The low density lipoprotein (LDL) ()receptor gene family includes the LDL receptor(1) , the very low density lipoprotein (VLDL) receptor(2) , the LDL receptor-related protein (LRP)(3) , and glycoprotein 330(4) . Together, these molecules have important roles in the catabolism of lipoproteins, proteinases, proteinase-inhibitor complexes, and matrix proteins (for reviews, see (5, 6, 7, 8) ). The members of this receptor family share structural motifs including cysteine-rich epidermal growth factor-like repeats, cysteine-rich ligand binding repeats, repeats containing the tetrapeptide sequence tyrosine-tryptophan-threonine-aspartic acid, and an asparagine-proline-X-tyrosine sequence within the cytoplasmic tail, which is responsible for endocytic signaling in coated pits.

The most recently identified member of this receptor family is the VLDL receptor(2) , so named because it appeared to specifically bind VLDL, probably via interaction with apolipoprotein E (apo E). At present, however, the physiological role of the VLDL receptor is uncertain. This receptor is most abundant in skeletal muscle, heart, adipose tissue, and brain(9, 10, 11) , tissues which metabolize fatty acids as an energy source. This fact, and the observation that the VLDL receptor recognizes apo E-containing lipoproteins, has led to the hypothesis that the VLDL receptor may play an important role in the delivery of triglyceride-rich lipoproteins to peripheral tissues(8) . Interestingly, a number of tissues that express high levels of the VLDL receptor also express lipoprotein lipase (LpL)(12) , a key enzyme in the metabolism of triglyceride-rich lipoproteins. It has been suggested that LpL may play an important role in conjunction with the VLDL receptor in the catabolism of lipoproteins(8) .

A chicken receptor has been identified that is responsible for the endocytosis of VLDL and vitellogenin(13) . The primary sequence of this receptor has a high degree of similarity with that of the mammalian VLDL receptors, indicating that it represents a chicken homologue. Insight into a function for the chicken VLDL receptor has been gained by identifying a mutant hen that is missing this receptor. Hens with this defect are characterized by hereditary hyperlipidemia and the absence of egg laying(14) . These observations indicate that the chicken VLDL receptor plays a critical role in mediating the transport of triglycerides into growing oocytes.

Recently, Battey et al.(15) discovered that a 39-kDa protein, termed the receptor-associated protein (RAP), binds with high affinity to the VLDL receptor and regulates its ligand binding properties. RAP was discovered when it copurified with LRP during ligand affinity chromatography(16, 17) . While the biological function of RAP remains unknown, it binds tightly to LRP, gp330, and the VLDL receptor and modulates their ligand binding activities(15, 18, 19, 20) . The localization of RAP within the endoplasmic reticulum (21) and studies in which the RAP gene was disrupted in mice (22) suggest that RAP may play an important role in the early processing of these receptors, perhaps in preventing association of the newly synthesized receptors with ligands or in regulating receptor transport or trafficking to the cell surface.

The high affinity interaction between RAP and the VLDL receptor suggested that the VLDL receptor, like LRP and gp330, may interact with additional ligands, and the present studies were undertaken to more fully define the ligand binding characteristics of this newly discovered receptor. These studies demonstrate that the VLDL receptor is a multiligand receptor and may play an important role in lipoprotein catabolism, by binding and internalizing both VLDL and lipoprotein lipase, and in proteinase catabolism, by mediating the cellular uptake of urokinase (uPA) complexed to its inhibitor, plasminogen activator inhibitor type I (PAI-1).

MATERIALS AND METHODS

Proteins

Cell Culture

Adenovirus Infection of PEA-13 Cells

Cell Internalization and Degradation Assays

Antibodies

Preparation of Cell Extracts

Immunoblotting and Ligand Blotting

RAP ligand blotting experiments were performed as described (15) using 25 nM RAP. RAP was detected using an affinity-purified anti-RAP IgG (R80, 1 µg/ml). As a control, RAP was omitted from the protocol. For the ligand blots using uPAPAI-1 complexes, nitrocellulose membranes were incubated with 50 nM uPAPAI-1 in the absence or presence of 1 µM RAP for 1 h at 25 °C. The bound uPAPAI-1 was detected by incubation with a monoclonal antibody against uPA (1 µg/ml), followed by incubation with a goat anti-mouse IgG horseradish peroxidase conjugate (Bio-Rad). The bands were visualized using the Renaissance chemiluminescence kit (DuPont NEN).

Solid Phase Binding Assay

where A is the absorbance at 650 nm, A is the absorbance value at saturation, A is the background absorbance in the absence of ligand, [L] is the molar concentration of free uPAPAI-1 complexes, and K is the dissociation constant. Since the free uPAPAI-1 concentration is unknown in these experiments, the use of this equation assumes that the amount of added ligand is greater than the amount of receptor bound to the microtiter wells. Under these conditions, the amount of free uPAPAI-1 is approximately equal to the total uPAPAI-1 concentration.

