Syndecan-1 mediates internalization of apoE-VLDL through a low density lipoprotein receptor-related protein (LRP)-independent, non-clathrin-mediated pathway

1476-511X-5-23 1476-511X Research Syndecan-1 mediates internalization of apoE-VLDL through a low density lipoprotein receptor-related protein (LRP)-independent, non-clathrin-mediated pathway Wilsie C Larissa lcwilsie@merck.com Gonzales M Amanda amgonzales@salud.unm.edu Orlando A Robert rorlando@salud.unm.edu

Department of Biochemistry and Molecular Biology, University of New Mexico, School of Medicine, MSC08 4670 1 University of New Mexico, Albuquerque, New Mexico, 87131, USA

Lipids in Health and Disease 1476-511X 2006 5 1 23 http://www.lipidworld.com/content/5/1/23 16945147 10.1186/1476-511X-5-23 09 8 2006 31 8 2006 31 8 2006 2006 Wilsie et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Triacylglyerol-rich very low density lipoprotein (VLDL) particles are the primary carriers of fatty acids in the circulation and as such serve as a rich energy source for peripheral tissues. Receptor-mediated uptake of these particles is dependent upon prior association with apolipoprotein E (apoE-VLDL) and is brought about by cell surface heparan sulfate proteoglycans (HSPG) in some cell types and by the low density lipoprotein receptor-related protein (LRP) in others. Although LRP's role in apoE-VLDL uptake has been well studied, the identity of the HSPG family member that mediates apoE-VLDL uptake has not been established. We investigated if syndecan-1 (Syn-1), a transmembrane cell surface HSPG, is able to mediate the internalization of apoE-VLDL and examined the relationship between Syn-1 and LRP toward apoE-VLDL uptake. For this study, we used a human fibroblast cell line (GM00701) that expresses large amounts of LRP, but possesses no LDL receptor activity to eliminate its contributions toward apoE-VLDL uptake.

Results

Although LRP in these cells is fully active as established by substantial α₂macroglobulin binding and internalization, uptake of apoE-VLDL is absent. Expression of human Syn-1 cDNA restored apoE-VLDL binding and uptake by these cells. Competition for this uptake with an LRP ligand-binding antagonist had little or no effect, whereas co-incubation with heparin abolished apoE-VLDL internalization. Depleting Syn-1 expressing cells of K⁺, to block clathrin-mediated endocytosis, showed no inhibition of Syn-1 internalization of apoE-VLDL. By contrast, treatment of cells with nystatin to inhibit lipid raft function, prevented the uptake of apoE-VLDL by Syn-1.

Conclusion

These data demonstrate that Syn-1 is able to mediate apoE-VLDL uptake in human fibroblasts with little or no contribution from LRP and that the endocytic path taken by Syn-1 is clathrin-independent and relies upon lipid raft function. These data are consistent with previous studies demonstrating Syn-1 association with lipid raft domains.

Background

Fatty acids, triacylglycerols, and cholesterol in plasma originate primarily from two sources; dietary intake and that which is synthesized and secreted by hepatocytes. Dietary lipids circulate in the form of chylomicrons which are synthesized by intestinal epithelial cells. They are relatively short lived particles during the postprandial period as they are rapidly metabolized to remnant lipoproteins and cleared from the circulation primarily by hepatocytes. By contrast, the liver synthesizes triacylglycerol-rich lipoproteins in the form of very low density lipoprotein (VLDL) particles which are much longer-lived fatty acid carriers than remnant lipoproteins. Because of this, VLDL particles provide the largest single source of fatty acids for peripheral tissues found in the circulation. These fatty acids are a rich energy source for cells with high metabolic rates and are also stored by adipose tissue for mobilization during periods of fasting. Previous studies have also shown that VLDL is highly atherogenic since excessive uptake of these lipoproteins by macrophages causes massive cholesterol accumulation and foam cell formation 123. Moreover, elevated levels of VLDL are found in the plasma of patients with type III hyperlipoproteinemia 4. It follows from these observations that cardiovascular health requires controlled levels of VLDL particles in the circulation, whether by decreased synthesis or increased uptake.

Considerable evidence has been presented suggesting that the mechanism for VLDL particle clearance involves cell surface binding and endocytic activities of either heparan sulfate proteoglycans (HSPG) 567 or the low density lipoprotein receptor-related protein (LRP) 89, or both receptors acting in a synergistic manner at the cell surface 10. Receptor-mediated association of VLDL with the cell surface also requires enrichment of the particle with apolipoprotein E (apoE) 611. Evidence supporting a role for LRP in apoE-VLDL uptake has been gathered by blocking LRP's endocytic function with a ligand binding antagonist or by tissue-specific gene ablation, both of which result in increased circulating levels of VLDL particles 1213. HSPG-mediated internalization of apoE-VLDL has been reported in several independent cell culture systems including fibroblasts 141516, CHO cells 17, HepG2 cells 1819, macrophages 20, and vascular smooth muscle cells 21. In each of these cell types, a significant reduction in apoE-VLDL internalization was demonstrated following the inhibition of HSPG activity either through a coincubation with heparin or heparinase treatment prior to ligand binding. Moreover, intravenous administration of heparinase into the portal circulation reduced hepatocyte-mediated VLDL uptake by 70% 710. Notably, in contrast to the studies examining LRP's role in apoE-VLDL clearance, studies on HSPG report that a coincubation of labeled apoE-VLDL with an LRP ligand binding antagonist showed minimal effects on lipoprotein uptake indicating little or no participation by this receptor. These results have created controversy as to the identity of the receptor used by VLDL particles for endocytic clearance. To distinguish which path, HSPG or LRP, serves as the primary uptake mechanism for apoE-VLDL, we have recently re-examined apoE-VLDL endocytosis following the inhibition of HSPG maturation using reagents that prevent glycosaminoglycan chain addition 16. In this study, we showed that apoE-VLDL uptake occurs primarily through an HSPG-mediated process with little or no contributions from LRP.

Identifying the HSPG that mediates apoE-VLDL clearance has been elusive. Specific HSPG, like those of the syndecan 2223, glypican 2324 and perlecan 2526 families, have primarily been studied in the context of their abilities to modulate growth factor responses. Although, syndecan 2728 and perlecan 2930 have been shown to internalize modified LDL, little information is available identifying the HSPG responsible for apoE-VLDL uptake. Syndecan-1 (Syn-1) was recently shown to colocalize with chylomicron remnants at the surface of hepatocytes suggesting that it may participate in the endocytic clearance of dietary lipids 31. In the present study, we investigated if Syn-1 is capable of mediating apoE-VLDL uptake and examined its functional relationship with LRP in lipoprotein handling at the cell surface.

