., and Havel, R. J. (1994) J. Lipid Res. 35, 709-720[Abstract]
44. Mulder, M., Lombardi, P., Jansen, H., van Berkel, T. J., Frants, R. R., and Havekes, L. M. (1993) J. Biol. Chem. 268, 9369-9375[Abstract/Free Full Text]
45. Orlando, R. A., and Farquhar, M. G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3161-3165[Abstract]
46. Czekay, R. P., Orlando, R. A., Woodward, L., Adamson, E. D., and Farquhar, M. G. (1995) J. Cell Sci. 108, 1433-1441[Abstract/Free Full Text]
47. Kerjaschki, D., Exner, M., Ullrich, R., Susani, M., Curtiss, L. K., Witztum, J. L., Farquhar, M. G., and Orlando, R. A. (1997) J. Clin. Invest. 100, 2303-2309[Abstract/Free Full Text]
48. Goldstein, J. L., Basu, S. K., and Brown, M. S. (1983) Methods Enzymol. 98, 241-260[Medline] [Order article via Infotrieve]
49. Czekay, R. P., Orlando, R. A., Woodward, L., Lundstrom, M., and Farquhar, M. G. (1997) Mol. Biol. Cell 8, 517-532[Abstract]
50. Ishibashi, S., Brown, M. S., Goldstein, J. L., Gerard, R. D., Hammer, R. E., and Herz, J. (1993) J. Clin. Invest. 92, 883-893[Medline] [Order article via Infotrieve]
51. Weaver, A. M., Hussaini, I. M., Mazar, A., Henkin, J., and Gonias, S. L. (1997) J. Biol. Chem. 272, 14372-14379[Abstract/Free Full Text]
52. Willnow, T. E. (1998) Biol. Chem. 379, 1025-1031[Medline] [Order article via Infotrieve]
53. Bu, G., and Schwartz, A. L. (1998) Trends Cell Biol. 8, 272-276[CrossRef][Medline] [Order article via Infotrieve]
54. Chappell, D. A., Inoue, I., Fry, G. L., Pladet, M. W., Bowen, S. L., Iverius, P. H., Lalouel, J. M., and Strickland, D. K. (1994) J. Biol. Chem. 269, 18001-18006[Abstract/Free Full Text]
55. Medh, J. D., Fry, G. L., Bowen, S. L., Pladet, M. W., Strickland, D. K., and Chappell, D. A. (1995) J. Biol. Chem. 270, 536-540[Abstract/Free Full Text]
56. Strickland, D. K., Kounnas, M. Z., and Argraves, W. S. (1995) FASEB J. 9, 890-898[Abstract/Free Full Text]
57. Lugemwa, F. N., Sarkar, A. K., and Esko, J. D. (1996) J. Biol. Chem. 271, 19159-19165[Abstract/Free Full Text]
58. Taylor, W. H., Sinha, A., Khan, I. A., McDaniel, S. T., and Esko, J. D. (1998) J. Biol. Chem. 273, 22260-22266[Abstract/Free Full Text]
59. Zeng, B. J., Mortimer, B. C., Martins, I. J., Seydel, U., and Redgrave, T. G. (1998) J. Lipid Res. 39, 845-860[Abstract/Free Full Text]
60. Huff, M. W., Miller, D. B., Wolfe, B. M., Connelly, P. W., and Sawyez, C. G. (1997) J. Lipid Res. 38, 1318-1333[Abstract]
61. Rinninger, F., Kaiser, T., Mann, W. A., Meyer, N., Greten, H., and Beisiegel, U. (1998) J. Lipid Res. 39, 1335-1348[Abstract/Free Full Text]
62. Chappell, D. A., and Medh, J. D. (1998) Prog. Lipid Res. 37, 393-422[CrossRef][Medline] [Order article via Infotrieve]
63. Lundstrom, M., Orlando, R. A., Saedi, M. S., Woodward, L., Kurihara, H., and Farquhar, M. G. (1993) Am. J. Pathol. 143, 1423-1435[Abstract]
64. Bu, G., Geuze, H. J., Strous, G. J., and Schwartz, A. L. (1995) EMBO J. 14, 2269-2280[Abstract]
65. Bu, G., Rennke, S., and Geuze, H. J. (1997) J. Cell Sci. 110, 65-73[Abstract/Free Full Text]
66. Obermoeller, L. M., Chen, Z., Schwartz, A. L., and Bu, G. (1998) J. Biol. Chem. 273, 22374-22381[Abstract/Free Full Text]
67. de Beer, F., Hendriks, W. L., van Vark, L. C., Kamerling, S. W., van Dijk, K. W., Hofker, M. H., Smelt, A. H., and Havekes, L. M. (1999) Arterioscler. Thromb. Vasc. Biol. 19, 633-637[Abstract/Free Full Text]
68. Vassiliou, G., and Stanley, K. K. (1994) J. Biol. Chem. 269, 15172-15178[Abstract/Free Full Text]
69. Hsueh, Y. P., Yang, F. C., Kharazia, V., Naisbitt, S., Cohen, A. R., Weinberg, R. J., and Sheng, M. (1998) J. Cell Biol. 142, 139-151[Abstract/Free Full Text]
70. Cohen, A. R., Woods, D. F., Marfatia, S. M., Walther, Z., Chishti, A. H., Anderson, J. M., and Wood, D. F. (1998) J. Cell Biol. 142, 129-138[Abstract/Free Full Text]
71. Trommsdorff, M., Borg, J. P., Margolis, B., and Herz, J. (1998) J. Biol. Chem. 273, 33556-33560[Abstract/Free Full Text]
72. Knauer, M. F., Orlando, R. A., and Glabe, C. G. (1996) Brain Res. 740, 6-14[CrossRef][Medline] [Order article via Infotrieve]
73. Rebeck, G. W., Moir, R. D., Mui, S., Strickland, D. K., Tanzi, R. E., and Hyman, B. T. (2001) Brain Res. Mol. Brain Res. 87, 238-245[Medline] [Order article via Infotrieve]
74. Ulery, P. G., Beers, J., Mikhailenko, I., Tanzi, R. E., Rebeck, G. W., Hyman, B. T., and Strickland, D. K. (2000) J. Biol. Chem. 275, 7410-7415[Abstract/Free Full Text]
75. Czekay, R. P., Kuemmel, T. A., Orlando, R. A., and Farquhar, M. G. (2001) Mol. Biol. Cell 12, 1467-1479[Abstract/Free Full Text]
76. Fitzgerald, M. L., Wang, Z., Park, P. W., Murphy, G., and Bernfield, M. (2000) J. Cell Biol. 148, 811-824[Abstract/Free Full Text]
77. Vlodavsky, I., Miao, H. Q., Medalion, B., Danagher, P., and Ron, D. (1996) Cancer Metastasis Rev. 15, 177-186[Medline] [Order article via Infotrieve]
78. van der Voort, R., Keehnen, R. M., Beuling, E. A., Spaargaren, M., and Pals, S. T. (2000) J. Exp. Med. 192, 1115-1124[Abstract/Free Full Text]
79. Baeg, G. H., and Perrimon, N. (2000) Curr. Opin. Cell Biol. 12, 575-580[CrossRef][Medline] [Order article via Infotrieve]


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