For the analysis of LPL and GST-LPLC binding to VLDL receptor, microtiter wells were coated with 100 µl of LPL or GST-LPLC (10 µg/ml) in 0.1 M sodium bicarbonate, pH 9.0, overnight at 4 °C. The wells were blocked as described above, and various concentrations of purified VLDL receptor in 0.075 M Tris, pH 8.0, 0.15 M NaCl, 5 mM CaCl, 0.05% Tween 20, 3% BSA was added to the wells or to BSA-coated wells. After an overnight incubation at 4 °C, the wells were washed and incubated with affinity-purified anti-VLDL receptor IgG (1 µg/ml) in the same buffer for 1 h at 25 °C. The bound antibody was detected with a goat anti-rabbit IgG conjugated to horseradish peroxidase using the substrate 3,3`,5,5`-tetramethylbenzidine and analyzed as mentioned above.

RESULTS

Infection of PEA13 Fibroblasts with Ad-VLDLR Results in the Expression of VLDL Receptor

Figure 1: Immunoblot analysis (A) and RAP ligand blot analysis (B) of cell extracts from murine PEA13 fibroblasts and adenovirus-infected fibroblasts. A, left panel, cell extracts prepared from PEA13 fibroblasts or from PEA13 fibroblasts infected with Ad-lacZ or Ad-VLDLR were subjected to SDS-PAGE on 4-12% gradient gels under non-reducing conditions, transferred to nitrocellulose, and incubated for 1 h with anti-VLDL receptor IgG (1 µg/ml). After washing, the blots were incubated with a goat anti-rabbit IgG-horseradish peroxidase conjugate. The bands were visualized by use of the Renaissance chemiluminescence kit. 14 µg of total protein were applied to each lane. Right panel, same as above, except the anti-VLDL receptor IgG was omitted from the blotting protocol. B, left panel, cell extracts prepared from PEA13 fibroblasts or from PEA13 fibroblasts infected with Ad-lacZ or Ad-VLDLR were subjected to SDS-PAGE on 4-12% gradient gels under non-reducing conditions, transferred to nitrocellulose, and incubated for 1 h with 25 nM RAP. After incubation, the blots were washed and incubated with 1 µg/ml anti-RAP IgG for 1 h at room temperature. After washing, the blots were incubated with a goat anti-rabbit IgG-horseradish peroxidase conjugate. The bands were visualized by use of the Renaissance chemiluminescence kit. 14 µg of total protein were applied to each lane. Right panel, same as above, except that RAP was omitted from the protocol.

The integrity of the expressed VLDL receptor was examined by RAP ligand blotting experiments on cell extracts. These experiments revealed that the VLDL receptor expressed in PEA13 fibroblasts following infection with Ad-VLDLR is able to bind RAP (Fig. 1B, left panel). It is of interest to note that the presumed VLDL receptor dimer appears unable to bind RAP. RAP binding proteins were not detected in parental PEA13 fibroblasts or in PEA13 fibroblasts infected with Ad-lacZ. The RAP blotting appears to be specific since no RAP (with the exception of trace amounts of endogenously produced protein) was detected when RAP was omitted from the procedure (Fig. 1B, right panel).

The VLDL Receptor Mediates the Cellular Catabolism of Lipoprotein Lipase

Figure 2: Time course for the internalization (A) and degradation (B) of I-labeled LpL by murine PEA13 fibroblasts infected with Ad-VLDLR. PEA13-fibroblasts infected with Ad-VLDLR () or PEA13 fibroblasts infected with Ad-lacZ () were plated into wells (1.5 10 cells/well), and 2 nMI-labeled LpL was added to each well. At indicated times, the extent of internalization (A) or degradation (B) was determined as described under ``Materials and Methods.'' In control experiments (open symbols), 1 µM RAP was included during the incubation. Each data point represents the average of duplicate determinations. , Ad-lacZ + RAP; , Ad-VLDLR + RAP.

The VLDL Receptor Binds to the Carboxyl-terminal Domain of Lipoprotein Lipase

Figure 3: The purified VLDL receptor binds LpL (A) and LpLC (B). Microtiter wells were coated with 100 µl of LpL (10 µg/ml) (A) or LpLC (B) (solid circles) overnight at 4 °C. The wells were then blocked for 1 h with 3% BSA at 25 °C. As a control, LpL and LpLC were omitted (open circles). Increasing concentrations of purified VLDL receptor were added to each well, and incubation was carried out overnight at 4 °C in 0.075 M Tris, pH 8.0, 0.15 M NaCl, 5 mM CaCl, 0.05% Tween 20, 3% BSA. After washing, the wells were incubated with 1 µg/ml anti-VLDL receptor IgG for 1 h at room temperature. Following washing, the amount of anti-VLDL receptor IgG bound to each well was detected using a goat anti-rabbit-IgG conjugated to horseradish peroxidase using the substrate 3,3`,5,5`-tetramethylbenzidine. The solid curves represent the best fit to determined by non-linear regression, with K = 1.0 and 1.2 nM for LpL and LpLC, respectively. Each data point represents the average of duplicate determinations.