Results

To determine if Syn-1 is capable of endocytic uptake of apoE-VLDL and to examine the relationship between Syn-1 and LRP toward apoE-VLDL clearance, we searched for a cell line that 1) expresses no detectable Syn-1 to permit transfection studies with human Syn-1, 2) expresses high levels of LRP to permit accurate and reliable measurements of its endocytic properties, and 3) expresses no LDL receptor activity to remove its contribution toward apoE-VLDL binding and clearance that might complicate our measurements of Syn-1 and LRP activities. Our search led us to the GM00701 human cell line which was obtained from a patient diagnosed with familial hypercholesterolemia and expresses LDL receptors with < 1% of normal activity. We compared LRP expression in this cell line to other cell lines known to express this receptor, including human SH-SY5Y neuroblastoma 32, mouse pre-adipocyte 3T3-L1 33 and human HT1080 fibrosarcoma 34. By semi-quantitative immunoblot analysis, we found that the GM00701 cells express more LRP than other cultured cells previously used to study LRP's cellular activities (Fig. 1). Interestingly, the relative molecular mass of LRP's light chain (~95 kDa) is slightly smaller in the cultured cell lines as compared to liver tissue, which is the in vivo location for the highest level of LRP expression. This is likely due to post-translational processing since no alternatively spliced transcripts for LRP have been reported.

Figure 1

Comparison of LRP expression in various cell lines

Comparison of LRP expression in various cell lines. (A) Cell lysates were prepared using 1% Triton X-100 detergent, proteins (20 μg/lane) were separated by 6% SDS-PAGE, and immunoblotted with anti-LRP pAb raised against a synthetic peptide derived from the cytoplasmic tail sequence [59]. Bound antibodies were detected by chemiluminescence imaging. Rat liver extract was used as a positive control for LRP expression. (B) Densitometric scans of the 95 kD light chain of LRP in (A).

Since GM00701 cells express large amounts of LRP, we anticipated that they would have a substantial capacity to bind and degrade the LRP specific ligand, α₂-macroglobulin (α₂M). To test this assumption, GM00701 cells were incubated at 4°C with ¹²⁵I-α₂M in the absence or presence of unlabeled α₂M or the LRP ligand binding antagonist, receptor associated protein (RAP). A significant amount of α₂M bound to GM00701 cells (Fig. 2A), of which 80–90% was specific as determined by competition with unlabeled α₂M and RAP. When the same experiment was performed at 37°C to measure LRP-specific ligand internalization and degradation, we found that GM00701 cells degraded more ¹²⁵I-α₂M than the binding capacity at the cell surface suggesting not only efficient LRP internalization, but also rapid recycling (Fig. 2B).

Figure 2

¹²⁵I-α₂M and ¹²⁵I-apoE-VLDL handling by GM00701 cells

¹²⁵I-α₂M and ¹²⁵I-apoE-VLDL handling by GM00701 cells. GM00701 cells were incubated with ¹²⁵I-α₂M (1 μg/ml) at 4°C for 3 h (A) or at 37°C for 1 h (B) in the absence or presence of recombinant human RAP-GST (50 μg/ml) or unlabeled α₂M (10 μg/ml). (C) GM00701 and 3T3-L1 cells were incubated with ¹²⁵I-apoE-VLDL (2 μg/ml) supplemented with 3 μg/ml apoE at 4°C for 3 h in the absence (solid bars) or presence (shaded bars) of unlabeled apoE-VLDL (100 μg/ml). After the indicated incubation time, ¹²⁵I-ligand bound to cells at 4°C was quantitated by scintillation counting following extraction of cells with 0.1 M NaOH. ¹²⁵I-α₂M degradation was quantitated following TCA precipitation as described in Methods. Error bars represent standard deviations of triplicate points. Inset, purified VLDL was separated by 7.5% SDS-PAGE and proteins were stained with Coomassie R. Molecular mass markers shown at left are in kD.

Since LRP has previously been reported to bind apoE-VLDL particles 89, we investigated if GM00701 cells were able to efficiently bind ¹²⁵I-apoE-VLDL with its high expression level of LRP. To accomplish this, we isolated VLDL particles from plasma obtained from New Zealand White rabbits maintained on a high fat, high cholesterol diet. Prior to radiolabeling, the VLDL preparation was analyzed by SDS-PAGE followed by Coomassie staining to confirm that these particles contain the expected apolipoproteins, B100 and E. As shown in Figure 2C (inset), the purified VLDL particles do indeed contain apoB100 (Mr, 512 kD) and apoE (Mr, 34 kD) indicating that they have the proper associated proteins necessary for efficient receptor binding and internalization. When GM00701 cells were incubated at 4°C with ¹²⁵I-apoE-VLDL, we found little or no specific binding (Fig. 2C) indicating that these cells are incapable of binding apoE-VLDL in spite of the presence of endocytically active LRP. Cell surface binding activity of our ¹²⁵I-apoE-VLDL preparation was confirmed by high level specific binding to mouse pre-adipocyte 3T3-L1 cells 35.

The inability of GM00701 fibroblasts to bind ¹²⁵I-apoE-VLDL and the lack of Syn-1 expression as determined by immunoblot and RT-PCR analysis (data not shown), established these cells as a viable model for us to investigate the functional relationship between Syn-1 and LRP toward apoE-VLDL binding and clearance. Toward this end, we constructed a mammalian expression vector containing the full length human Syn-1 cDNA (pMH/Syn-1-HA) and carried out transient transfection studies with GM00701 cells. Immunoblot analysis of cells transfected with vector alone (Fig. 3A, lane 1) showed no endogenous expression of Syn-1. By contrast, cells transfected with pMH/Syn-1-HA (lane 2) demonstrated efficient Syn-1 expression (Mr ~77kDa). The 77 kDa protein identified by immunoblotting represents the core protein of Syn-1 3637. Mature Syn-1 demonstrates a relative molecular mass of > 250 kD as a result of its multiple glycosaminoglycan additions 38.

Figure 3

Expression of syndecan-1 in GM00701 cells

Expression of syndecan-1 in GM00701 cells. (A) GM00701 cells were transfected with pMH alone (lane 1) or pMH/Syn-1-HA (lane 2). Forty eight hours after transfection, total protein was extracted from cells with 1% Triton X-100 and equal amounts of protein (20 μg/lane) were separated by 7.5% SDS-PAGE, transferred to PVDF membrane and immunoblotted with anti-Syn-1 polyclonal antisera (1:1000) that was raised against a recombinant his-tagged fusion protein representing full length human Syn-1. Chemiluminescence detection was used to visualize bound antibodies. (B) GM00701 cells were transfected with pMH/Syn-1-HA and cells were selected that stably express Syn-1 (GM00701/Syn-1-HA). Cells were incubated with either TBS (lane 1), 8 M urea (lane 2), or 1% Triton X-100 (lane 3). Insoluble material was pelleted by centrifugation and soluble proteins were separated by 7.5% SDS-PAGE and immunoblotted with anti-Syn-1 antisera as described in (A). (C) GM00701/Syn-1-HA cells were incubated without (lane 1) or with (lane 2) heparinase (1 unit/ml) for 5 h at 37°C. Total cellular proteins were extracted with 8 M urea, separated by 6% SDS-PAGE and immunoblotted with anti-Syn-1 antisera as described in (A). Asterisks indicate the 77 kD, non-glycosylated Syn-1 core protein [36, 37].

In order to confirm proper maturation of Syn-1 in transfected cells as defined by heparan sulfate glycosaminoglycan addition, we transfected GM00701 cells with pMH/Syn-1-HA vector and enriched for those cells stably expressing Syn-1 using antibiotic selection. We then immunoblotted protein extracts from GM00701/Syn-1-HA cells prepared with 8 M urea. Urea extraction is often necessary to release mature Syn-1 from cytoskeletal interactions that are resistant to mild detergents such as Triton X-100 3739. As shown in Figure 3B, a high molecular mass protein (Mr, > 250 kD) was identified by anti-Syn-1 antibodies following urea extraction (lane 2), but not after extraction with Triton X-100 (lane 3). As expected, treatment with Tris-buffered saline was unable to extract Syn-1 from cells (lane 1) since Syn-1 is an integral membrane protein.

To verify that the large molecular mass change in Syn-1 as seen by immunoblotting is due to heparan sulfate glycosaminoglycan addition, we investigated the effect of heparinase treatment on the electrophoretic mobility of Syn-1 from GM00701/Syn-1-HA cells. Without heparinase treatment, mature Syn-1 migrated to its expected relative molecular mass of > 250 kDa (Fig. 3C, lane 1). With heparinase treatment, a significant increase in Syn-1 core protein (Mr, 77 kDa) was observed that directly corresponded to a decrease in mature Syn-1 (Mr, > 250 kDa) (lane 2). We interpret these data to indicate that mature Syn-1 expressed by GM00701/Syn-1-HA cells contains heparan sulfate glycosaminoglycan chains and undergoes normal post-translational modifications.

Since GM00701 cells are unable to bind apoE-VLDL even with high expression levels of LRP, we next asked if introducing Syn-1 expression is able to rescue apoE-VLDL uptake by these cells. To address this, GM00701 and GM00701/Syn-1-HA cells were incubated with DiI-labeled apoE-VLDL at 37°C and examined by fluorescence microscopy for uptake of the labeled ligand. Consistent with the results we obtained in 4°C binding studies with ¹²⁵I-apoE-VLDL, GM00701 cells were unable to bind or internalize DiI-apoE-VLDL (Fig. 4, panel C). However, GM00701/Syn-1-HA cells were able to readily take up DiI-apoE-VLDL as evidenced by labeled apoE-VLDL within intracellular vesicles (Fig. 4, panel D). These data clearly show that expression of Syn-1 rescues apoE-VLDL internalization by these cells. To measure the increase in apoE-VLDL binding resulting from Syn-1 expression, we incubated GM00701 and GM00701/Syn-1-HA cells with ¹²⁵I-apoE-VLDL at 4°C in the absence or presence of unlabeled apoE-VLDL and quantitated specific ligand binding. Expression of Syn-1 in GM00701/Syn-1-HA cells resulted in a 5-fold increase in specific apoE-VLDL binding as compared to control GM00701 cells (Fig. 5).

Figure 4

Expression of Syn-1 restores apoE-VLDL uptake in GM00701 cells

Expression of Syn-1 restores apoE-VLDL uptake in GM00701 cells. GM00701 (panels A and C) and GM00701/Syn-1-HA (panels B and D) cells were incubated with DiI-labeled apoE-VLDL (4 μg/ml) for 3 h at 37°C. Unassociated ligand was removed by rinsing, cells were fixed with paraformaldehyde and observed by fluorescence microscopy (panels C and D, 550 nm excitation-573 nm emission). Corresponding phase contrast images are shown in panels A and B. Magnification, 630X.

Figure 5

Comparison of ¹²⁵I-apoE-VLDL binding to GM00701 and GM00701/Syn-1-HA

Comparison of ¹²⁵I-apoE-VLDL binding to GM00701 and GM00701/Syn-1-HA. GM00701 or GM00701/Syn-HA cells were incubated with ¹²⁵I-apoE-VLDL (2 μg/ml) for 3 h at 4°C in the absence or presence of unlabeled apoE-VLDL (100 μg/ml). Unbound ligand was removed by rinsing, cells were solubilized with 0.1 M NaOH and subjected to scintillation counting. Values shown represent specific ¹²⁵I-apoE-VLDL binding and were obtained by subtracting amounts of ¹²⁵I-apoE-VLDL bound in the presence of unlabeled apoE-VLDL from total ¹²⁵I-apoE-VLDL binding. Error bars represent standard deviations of triplicate points.

The question that arises from these observations asks if LRP plays a role with Syn-1 in the binding and uptake of apoE-VLDL. To address this question, we incubated GM00701/Syn-1-HA cells with DiI-apoE-VLDL in the absence or presence of either RAP-GST (to block LRP function) or heparin (to block Syn-1 function) and visualized ligand uptake by fluorescence microscopy following a 37°C incubation. RAP is known to prevent the binding of all ligands to LRP 40, including VLDL 41, and has proven invaluable as a pharmacological antagonist for characterizing LRP's ligand binding and uptake properties. With no co-incubation by ligand binding antagonists, DiI-apoE-VLDL is readily taken up by GM00701/Syn-1-HA cells as was originally identified in Fig. 4 (Fig. 6, panel A). Co-incubation with heparin blocked most, if not all, DiI-apoE-VLDL uptake (Fig. 6, panel C) demonstrating the requirement for heparan sulfate glycosaminoglycans on Syn-1 for DiI-apoE-VLDL uptake. However, RAP-GST demonstrated only marginal inhibition of uptake (Fig. 6, panel B), suggesting that Syn-1 is necessary for apoE-VLDL uptake and is able to internalize apoE-VLDL independent from LRP's endocytic activity.

Figure 6

Heparin, but not RAP-GST, competes for DiI-apoE-VLDL uptake by GM00701/Syn-1-HA cells

Heparin, but not RAP-GST, competes for DiI-apoE-VLDL uptake by GM00701/Syn-1-HA cells. GM00701/Syn-1-HA cells were cultured on glass coverslips and incubated with DiI-labeled apoE-VLDL (4 μg/ml) in the absence (panel A) or presence of recombinant human RAP-GST (50 μg/ml, panel B) or heparin (200 μg/ml, panel C) at 37°C for 3 h. Cells were fixed and processed for fluorescence microscopy. Magnification, 630X.