The VLDL Receptor Binds Pro-uPA and uPAPAI-1 Complexes and Mediates Their Cellular Catabolism

Figure 4: Binding of uPAPAI-1 complexes to the VLDL receptor measured by enzyme-linked immunosorbent assay (A) and ligand blot analysis (B). A, microtiter wells were coated with anti-VLDL receptor IgG (20 µg/ml) overnight at 4 °C. The wells were then blocked with 3% BSA for 1 h at 25 °C, and purified VLDL receptor was then incubated with the coated microtiter wells overnight at 4 °C (closed circles). In control experiments, BSA was used to coat the microtiter wells (open circles). Increasing concentrations of uPAPAI-1 complexes were incubated with each well overnight at 4 °C in 0.05 M Tris, pH 7.4, 0.15 M NaCl, 5 mM CaCl, 3% BSA, pH 7.4. Following washing, the wells were incubated with a mouse monoclonal anti-uPA IgG (1 µg/ml) for 1 h at 25 °C. The amount of IgG bound to each well was detected using a goat anti-mouse-IgG conjugated to horseradish peroxidase using the substrate 3,3`,5,5`-tetramethylbenzidine. The solid curves represent the best fit to determined by non-linear regression, with K = 15 nM. Each data point represents the average of duplicate determinations. B, purified VLDL receptor was subjected to SDS-PAGE under non-reducing conditions, transferred to nitrocellulose, blocked with 3% milk, and incubated with 50 nM uPAPAI-1 complex in the absence (left panel) or presence of 1 µM RAP (right panel). Following an overnight incubation at 4 °C, the blot was incubated with an anti-uPA monoclonal antibody (2 µg/ml). Binding was detected with a goat anti-mouse IgG-horseradish peroxidase conjugate. The bands were visualized by use of the Renaissance chemiluminescence kit.

The interaction between uPAPAI-1 complexes and the VLDL receptor was also confirmed by ligand blotting experiments. For these experiments, purified VLDL receptor was subjected to SDS-PAGE, transferred to nitrocellulose, and incubated with uPAPAI-1 complexes. The binding of uPAPAI-1 complexes to the immobilized VLDL receptor was visualized using an anti-uPA IgG. The results demonstrate that uPAPAI-1 complexes bind to the VLDL receptor (Fig. 4B, left panel). In the presence of excess RAP, the binding was completely inhibited (Fig. 4B, right panel). Together, these in vitro binding experiments document that uPAPAI-1 complexes interact with the VLDL receptor with high affinity and that RAP blocks ligand binding.

The ability of PEA13 fibroblast infected with Ad-VLDLR to mediate the cellular uptake and degradation of I-uPAPAI-1 complexes was next investigated. The results of a representative experiment are shown in Fig. 5and demonstrate that while PEA-13 fibroblasts infected with Ad-lacZ are unable to internalize or degrade I-uPAPAI-1 complexes, PEA13 fibroblasts that express the VLDL receptor following infection with Ad-VLDLR are effective in internalizing and degrading I-uPAPAI-1 complexes. The cellular internalization and degradation of these complexes are completely blocked by the addition of exogenous RAP. Parental PEA13 fibroblasts, like those infected with Ad-lacZ, are also unable to internalize or degrade I-uPAPAI-1 complexes (data not shown). Together, these data provide compelling evidence that the VLDL receptor mediates the cellular uptake of uPAPAI-1 complexes leading to their degradation.

Figure 5: Internalization of I-labeled uPAPAI-1 complexes by murine PEA13 fibroblasts infected with Ad-VLDLR. PEA13 fibroblasts infected with Ad-VLDLR () or PEA13 fibroblasts infected with Ad-lacZ () were plated into wells (1.5 10 cells/well), and 2 nMI-labeled uPAPAI-1 complexes were added to each well (closed symbols). At indicated times, the extent of internalization (A) or degradation (B) was determined as described under ``Materials and Methods.'' In control experiments, 1 µM RAP was included during the incubation (open symbols). Each data point represents the average of duplicate determinations. , Ad-lacZ + RAP; , Ad-VLDLR + RAP.

Since LRP is known to also directly bind pro-uPA and mediate its internalization(28) , it was of interest to examine the potential role of the VLDL receptor in the catabolism of this molecule. Fig. 6demonstrates that cells infected with Ad-VLDLR but not with Ad-lacZ mediate the cellular internalization (Fig. 6A) and degradation (Fig. 6D) of I-pro-uPA. This process is blocked when RAP is included during the incubation. As a control, mouse embryo fibroblasts that express LRP were also utilized, and these cells also mediate the cellular internalization (Fig. 6A) and degradation (Fig. 6D) of pro-uPA in a process that is antagonized by RAP. These data are consistent with previous studies(28) , demonstrating that LRP mediates the internalization of pro-uPA. The amount of I-pro-uPA internalized by cells expressing the VLDL receptor or LRP represents about 7% of the amount of I-uPAPAI-1 complexes that are internalized by these cells (compare Fig. 6A with 6B). These results indicate that the preferred ligand for both of these receptors is the uPAPAI-1 complex.