We 16 and others 1418192021 have previously shown that internalization of apoE-VLDL by cells is partially mediated by HSPG and in the present study we show that Syn-1 is capable of serving the role of this HSPG. These data now raise important questions as to the endocytic pathway used for internalization. LRP, like other members of the LDL receptor family, contains the clathrin-mediated internalization signal, NPXY amino acid sequence in their cytoplasmic tails. However, transmembrane HSPG such as Syn-1 do not contain this sequence and are likely internalized by cells through a non-clathrin mediated pathway. Previous studies have shown that antibody induced clustering of cell surface Syn-1 promotes its association with detergent-insoluble lipid rafts 28. In addition, cholesterol depletion of cells prevented Syn-1 from associating with these lipid rafts. Together, these observations suggest a role for non-clathrin-mediated pathways for Syn-1 internalization. To examine the pathway taken by Syn-1 for the internalization of apoE-VLDL, we measured uptake of ¹²⁵I-apoE-VLDL and ¹²⁵I-α₂M by GM00701/Syn-1-HA cells under conditions of K⁺depletion. Larkin, et al., have previously shown that K⁺depletion specifically inhibits clathrin-mediated internalization, but has little or no affect on non-clathrin mediated endocytosis 42. As shown in Figure 7, K⁺depletion significantly inhibited ¹²⁵I-α₂M uptake by LRP as expected since LRP endocytosis is clathrin-mediated. However, ¹²⁵I-apoE-VLDL internalization was unaffected by this treatment indicating that Syn-1 with bound apoE-VLDL is endocytosed through a non-clathrin mediated pathway.

Figure 7

¹²⁵I-α₂M internalization, but not ¹²⁵I-apoE-VLDL, is inhibited by K⁺depletion of cells

¹²⁵I-α₂M internalization, but not ¹²⁵I-apoE-VLDL, is inhibited by K⁺depletion of cells. GM00701/Syn-1-HA cells were treated with K⁺-depleted buffers (open symbols), or K⁺-containing serum-free media (closed symbols), as described in Methods. Cells were incubated with either ¹²⁵I-apoE-VLDL (2 μg/ml) (circles) or ¹²⁵I-α₂M (0.5 μg/ml) (triangles) for 20 min at 37°C. Specificity of uptake was determined by a parallel incubation with either unlabeled apoE-VLDL (100 μg/ml) or unlabeled α₂M (5 μg/ml), respectively. After the 37°C incubation, cells were chilled to 4°C and acid stripped with 50 mM glycine, pH 3, 100 mM NaCl for 5 min to remove surface bound ligand (that which was not internalized). Internalized ligand was quantitated by solubilizing cells with 0.1 N NaOH and scintillation counting.

To determine if Syn-1 with bound apoE-VLDL internalizes through lipid raft domains, we examined the effect of nystatin on uptake of DiI-apoE-VLDL in GM00701/Syn-1-HA cells. Nystatin is a sterol-binding antibiotic that sequesters membrane-associated cholesterol thereby disrupting plasma membrane microdomains such as lipid rafts 434445. As shown in Figure 8, nystatin had no effect on clathrin-mediated endocytosis of a₂M (lower panels). By contrast, little or no uptake of DiI-apoE-VLDL occurred in the presence of nystatin (upper panels) indicating that Syn-1 internalizes apoE-VLDL through a lipid raft microdomain.

Figure 8

Syn-1 internalizes DiI-apoE-VLDL through a lipid raft-mediated pathway

Syn-1 internalizes DiI-apoE-VLDL through a lipid raft-mediated pathway. GM00701/Syn-1-HA cells were incubated at 37°C in the absence or presence of nystatin (25 μg/ml) for 30 min prior to a 60 min incubation with DiI-apoE-VLDL (upper panels) or 488-α₂M (lower panels) (5 μg/ml). Cells were fixed and visualized by fluorescence microscopy (488-α₂M, FITC filter set; DiI-apoE-VLDL, rhodamine filter set). Magnification, 630X.

Discussion

Our previous study demonstrated that HSPG are able to bind and internalize apoE-VLDL with little or no contributions from LRP 16. In the present study, we now explore if Syn-1 represents a class of HSPG that is able to provide a path for apoE-VLDL binding and internalization, and investigate its relationship with LRP in this function. Using GM00701 cells, a human fibroblast cell line that expresses abundant amounts of LRP and no detectable Syn-1, we show that these cells are unable to bind or internalize apoE-VLDL even with active LRP receptor function, which was confirmed by α₂M binding and degradation. However, apoE-VLDL binding and internalization by these cells was rescued following the introduction of stable Syn-1 expression indicating that this HSPG is fully capable of mediating lipoprotein binding and uptake. We also show that apoE-VLDL internalization by the transfected cells is blocked by heparin co-incubation, but unaffected by a LRP ligand binding antagonist demonstrating that Syn-1 mediates uptake in an LRP-independent manner. To the best of our knowledge, this is the first example of a member of the syndecan family of HSPG serving as a clearance receptor for apoE-VLDL particles.

A previous study by Fuki, et al. 27, demonstrated that Syn-1 internalizes lipoprotein lipase-enriched LDL through a pathway that is distinct from clathrin coated pits and that internalization proceeds after clustering Syn-1 with a multivalent ligand. Since LRP apparently plays little role in the uptake of apoE-VLDL in GM00701 cells, we examined if internalization of apoE-VLDL by Syn-1 occurs through a clathrin-dependent or clathrin-independent pathway. We found that K⁺depletion significantly inhibited α₂M uptake, which internalizes via LRP through a clathrin-mediated pathway, but uptake of apoE-VLDL was unaffected by this treatment. By contrast, treatment of cells with nystatin, which disrupts lipid raft function 434445, prevented apoE-VLDL uptake by Syn-1 indicating that Syn-1-mediated clearance of apoE-VLDL proceeds through a non-clathrin pathway. This conclusion is also consistent with the fact that the cytoplasmic tail of Syn-1 does not contain the clathrin-mediated internalization signal sequence (NPXY). Additional evidence for non-clathrin-mediated internalization for Syn-1 was obtained through studies using a chimeric receptor consisting of the ectodomain of the Fc receptor Ia linked to the transmembrane and cytoplasmic domains of Syn-1 28. Upon clustering this chimera with IgG, it was found to redistribute into detergent insoluble lipid rafts. Pretreatment of cells with cholesterol depleting reagents prevented detergent insolubility of the Fc-Syn-1 chimera upon clustering and inhibited its internalization. From these data it appears that the transmembrane domain and cytoplasmic tail sequences of Syn-1 are sufficient for partitioning the receptor into lipid raft domains. Recently, it was shown that Syn-4 also redistributes into lipid rafts upon clustering 46, suggesting that this may be a conserved feature among all members of the syndecan family.

The mechanism of endocytosis by means of lipid raft domains remains a topic of ongoing study; however, information is becoming available as to the initial partitioning of proteins into lipid rafts 4748. Possible mechanisms to segregate plasma membrane proteins include hydrophobic modifications, such as lipidation, protein-protein interactions with lipid-raft resident proteins, or targeting signals that are encoded within the polypeptide domain of the membrane protein. Although Syn-1 has not been reported to undergo lipid modification or directly interact with lipid raft residents, such as caveolin, annexin, or flotillin, it may utilize intrinsic targeting information within its transmembrane domain or cytoplasmic tail in a similar fashion as the epidermal growth factor receptor 49. Once receptors internalize with their bound ligand through non-clathrin mediated pathways, they can deliver their cargo to classic endocytic compartments, such as recycling endosomes 50. As a result of this crosstalk between non-clathrin pathways and classical endocytic pathways, Syn-1 would be able to deliver apoE-VLDL to lysosomal acid lipases for release of fatty acids for cellular utilization 51. Whether Syn-1 recycles to the cell surface after delivering its cargo to endosomes remains to be determined.

LRP was originally identified as a lipoprotein clearance receptor by virtue of its structural homology with the LDL receptor, its high level expression in liver and its function in hepatic remnant metabolism elegantly demonstrated using genetically modified mice 1213. However, the question remains as to why in certain cell types, HSPG, and as we show here, Syn-1, serves as the primary clearance receptor for apoE-VLDL and LRP plays little or no role in the uptake of this ligand. Answers to this question may come from more recent studies investigating alternative functions for LRP (reviewed in 525354). LRP is now known to carry out dual roles; serving as an endocytic receptor for many diverse, structurally different ligands and as a signaling receptor that is able to modulate activities of other cell surface proteins. Whether LRP functions as a cargo receptor or signaling receptor may be determined by the state of receptor phosphorylation, post-translational processing, or complement of cytosolic messengers/adaptors that might dictate the preference between these dual functions. Thus, studies on hepatocytes have demonstrated that LRP functions as a key element in lipoprotein handling in vivo, whereas in fibroblasts 5556 or cells of neuronal lineage 57, LRP may be acting primarily as a co-receptor modulating signal transduction events that control cell behavior.

Methods

Materials

Human α₂-macroglobulin (α₂M), heparin, and heparinase were purchased from Sigma-Aldrich (St. Louis, MO). Na¹²⁵I was purchased from Perkin-Elmer (Boston, MA). Pre-cast polyacrylamide electrophoresis (PAGE) gels and goat anti-rabbit horseradish peroxidase-conjugated secondary antibody were from BioRad (Hercules, CA). All oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA). All restriction enzymes were from New England Biolabs (Ipswich, MA). RAP-GST fusion protein was purified as previously described 58. Anti-LRP polyclonal antibody was raised against an 18 amino acid peptide from the cytoplasmic tail of human LRP 59. Tissue culture plastics were purchased from Corning (Corning, NY). Buffers, salts, and detergents were obtained from either Sigma-Aldrich or Calbiochem (La Jolla, CA).

Cell culture

GM00701 human fibroblasts (Coriell Institute, Camden, NJ) were obtained from a patient diagnosed with familial hypercholesterolemia. This cell line expresses < 1% of normal activity for the LDL receptor. GM00701 cells were cultured in Minimal Essential Media, Eagle modification with Earle's balanced salt solution (MEM-Eagle's, Life Technologies/Invitrogen, Carlsbad, CA). Media was supplemented with 2X concentration of essential and non-essential amino acids and vitamins, 15% (v/v) fetal bovine serum (Irvine Scientific, Santa Ana, CA), 100 μg/ml streptomycin sulfate, and 100 units/ml penicillin G. 3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) fetal calf serum (Irvine Scientific, Santa Ana, CA), 1 mM sodium pyruvate, 100 μg/ml streptomycin sulfate, and 100 units/ml penicillin. Cells were cultured at 37°C with 5% CO₂for GM00701 cells and 10% CO₂for preadipocyte 3T3-L1 cells. Cells were passaged twice weekly.

Immunoblotting

Cell lysates were prepared with 20 mM Tris pH 7.4, 150 mM NaCl (TBS) containing either 1% (v/v) Triton-X100 or 8 M urea. Solubilized proteins were mixed with SDS-PAGE sample buffer supplemented with 2% (v/v) β-mercaptoethanol, heated to 95°C, separated by SDS-PAGE and transferred to Immobilon-P (Millipore, Billerica, MA) using a wet tank transfer system (BioRad, Hercules, CA). Membranes were blocked with TBS, 0.1% (v/v) Tween-20, 5% (w/v) non-fat dry milk for 20 minutes at 23°C and incubated with the indicated primary antibody for 2 h at 23°C. Membranes were washed three times (10 min each) with TBS, 0.1% (v/v) Tween-20, and bound antibodies were detected with species-specific HRP-conjugated secondary antibodies (1:3000, BioRad) followed by chemiluminescence detection according to the manufacturer's instructions (Pierce, Rockford, IL). Images were captured using a Syngene GeneGnome system equipped with a Peltier-cooled 16-bit CCD camera and saturation detection. Densitometry of scanned images was performed using Scion Image software version 4.0.2.

Activation of α₂-macroglobulin and labeling procedures

α₂M 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). For radioiodinations, both apoE-VLDL and α₂M were labeled with ¹²⁵I (Perkin-Elmer) using Iodo-Beads (Pierce) as previously described 60. Specific activities were routinely between 3000 and 5000 cpm/ng protein. Using the Bligh-Dyer lipid extraction procedure, we determined that approximately 10% of ¹²⁵I-label was incorporated into lipid of the VLDL particle while the remaining 90% of label was found coupled to protein. α₂M was labeled for fluorescence detection using Alexa Fluor^®488 Protein labeling kit (Molecular Probes, Eugene, OR).

Cell surface 4°C binding assay

Cells were grown on tissue culture plates precoated with 1% gelatin and used when confluent. 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-apoE-VLDL (2 μg/ml) or ¹²⁵I-α₂M (1 μg/ml) at 4°C in the absence or presence of the indicated unlabeled competitors. ¹²⁵I-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 ¹²⁵Iodine). 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 non-specific binding (non-competable) 61. The actual amount of ligand bound to cells was calculated as cpm ÷ the specific activity of ¹²⁵I-labeled ligand. All data points represent averages of replicate points with standard errors of < 5%.

37°C ligand degradation assay

Cells were incubated at 37°C/5% CO₂with ¹²⁵I-α₂M (1 μg/ml) diluted into MEM-Eagle's containing 1% BSA in the absence or presence of unlabeled α₂M (10 μg/ml). At the indicated times, media was removed and processed for trichloroacetic acid (TCA) precipitation 62. TCA soluble material was added to EcoLume and subjected to scintillation counting. Degradation was calculated as TCA-soluble cpm ÷ specific activity of the ¹²⁵I-α₂M.

pMH/Syn-1-HA construction and GM00701 cell transfections

The cDNA for full length human Syn-1 was obtained from the I.M.A.G.E. Consortium (clone no. 3347793 in pOTB7 vector, accession no. BE272506) and amplified by PCR using the following conditions: 30 cycles, 94°C for 1 min, 60°C for 1 min, 72°C for 3 min; 1 cycle, 72°C for 7 min. Primer sequences used: upstream, 5'-CCGGGCAGCATGGGGCGCGCG-3'; downstream, 5'-TTGGCATAGAATTCCTCCTGTTTGG-3'. The cDNA was cloned into pMH vector (Roche, Indianapolis, IN) using restriction sites HindIII and Not1, and placed in frame with a hemagglutinin (HA) peptide-encoding sequence at its 3' end. pMH/Syn-1-HA vector was introduced into DH5α E. coli, amplified by overnight growth, and purified for mammalian transfections using an endotoxin-free plasmid purification kit (Qiagen, Valencia, CA). For transfections, GM00701 cells were plated onto 10 cm tissue culture dishes and incubated with 8 μg vector/dish together with Lipofectamine Plus according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). Forty eight hours post-transfection, cell growth media was replaced with fresh media containing 1 mg/ml neomycin for selection of stably transfected cells.