Figure 6: Cellular internalization of I-labeled pro-uPA, uPAPAI-1 complexes, and methylamine-activated M (M*) by murine fibroblasts. Murine PEA13 fibroblasts infected with Ad-lacZ or Ad-VLDLR and mouse embryonic fibroblasts (MEF) were plated into culture wells (1.5 10 cells/well). I-Labeled pro-uPA (2 nM) (panels A and D), I-labeled uPAPAI-1 complex (2 nM) (panels B and E), and I-labeled M* (1 nM) (panels C and F) were added (crosshatch bars), and the extent of cellular internalization (panels A, B, and C) and degradation (panels D, E, and F) was determined after 10 h of incubation as described under ``Materials and Methods.'' In control experiments, 1 µM RAP was included (open bars).

The VLDL Receptor Does Not Mediate the Cellular Uptake of Activated M

The VLDL Receptor Is Expressed in Vascular Cells

Figure 7: RAP ligand blot and immunoblot analysis of cell extracts derived from vascular cells. Left panel, cell extracts from human aortic endothelial cells (HAEC), human umbilical vein endothelial cells (HUVEC), and human aortic smooth muscle cells (HASMC) were subjected to SDS-PAGE on 4-12% gradient gels under non-reducing conditions, transferred to nitrocellulose, and incubated with 25 nM RAP. After 1 h at room temperature, the nitrocellulose was washed and incubated with 1 µg/ml rabbit anti-RAP IgG for 1 h at room temperature, followed by incubation with a goat anti-rabbit IgG-horseradish peroxidase conjugate. The bands were visualized by use of the Renaissance chemiluminescence kit. 40 µg of protein were loaded on each lane. Middle panel, nitrocellulose blots were incubated with anti-LRP IgG (R777) (1 µg/ml) and processed as described above. Right panel, cell extracts from human umbilical vein endothelial cells were applied to RAP-Sepharose. After washing, the column was eluted with 10 mM glycine, pH 2.0, containing 150 mM NaCl, 0.05% Tween 20, 0.05% Triton X-100. The pH was immediately adjusted to 8.0 by the addition of 1 M Tris, pH 8.0. An aliquot was subjected to SDS-PAGE, transferred to nitrocellulose, and incubated with an anti-VLDL receptor IgG. The IgG was visualized as described above.

Immunoblotting experiments of the cell extracts using an anti-VLDL receptor IgG failed to detect any protein. This is most likely due to the low sensitivity of this technique when compared to RAP ligand blotting approaches. To confirm that the 130-kDa polypeptide represents the VLDL receptor, an affinity chromatography approach was utilized. Detergent extracts from human umbilical vein endothelial cells were prepared and applied to RAP-Sepharose to concentrate the VLDL receptor. The eluted protein was subjected to SDS-PAGE and transferred to nitrocellulose, and the VLDL receptor was identified by immunoblot analysis using an anti-VLDL receptor IgG. The results of this experiment are shown in Fig. 7(right panel) and demonstrate that the 130- and 105-kDa polypeptides are detected with the anti-VLDL receptor IgG. As a control, molecular weight standards were incubated with the anti-VLDL receptor IgG to demonstrate the specificity of this antisera. The immunoreactive material detected at approximately 260 kDa is presumed to represent a dimer of the VLDL receptor and has been previously observed in extracts from cells transfected with the VLDL receptor cDNA(15) . The immunoblotting results confirm that human umbilical vein endothelial cells express the VLDL receptor. These studies are supported by in situ hybridization studies, which have detected VLDL receptor mRNA in human endothelium (34) and by Northern analysis(29) .

To determine if the VLDL receptor is functional in endothelial cells, the ability of these cells to mediate the cellular internalization and degradation of I-labeled RAP was investigated. Fig. 8demonstrates that human umbilical vein endothelial cells rapidly internalize I-labeled RAP, leading to its degradation, and this is consistent with the expression of functional VLDL receptors in these cells.

Figure 8: Time course of internalization and degradation of I-labeled RAP by human umbilical vein endothelial cells. Human umbilical vein endothelial cells, plated in 6-well plates at 2.4 10 cells per well, were incubated with 1 nMI-labeled RAP. At the indicated times, the cells were washed with cold phosphate-buffered saline, and the extent of internalization () and degradation () was determined as described under ``Materials and Methods.''

The internalization and degradation of I-uPAPAI-1 complexes by human endothelial cells were also investigated. The results of this experiment, shown in Fig. 9, demonstrate that human endothelial cells internalize (Fig. 9A) and degrade (Fig. 9B) uPAPAI-1 complexes. To assess the contribution of the VLDL receptor to this process, RAP was included during the incubation and was found to block approximately 45% of the specific degradation (Fig. 9B). This suggests that a RAP-sensitive receptor, most likely the VLDL receptor, is contributing to the uptake and degradation of this ligand. Thus, these experiments reveal that the VLDL receptor likely plays a major role in regulating uPAPAI-1 levels on the endothelial cell surface. It is apparent that additional RAP-insensitive mechanisms also exist for the cellular uptake of uPAPAI-1 complexes.