Anti-Syn-1 polyclonal antibody

The cDNA for full length human Syn-1 was amplified by PCR using the conditions described above and cloned into pET28 vector (EMD Biosciences, San Diego, CA) using restriction sites HindIII and XhoI. The cDNA was placed in frame with a hexa-histidine encoding sequence at its 3' end (Syn-1-His₆). Primer sequences used: upstream, 5'-GCAAGCTTCAAATTGTGGCTACTAATTTGCCC-3'; downstream, 5'-CGCTCGAGGGCATAGAATTCCTCCTGTTTGG-3'. Syn-1-His₆was purified by nickel-affinity chromatography and purity (> 95%) was confirmed by Coomassie staining following SDS-PAGE. Protein identity was also confirmed by mass spectrometry analysis (University of New Mexico Proteomics Core Facility). Purified Syn-1-His₆(500 μg) was thoroughly mixed with an equal volume of Freund's Complete adjuvant and injected subcutaneously into New Zealand White rabbits. Rabbits were boosted with 100 μg Syn-1-His₆mixed with Freund's Incomplete adjuvant at three week intervals.

Preparation of Di-I-labeled apoE-VLDL

New Zealand White rabbits were placed on a high-fat chow diet (10% peanut oil/1% cholesterol) for a minimum of 4 d, blood was drawn into 1 mM EDTA and centrifuged at 2000 × g, 15 min to remove cells. Chylomicrons were floated by centrifuging plasma at 100,000 × g for 10 min and removed by pipetting. Plasma was then mixed with OptiPrep™ (12% iodixanol final concentration, Oslo, Norway) and centrifuged at 350,000 × g for 20 h (SW55Ti rotor) with slow acceleration and deceleration. VLDL particles (density of 1.006 g/ml) were removed from the top of the gradient by pipetting and analyzed by SDS-PAGE and Coomassie R staining to confirm the presence of apoB100 (Mr, 512 kDa) and apoE (Mr, 34 kDa). Animal protocol (#0350) was approved by the University of New Mexico, Health Sciences Center Laboratory Animal Care and Use Committee. For DiI (3,3'-dioctadecylindocarbocyanine, Molecular Probes, Invitrogen) labeling, a working stock of 3 mg/ml was made in dimethylsulfoxide and 0.15 mg was slowly added to 1.67 mg apoE-VLDL (in 1.9 ml) with vortexing to rapidly mix. The mixture was then wrapped in foil and incubated for 8 h at 37°C. Unincorporated DiI was removed from DiI-labeled apoE-VLDL by OptiPrep™ gradient centrifugation as described above.

DiI-labeled apoE-VLDL uptake assay

GM00701 or GM00701/Syn-1-HA cells were plated on glass coverslips, rinsed twice with MEM-Eagle's and incubated with DiI-apoE-VLDL (4 μg/ml) diluted into MEM-Eagle's containing 1% bovine serum albumin in the absence or presence of heparin (200 μg/ml) or RAP-GST (50 μg/ml). After 1 h at 37°C, cells were rinsed with phosphate buffered saline, fixed with 3% (w/v) paraformaldehyde for 20 min, and mounted using Vectashield (Vector Labs, Burlingame, CA). Cells were observed with a Zeiss Axioskop microscope equipped for epifluorescence. Images were capture with a Hamamatsu digital/video camera and AxioVision software.

K⁺depletion studies

K⁺depletion studies were carried out according to Larkin, et al. 42. Briefly, GM00701/Syn-1-HA cells were rinsed with K⁺-free buffer (50 mM Hepes, pH 7.4, 100 mM NaCl) and subjected to hypotonic shock by incubation at 37°C for 5 min with a 1:1 ratio of MEM-Eagle's media:dH₂O. Hypotonic shock buffer was then replaced with K⁺-free buffer for 10 min at 37°C. Control, non-treated cells were incubated in parallel with serum-free, MEM-Eagle's media. Cells were then incubated at 37°C with either ¹²⁵I-apoE-VLDL or ¹²⁵I-α₂M diluted into K⁺-free buffer (treated cells) or serum-free, MEM-Eagle's media (control cells). At the indicated times, cells were chilled on ice and rinsed (3X) with ice cold 20 mM Hepes, pH 7.4, 150 mM NaCl. Bound ligand that had not undergone internalization was dissociated by a 5 min incubation with 20 mM NaOAc, pH 3, 150 mM NaCl. Internalized ¹²⁵I-ligand was then extracted by solubilizing cells with 0.1 N NaOH, added to EcoLume and quantitated by scintillation counting. In all cases, specificity of ligand internalization was determined by a parallel co-incubation with > 10-fold molar excess of unlabeled ligand.

Abbreviations

ApoE = apolipoprotein E

HSPG = heparan sulfate proteoglycan

VLDL = very low density lipoprotein

LRP = low density lipoprotein receptor-related protein

Syn-1 = syndecan-1

RAP = receptor associated protein

LPL = lipoprotein lipase

DiI = 1,1'-dictadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

LCW carried out the majority of studies. AMG contributed to ligand uptake studies and manuscript preparation. RAO provided the original conceptual framework for the study, carried out pilot experiments, organized experimental design and finalized the manuscript for submission. All authors read and approved the final version.

Acknowledgements

This work was supported by the National Institutes of Health Grant HL63291 and a Grant-In-Aid award from the American Heart Association (to R.A.O.). The images presented in this study were generated in the Fluorescence Microscopy Facility which is supported by NCRR 1 S10 RR14668, NSF MCB9982161, NCRR P20 RR11830, NCI R24 CA88339, NCRR S10 RR19287, NCRR S10 RR016918, the University of New Mexico Health Sciences Center, and the University of New Mexico Cancer Center.