Figure 9: Effect of RAP on uptake and internalization of I-uPAPAI-1 complexes by human umbilical vein endothelial cells. Human umbilical vein endothelial cells were plated in 6-well plates at 1.9 10 cells/well, and 5 nMI-labeled uPAPAI-1 complexes were added to each well (). At indicated times, the extent of internalization (A) or degradation (B) was determined as described under ``Materials and Methods.'' In control experiments, 500 nM RAP was included during the incubation (). Each data point represents the average of duplicate determinations.

The catabolism of bovine LpL by porcine endothelial cells has been previously investigated(35) . Saxena et al.(35) found that LpL bound to the cell surface and was rapidly internalized by cultured endothelial cells. This process was inhibited when heparin was included during the incubation. Interestingly, it was observed (35) that the LpL was not degraded in these experiments but rather was recycled back to the cell surface and could be recovered from the medium. In several of our studies utilizing human umbilical vein endothelial cells, we noted that I-labeled LpL was internalized but not degraded. The internalization was not prevented by RAP. In other experiments, however, we noted extensive degradation of I-labeled LpL that was prevented by RAP. The reason for this discrepancy is not readily apparent but may relate to variable expression of the VLDL receptor in endothelial cells. The results of these studies confirm that the regulation of LpL levels on the endothelium is a complex process that likely involves several cell surface molecules.

DISCUSSION

The VLDL receptor is a newly discovered member of the LDL receptor family whose domain organization is remarkably similar to that of the LDL receptor, with the exception that the VLDL receptor contains an additional copy of a cysteine-rich ligand binding repeat. Despite similarities in the structure of these two receptors, a notable difference in their ligand binding properties exists. The LDL receptor binds and internalizes apo B-100 (LDL) or apo E-containing lipoproteins such as intermediate density lipoprotein, -migrating VLDL, and VLDL (5) . On the other hand, the VLDL receptor recognizes apo E-containing lipoproteins but only binds weakly to LDL(2) . The differences in ligand binding properties of these two receptors were further highlighted when recent studies found that RAP binds with high affinity to the VLDL receptor (K = 0.7 nM) (15) but binds weakly to the LDL receptor (K = 300 nM)(36) . Since the biological role of the VLDL receptor is not fully understood, the present investigation was initiated to gain insight into its function by further characterizing the ligand binding properties of this receptor.

The strategy that was employed to measure the capacity of the VLDL receptor to mediate the cellular internalization of ligands involved introducing the VLDL receptor gene into a cell line deficient in this receptor and demonstrating an enhanced uptake of ligands in those cells expressing the VLDL receptor. A murine fibroblast cell line (26) genetically deficient in LRP was utilized for this purpose. An adenoviral vector was chosen to introduce the gene for the VLDL receptor into these cells since adenovirus-mediated gene transfer to mammalian cells in culture has proven to be a highly effective means for introducing genes into a variety of cells(37) . Immunoblotting and RAP ligand blotting experiments confirmed that infection of the PEA13 fibroblasts with Ad-VLDLR led to high levels of expression of this receptor.

Using fibroblasts infected with Ad-VLDLR, we documented the ability of the VLDL receptor to mediate the cellular internalization and subsequent degradation of LpL. In this regard, the VLDL receptor is similar to other members of the LDL receptor family, such as LRP and gp330, both of which bind LpL and mediate its cellular catabolism(31, 38) . Interestingly, Takahashi et al. (39) recently demonstrated that both LpL and apo E enhance the binding of triglyceride-rich lipoproteins to the VLDL receptor, an effect that has also been noted on LRP-mediated uptake and degradation of triglyceride-rich lipoproteins(40) . LpL is a key enzyme involved in lipoprotein metabolism and is synthesized by parenchymal cells, such as adipocytes(41) . A significant portion of newly synthesized LpL appears to be degraded(42) , while the remainder is secreted and transferred by an unknown mechanism to nearby vascular endothelium, where it remains bound through interaction with membrane-associated heparan sulfate chains(43, 44) . Triglyceride-rich lipoproteins bind transiently to LpL at the vascular endothelium, and the enzyme rapidly hydrolyzes triglycerides enabling tissues to utilize fatty acids from the lipoproteins, thereby transforming large lipoproteins, such as chylomicrons and VLDL into cholesterol-rich remnant lipoproteins, which can be taken up by the liver.