Cholesteryl ester accumulation in macrophages resulting from receptor-mediated uptake and degradation of hypercholesterolemic canine beta-very low density lipoproteins Goldstein JL Ho YK Brown MS Innerarity TL Mahley RW Journal of Biological Chemistry 1980 255 5 1839 1848 7354064 Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages Van Lenten BJ Fogelman AM Hokom MM Benson L Haberland ME Edwards PA Journal of Biological Chemistry 1983 258 8 5151 5157 6300118 Lipoproteins activate acyl-coenzyme A:cholesterol acyltransferase in macrophages only after cellular cholesterol pools are expanded to a critical threshold level Xu XX Tabas I Journal of Biological Chemistry 1991 266 26 17040 17048 1894601 Pathogenesis of type III hyperlipoproteinemia (dysbetalipoproteinemia). Questions, quandaries, and paradoxes Mahley RW Huang Y Rall SCJ Journal of Lipid Research 1999 40 11 1933 1949 10552997 Functions of cell surface heparan sulfate proteoglycans Bernfield M Gotte M Park PW Reizes O Fitzgerald ML Lincecum J Zako M Annual Review of Biochemistry 1999 68 729 777 10.1146/annurev.biochem.68.1.729 10872465 Role of heparan sulfate proteoglycans in the binding and uptake of apolipoprotein E-enriched remnant lipoproteins by cultured cells Ji ZS Brecht WJ Miranda RD Hussain MM Innerarity TL Mahley RW J Biol Chem 1993 268 14 10160 10167 7683668 Intravenous heparinase inhibits remnant lipoprotein clearance from the plasma and uptake by the liver: in vivo role of heparan sulfate proteoglycans Ji ZS Sanan DA Mahley RW J Lipid Res 1995 36 3 583 592 7539827 Lipoprotein and receptor interactions in vivo Herz J Willnow TE Curr Opin Lipidol 1995 6 2 97 103 7773575 Role of heparan sulfate proteoglycans and the LDL receptor-related protein in remnant lipoprotein metabolism Mahley RW Ji ZS Brecht WJ Miranda RD He D Ann N Y Acad Sci 1994 737 39 52 7944147 Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E Mahley RW Ji ZS Journal of Lipid Research 1999 40 1 1 16 9869645 Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein Kowal RC Herz J Weisgraber KH Mahley RW Brown MS Goldstein JL Journal of Biological Chemistry 1990 265 18 10771 10779 2355022 Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants Rohlmann A Gotthardt M Hammer RE Herz J J Clin Invest 1998 101 3 689 695 508614 9449704 Inhibition of hepatic chylomicron remnant uptake by gene transfer of a receptor antagonist Willnow TE Sheng Z Ishibashi S Herz J Science 1994 264 5164 1471 1474 7515194 Heparan sulfate proteoglycan-mediated uptake of apolipoprotein E-triglyceride-rich lipoprotein particles: a major pathway at physiological particle concentrations Al-Haideri M Goldberg IJ Galeano NF Gleeson A Vogel T Gorecki M Sturley SL Deckelbaum RJ Biochemistry 1997 36 42 12766 12772 10.1021/bi9631024 9335533 Lipoprotein lipase-mediated uptake of lipoprotein in human fibroblasts: evidence for an LDL receptor-independent internalization pathway Fernandez-Borja M Bellido D Vilella E Olivecrona G Vilaro S J Lipid Res 1996 37 3 464 481 8728311 The low density lipoprotein receptor-related protein complexes with cell surface heparan sulfate proteoglycans to regulate proteoglycan-mediated lipoprotein catabolism Wilsie LC Orlando RA Journal of Biological Chemistry 2003 278 18 15758 15764 10.1074/jbc.M208786200 12598530 Heparan sulfate proteoglycans are primarily responsible for the maintenance of enzyme activity, binding, and degradation of lipoprotein lipase in Chinese hamster ovary cells Berryman DE Bensadoun A J Biol Chem 1995 270 41 24525 24531 10.1074/jbc.270.41.24525 7592670 Heparan sulfate proteoglycans participate in hepatic lipaseand apolipoprotein E-mediated binding and uptake of plasma lipoproteins, including high density lipoproteins Ji ZS Dichek HL Miranda RD Mahley RW J Biol Chem 1997 272 50 31285 31292 10.1074/jbc.272.50.31285 9395455 Chylomicron remnant uptake is regulated by the expression and function of heparan sulfate proteoglycan in hepatocytes Zeng BJ Mortimer BC Martins IJ Seydel U Redgrave TG J Lipid Res 1998 39 4 845 860 9555948 Heparan sulfate proteoglycans mediate internalization and degradation of beta-VLDL and promote cholesterol accumulation by pigeon macrophages Seo T St Clair RW Journal of Lipid Research 1997 38 4 765 779 9144091 LDL receptor family-dependent and -independent pathways for the internalization and digestion of lipoprotein lipase-associated beta-VLDL by rat vascular smooth muscle cells Weaver AM Lysiak JJ Gonias SL J Lipid Res 1997 38 9 1841 1850 9323593 The cell surface proteoglycan syndecan-1 mediates fibroblast growth factor-2 binding and activity Filla MS Dam P Rapraeger AC J Cell Physiol 1998 174 3 310 321 10.1002/(SICI)1097-4652(199803)174:3<310::AID-JCP5>3.0.CO;2-R 9462693 Stimulation of fibroblast growth factor receptor-1 occupancy and signaling by cell surface-associated syndecans and glypican Steinfeld R Van Den Berghe H David G J Cell Biol 1996 133 2 405 416 10.1083/jcb.133.2.405 8609172 Hepatocyte growth factor-mediated renal epithelial branching morphogenesis is regulated by glypican-4 expression Karihaloo A Kale S Rosenblum ND Cantley LG Mol Cell Biol 2004 24 19 8745 8752 516744 15367691 10.1128/MCB.24.19.8745-8752.2004 The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7 Mongiat M Taylor K Otto J Aho S Uitto J Whitelock JM Iozzo RV J Biol Chem 2000 275 10 7095 7100 10.1074/jbc.275.10.7095 10702276 Epidermal transformation leads to increased perlecan synthesis with heparin-binding-growth-factor affinity Tapanadechopone P Tumova S Jiang X Couchman JR Biochem J 2001 355 Pt 2 517 527 1221765 11284741 10.1042/0264-6021:3550517 The syndecan family of proteoglycans. Novel receptors mediating internalization of atherogenic lipoproteins in vitro Fuki IV Kuhn KM Lomazov IR Rothman VL Tuszynski GP Iozzo RV Swenson TL Fisher EA Williams KJ J Clin Invest 1997 100 6 1611 1622 508343 9294130 Transmembrane and cytoplasmic domains of syndecan mediate a multi-step endocytic pathway involving detergent-insoluble membrane rafts Fuki IV Meyer ME Williams KJ Biochemical Journal 2000 351 Pt 3 607 612 1221399 11042114 10.1042/0264-6021:3510607 Delayed catabolism of apoB-48 lipoproteins due to decreased heparan sulfate proteoglycan production in diabetic mice Ebara T Conde K Kako Y Liu Y Xu Y Ramakrishnan R