In situ hybridization studies (34) have detected VLDL receptor mRNA in human endothelium. These results have been confirmed by Northern blot analysis (29) of mRNA isolated from human umbilical vein endothelial cells. The present studies used RAP ligand blotting and immunoblotting techniques on cell extracts to confirm that the VLDL receptor is expressed in human endothelial cells and smooth muscle cells. To assess the function of the VLDL receptor in mediating the internalization of ligands in human umbilical vein endothelial cells, I-labeled RAP was utilized as a ligand. These experiments revealed that the VLDL receptor appears to be functional in these cells since they rapidly internalize and degrade RAP. However, the role of the VLDL receptor in regulating levels of LpL on the endothelium at this time remains ambiguous, since variable results were obtained in our experiments. Possibly, this results from variable expression of the VLDL receptor in endothelial cells and the involvement of other cell surface molecules that bind LpL.

In addition to its role in the catabolism of LpL and apoE-containing lipoproteins, the VLDL receptor may also play an important role in proteinase catabolism by binding and mediating the cellular internalization of uPAPAI-1 complexes. uPA is synthesized by endothelial cells as a single chain zymogen, pro-uPA, that is converted to the active two chain enzyme (two chain-uPA) by proteolysis. The conversion of pro-uPA to active two chain-uPA is enhanced upon interaction with the urokinase plasminogen activator receptor (uPAR). This molecule is a 55-kDa glycosyl-phosphatidylinositol-anchored cell surface protein (45, 46) that is localized on many cell types, including endothelial cells(47) . In addition to facilitating activation of pro-uPA, binding of u-PA to uPAR acts to localize uPA activity on the cell surface(48) , where it has been implicated in the process of pericellular proteolysis, cell migration, and tissue remodeling(49) . uPA activity is regulated by PAI-1, a rapidly acting inhibitor that is also produced by the endothelium(50) .

Once a complex between uPA and PAI-1 forms, it is rapidly internalized and degraded in a process mediated by LRP(33, 51) . The results of the present investigation confirm that the VLDL receptor, like LRP, can also mediate the cellular catabolism of uPAPAI-1 complexes. This conclusion is supported by in vitro binding studies, which document a high affinity interaction between uPAPAI-1 complexes and the purified VLDL receptor. RAP was shown to antagonize the binding. Further, cultured fibroblasts expressing the VLDL receptor following infection with Ad-VLDLR mediate cellular uptake of I-labeled uPAPAI-1 complexes leading to their degradation. Thus, the VLDL receptor, like LRP (51) and gp330 (52) , binds to uPAPAI-1 complexes and mediates their cellular uptake and degradation. This conclusion is supported by recent findings of Heegaard et al.(53) . The presence of the VLDL receptor on the vascular endothelium suggests a role for this receptor in regulating fibrinolysis, and our experiments suggest a major role for this receptor on the endothelium in regulating uPAPAI-1 levels. However, it is apparent that other RAP-insensitive mechanisms exist on the vascular endothelium that contribute to the internalization of uPAPAI-1 complexes.

Both LRP and the VLDL receptor are able to mediate the cellular uptake of pro-uPA directly, although much less pro-uPA is internalized by either receptor when compared with uPAPAI-1 complexes. This might relate to a decreased affinity of these receptors for pro-uPA when compared with uPAPAI-1 complexes(28) . Nykjaer et al.(54) found that soluble uPAR blocked the binding of pro-uPA to LRP, suggesting that uPAR may protect pro-uPA from LRP-mediated internalization. This observation may also extend to the VLDL receptor and stresses that a major function of uPAR is to protect uPA from being internalized and subsequently degraded.

In summary, the present studies have found that the VLDL receptor, like other members of the LDL receptor family, is a multiligand receptor and, in addition to apo E-containing lipoproteins, also binds and mediates the cellular catabolism of LpL as well as uPAPAI-1 complexes. The present studies detected the VLDL receptor in endothelial cells, and cell uptake experiments suggest that the VLDL receptor plays an important role, along with other molecules, in the regulation of uPAPAI-1 levels on the vascular endothelium.

FOOTNOTES

*

This work was supported by Grants HL50787 and GM42581 (to D. K. S.), HL49264 (to D. A. C.), HL50827 (to K. R. M.), and HD29946 (to J. F. S.), a grant-in-aid from SEPA American Heart Association, and the W. W. Smith Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0726; Fax: 301-738-0794; strickla@hlsun.red-cross.org.

()

The abbreviations used are: LDL, low density lipoprotein; LRP, low density lipoprotein receptor-related protein; VLDL, very low density lipoprotein; RAP, receptor-associated protein; apo E, apolipoprotein E; LpL, lipoprotein lipase; LpLC, carboxyl-terminal domain (residues 313-448) of lipoprotein lipase; uPA, urokinase; pro-uPA, pro-urokinase; PAI-1, plasminogen activator inhibitor type I; Ad-lacZ, adenovirus containing lacZ cDNA; Ad-VLDLR, adenovirus containing VLDL receptor cDNA; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; uPAR, urokinase plasminogen activator receptor.