Goldberg IJ Shachter NS J Clin Invest 2000 105 12 1807 1818 378502 10862796 Perlecan heparan sulfate proteoglycan: a novel receptor that mediates a distinct pathway for ligand catabolism Fuki IV Iozzo RV Williams KJ Journal of Biological Chemistry 2000 275 33 25742 25750 10.1074/jbc.M909173199 10818109 LDL receptor-related protein mediates cell-surface clustering and hepatic sequestration of chylomicron remnants in LDLR-deficient mice Yu KC Chen W Cooper AD Journal of Clinical Investigation 2001 107 11 1387 1394 209318 11390420 Neuronal apoptosis by apolipoprotein E4 through low-density lipoprotein receptor-related protein and heterotrimeric GTPases Hashimoto Y Jiang H Niikura T Ito Y Hagiwara A Umezawa K Abe Y Murayama Y Nishimoto I J Neurosci 2000 20 22 8401 8409 11069947 The insulin-stimulated cell surface presentation of low density lipoprotein receptor-related protein in 3T3-L1 adipocytes is sensitive to phosphatidylinositide 3-kinase inhibition Ko KW Avramoglu RK McLeod RS Vukmirica J Yao Z Biochemistry 2001 40 3 752 759 10.1021/bi001797+ 11170392 Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity Czekay RP Kuemmel TA Orlando RA Farquhar MG Molecular Biology of the Cell 2001 12 5 1467 1479 34598 11359936 Cell surface heparan sulfate proteoglycans contribute to intracellular lipid accumulation in adipocytes Wilsie LC Chanchani S Navaratna D Orlando RA Lipids Health Dis 2005 4 1 2 545935 15636641 10.1186/1476-511X-4-2 Core protein structure and sequence determine the site and presence of heparan sulfate and chondroitin sulfate on syndecan-1 Kokenyesi R Bernfield M J Biol Chem 1994 269 16 12304 12309 8163535 Analysis of transport and targeting of syndecan-1: effect of cytoplasmic tail deletions Miettinen HM Edwards SN Jalkanen M Mol Biol Cell 1994 5 12 1325 1339 301161 7696713 Syndecans: multifunctional cell-surface co-receptors Carey DJ Biochemical Journal 1997 327 Pt 1 1 16 1218755 9355727 The cytoplasmic domain of syndecan-1 is required for cytoskeleton association but not detergent insolubility. Identification of essential cytoplasmic domain residues Carey DJ Bendt KM Stahl RC Journal of Biological Chemistry 1996 271 25 15253 15260 10.1074/jbc.271.25.15253 8662979 Receptor-associated protein (RAP): a specialized chaperone for endocytic receptors Willnow TE Biological Chemistry 1998 379 8-9 1025 1031 9792434 The 39-kDa receptor-associated protein modulates lipoprotein catabolism by binding to LDL receptors Medh JD Fry GL Bowen SL Pladet MW Strickland DK Chappell DA J Biol Chem 1995 270 2 536 540 10.1074/jbc.270.2.536 7822276 Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts Larkin JM Brown MS Goldstein JL Anderson RG Cell 1983 33 1 273 285 10.1016/0092-8674(83)90356-2 6147196 How do the polyene macrolide antibiotics affect the cellular membrane properties? Bolard J Biochim Biophys Acta 1986 864 3-4 257 304 3539192 Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover Di Guglielmo GM Le Roy C Goodfellow AF Wrana JL Nat Cell Biol 2003 5 5 410 421 10.1038/ncb975 12717440 The effects of the polyene antibiotics nystatin and amphotericin B on thin lipid membranes Holz RW Ann N Y Acad Sci 1974 235 0 469 479 4528030 Clustering induces redistribution of syndecan-4 core protein into raft membrane domains Tkachenko E Simons M J Biol Chem 2002 277 22 19946 19951 10.1074/jbc.M200841200 11889131 Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling Le Roy C Wrana JL Nat Rev Mol Cell Biol 2005 6 2 112 126 10.1038/nrm1571 15687999 Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells Zacharias DA Violin JD Newton AC Tsien RY Science 2002 296 5569 913 916 10.1126/science.1068539 11988576 Second cysteine-rich region of epidermal growth factor receptor contains targeting information for caveolae/rafts Yamabhai M Anderson RG J Biol Chem 2002 277 28 24843 24846 10.1074/jbc.C200277200 12023273 GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway Sabharanjak S Sharma P Parton RG Mayor S Dev Cell 2002 2 4 411 423 10.1016/S1534-5807(02)00145-4 11970892 Lysosomal acid lipase and atherosclerosis Du H Grabowski GA Curr Opin Lipidol 2004 15 5 539 544 10.1097/00041433-200410000-00007 15361789 Low density lipoprotein receptor-related protein: regulation of the plasma membrane proteome Gonias SL Wu L Salicioni AM Thromb Haemost 2004 91 6 1056 1064 15175790 LRP: a multifunctional scavenger and signaling receptor Herz J Strickland DK J Clin Invest 2001 108 6 779 784 200939 11560943 10.1172/JCI200113992 LDL receptor relatives at the crossroad of endocytosis and signaling Schneider WJ Nimpf J Cell Mol Life Sci 2003 60 5 892 903 10.1007/s00018-003-2183-Z 12827279 Platelet-derived growth factor receptor-beta (PDGFR-beta) activation promotes its association with the low density lipoprotein receptor-related protein (LRP). Evidence for co-receptor function Newton CS Loukinova E Mikhailenko I Ranganathan S Gao Y Haudenschild C Strickland DK J Biol Chem 2005 280 30 27872 27878 10.1074/jbc.M505410200 15944146 Low density lipoprotein receptor-related protein 1 (LRP1) controls endocytosis and c-CBL-mediated ubiquitination of the platelet-derived growth factor receptor beta (PDGFRbeta) Takayama Y May P Anderson RG Herz J J Biol Chem 2005 280 18 18504 18510 10.1074/jbc.M410265200 15753096 LRP and Alzheimer's disease Zerbinatti CV Bu G Rev Neurosci 2005 16 2 123 135 15959937 Functional domains of the receptor-associated protein (RAP) Orlando RA Farquhar MG Proc Natl Acad Sci U S A 1994 91 8 3161 3165 43535 7512726 10.1073/pnas.91.8.3161 The expression of megalin (gp330) and LRP diverges during F9 cell differentiation Czekay RP Orlando RA Woodward L Adamson ED Farquhar MG J Cell Sci 1995 108 Pt 4 1433 1441 7615664 Pathogenic antibodies inhibit the binding of apolipoproteins to megalin/gp330 in passive Heymann nephritis Kerjaschki D Exner M Ullrich R Susani M Curtiss LK Witztum JL Farquhar MG Orlando RA J Clin Invest 1997 100 9 2303 2309 508426 9410908 Receptor-mediated endocytosis of low-density lipoprotein in cultured cells Goldstein JL Basu SK Brown MS Methods in Enzymology 1983 98 241 260 6321901 Endocytic trafficking of megalin/RAP complexes: dissociation of the complexes in late endosomes Czekay RP Orlando RA Woodward L Lundstrom M Farquhar MG Mol Biol Cell 1997 8 3 517 532 276101 9188102