ACKNOWLEDGEMENTS

REFERENCES

1. Yamamoto, T., Davis, G. C., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. L., and Russel, D. W. (1984) Cell 39, 27-38 [CrossRef][Medline] [Order article via Infotrieve]
2. Takahashi, S., Kawarabayasi, Y., Nakai, T., Sakai, J., and Yamamoto, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9252-9256 [Abstract/Free Full Text]
3. Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988) EMBO J. 7, 4119-4127 [Medline] [Order article via Infotrieve]
4. Saito, A., Pietromonaco, S., Loo, A. K. C., and Farquhar, M. G. (1994) Proc. Natl. Acad Sci. U. S. A. 91, 9725-9729 [Abstract/Free Full Text]
5. Brown, M. S., and Goldstein, J. L. (1986) Science 232, 34-47 [Free Full Text]
6. Strickland, D. K., Kounnas, M. Z., and Argraves, W. S. (1995) FASEB J. 9, 890-898 [Abstract]
7. Krieger, M., and Herz, J. (1994) Annu. Rev. Biochem. 63, 601-637 [Medline] [Order article via Infotrieve]
8. Yamamoto, T., Takahashi, S., Sakai, J., and Kawarabayasi, Y. (1993) Trends Cardiovasc. Med. 3, 144-148
9. Gåfvels, M. E., Caird, M., Britt, D., Jackson, C. L., Patterson, D., and Strauss, J. F., III (1993) Somat. Cell Mol. Genet. 19, 557-569 [CrossRef][Medline] [Order article via Infotrieve]
10. Sakai, J., Hoshino, A., Takahashi, S., Miura, Y., Ishii, H., Suzuki, H., Kawarabayasi, Y., and Yamamoto, T. (1994) J. Biol. Chem. 269, 2173-2182 [Abstract/Free Full Text]
11. Webb, J. C., Sun, X.-M., Patel, D. D., McCarthy, S. N., Knight, B. L., and Soutar, A. K. (1992) J. Lipid Res. 33, 689-698 [Abstract]
12. Kirchgessner, T. G., LeBoeuf, R. C., Langner, C. A., Zollman, S., Chang, C. H., Taylor, B. A., Schotz, M. C., Gordon, J. I., and Lusis, A. J. (1989) J. Biol. Chem. 264, 1473-1482 [Abstract/Free Full Text]
13. Bujo, H., Hermann, M. O., Kaderli, M., Jacobsen, L., Surawara, S., Yamamoto, T., and Schneider, W. J. (1994) EMBO J. 13, 5165-5175 [Medline] [Order article via Infotrieve]
14. Nimpf, J., Radosavljevic, M. J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 1393-1398 [Abstract/Free Full Text]
15. Battey, F., Gåfvels, M. E., Fitzgerald, D. J., Argraves, W. S., Chappell, D. A., Strauss, J. F., III, and Strickland, D. K. (1994) J. Biol. Chem. 269, 23268-23273 [Abstract/Free Full Text]
16. Ashcom, J. D., Tiller, S. E., Dickerson, K., Cravens, J. L., Argraves, W. S., and Strickland, D. K. (1990) J. Cell Biol. 110, 1041-1048 [Abstract/Free Full Text]
17. Jensen, P. H., Moestrup, S. K., and Gliemann, J. (1989) FEBS Lett. 255, 275-280 [CrossRef][Medline] [Order article via Infotrieve]
18. Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S. (1991) J. Biol. Chem. 266, 21232-21238 [Abstract/Free Full Text]
19. Williams, S. E., Ashcom, J. D., Argraves, W. S., and Strickland, D. K. (1992) J. Biol. Chem. 267, 9035-9040 [Abstract/Free Full Text]
20. Kounnas, M. Z., Argraves, W. S., and Strickland, D. K. (1992) J. Biol. Chem. 267, 21162-21166 [Abstract/Free Full Text]
21. Biemesderfer, D., Dekan, G., Aronson, P. S., and Farquhar, M. G. (1993) Am. J. Physiol. 264, F1011-F1020
22. Willnow, T. E., Armstrong, S. A., Hammer, R. E., and Herz, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4537-4541 [Abstract/Free Full Text]
23. Bradford, M. M. (1976) Anal. Biochem. 72, 248-255 [CrossRef][Medline] [Order article via Infotrieve]
24. Iverius, P., and Ostlund-Lindqvist, A. M. (1976) J. Biol. Chem. 251, 7791-7795 [Abstract/Free Full Text]
25. Williams, S. E., Inoue, I., Tran, H., Fry, G. L., Pladet, M. W., Iverius, P., Lalouel, J., Chappell, D. A., and Strickland, D. K. (1994) J. Biol. Chem. 269, 8653-8658 [Abstract/Free Full Text]
26. Willnow, T. E., and Herz, J. (1994) J. Cell Sci. 107, 719-726 [Abstract]
27. Berkner, K. L. (1988) BioTechniques 6, 616-629 [Medline] [Order article via Infotrieve]
28. Kounnas, M. Z., Henkin, J., Argraves, W. S., and Strickland, D. K. (1993) J. Biol. Chem. 268, 21862-21867 [Abstract/Free Full Text]
29. Wittmaack, F. M., Gafvels, M. E., Bronner, M., Matsuo, H., McCrae, K. R., Tomaszewski, J. E., Robinson, S. L., Strickland, D. K., and Strauss, J. F., III (1995) Endocrinology 136, 340-348 [Abstract]
30. Kounnas, M. Z., Morris, R. E., Thompson, M. R., Fitzgerald, D. J., Strickland, D. K., and Saelinger, C. B. (1992) J. Biol. Chem. 267, 12420-12423 [Abstract/Free Full Text]
31. 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]
32. Jacobsen, L., Hermann, M., Vieira, P. M., Schneider, W. J., and Nimpf, J. (1995) J. Biol. Chem. 270, 6468-6475 [Abstract/Free Full Text]
33. Herz, J., Clouthier, D. E., and Hammer, R. E. (1992) Cell 71, 411-421 [CrossRef][Medline] [Order article via Infotrieve]
34. Multhaupt, H. A. B., Jin, H., Arenas-Elliot, C., Strauss, J. F., III, Gafvels, M., Warhol, M. J., and McCrae, K. P. (1994) Circulation 90, 1-2 [Free Full Text]
35. Saxena, U., Klein, M. G., and Goldberg, I. J. (1990) J. Biol. Chem. 265, 12880-12886 [Abstract/Free Full Text]
36. 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]
37. Becker, T. C., Noel, R. J., Coats, W. S., Gomez-Foix, A. M., Alam, T., Gerard, R. D., and Newgard, C. B. (1994) Methods Cell Biol. 43, 161-189
38. Kounnas, M. Z., Chappell, D. A., Strickland, D. K., and Argraves, W. S. (1993) J. Biol. Chem. 268, 14176-14181 [Abstract/Free Full Text]
39. Takahashi, S., Suzuki, J., Kohno, M., Oida, K., Tamai, T., Miyabo, S., Yamamoto, T., and Nakai, T. (1995) J. Biol. Chem. 270, 15747-15754 [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. Olivecrona, T., Chernick, S. S., Bengtsson-Olivecrona, G., Garrison, M., and Scow, R. O. (1987) J. Biol. Chem. 262, 10748-10759 [Abstract/Free Full Text]
42. Cisar, L., Hoogewerf, A. J., Cupp, M., Rapport, C. A., and Bensadoun, A. (1989) J. Biol. Chem. 264, 1767-1774 [Abstract/Free Full Text]
43. Bensadoun, A. (1991) Annu. Rev. Nutr. 11, 217-237 [CrossRef][Medline] [Order article via Infotrieve]
44. Stins, M. F., Sivaram, P., Sasaki, A., and Goldberg, I. J. (1993) J. Lipid Res. 34, 1853-1861 [Abstract]
45. Roldan, A. L., Cubellis, M. V., Masucci, M. T., Behrendt, N., Lund, L. R., Dano, K., Appella, E., and Blasi, F. (1990) EMBO J 9, 467-474 [Medline] [Order article via Infotrieve]
46. Ploug, M., Ronne, E., Behrendt, N., Jensen, A. L., Blasi, F., and Dano, K. (1991) J. Biol. Chem. 266, 1926-1933 [Abstract/Free Full Text]
47. Haddock, R. C., Spell, M. L., Baker, C. D., Grammer, J. R., Parks, J. M., Speidel, M., and Booyse, F. M. (1991) J. Biol. Chem. 266, 21466-21473 [Abstract/Free Full Text]
48. Ellis, V., and Dano, K. (1992) Fibrinolysis 6, Suppl. 4, 27-34
49. Blasi, F. (1993) BioEssays 15, 105-111 [CrossRef][Medline] [Order article via Infotrieve]
50. Schleef, R. R., and Loskutoff, D. J. (1988) Haemostasis 18, 328-341 [Medline] [Order article via Infotrieve]
51. Nykjaer, A., Petersen, C. M., Moller, B., Jensen, P. H., Moestrup, S. K., Holtet, T. L., Etzerodt, M., Thogersen, H. C., Munch, M., Andreasen, P. A., and Gliemann, J. (1992) J. Biol. Chem. 267, 14543-14546 [Abstract/Free Full Text]
52. Moestrup, S. K., Nielsen, S., Andreasen, P., Jorgensen, K. E., Nykjaer, A., Roigaard, H., Gliemann, J., and Christensen, E. I. (1993) J. Biol. Chem. 268, 16564-16570 [Abstract/Free Full Text]
53. Heegaard, C. W., Simonsen, A. C. W., Oka, K., Kjoller, L., Christensen, A., Madsen, B., Ellgaard, L., Chan, L., and Andreasen, P. A. (1995) J. Biol. Chem. 270, 20855-20861 [Abstract/Free Full Text]
54. Nykjaer, A., Kjoller, L., Cohen, R. L., Lawrence, D. A., Garni-Wagner, B. A., Todd, R. F., III, and van Zonneveld, A. (1994) J. Biol. Chem. 269, 25668-25676 [Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles: