©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Very Low Density Lipoprotein Receptor Mediates the Cellular Catabolism of Lipoprotein Lipase and Urokinase-Plasminogen Activator Inhibitor Type I Complexes (*)

(Received for publication, April 14, 1995; and in revised form, August 10, 1995)

Kelley M. Argraves (1) Frances D. Battey (1) Colin D. MacCalman (2) Keith R. McCrae (3) Mats Gåfvels (4) Karen F. Kozarsky (6) David A. Chappell (5) Jerome F. Strauss , III (2) Dudley K. Strickland (1)(§)

From the  (1)Holland Laboratory, Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, (2)Departments of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6142, (3)Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, (4)Metabolism and Molecular Nutrition Units, Departments of Internal Medicine, Karolinska Institute and Huddinge University Hospital, Huddinge, S-14186 Sweden, (5)Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, and the (6)Institute of Human Gene Therapy and Department of Molecular and Cellular Engineering, University of Pennsylvania, and the Wistar Institute, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The very low density lipoprotein (VLDL) receptor binds apolipoprotein E-rich lipoproteins as well as the 39-kDa receptor-associated protein (RAP). Ligand blotting experiments using RAP and immunoblotting experiments using an anti-VLDL receptor IgG detected the VLDL receptor in detergent extracts of human aortic endothelial cells, human umbilical vein endothelial cells, and human aortic smooth muscle cells. To gain insight into the role of the VLDL receptor in the vascular endothelium, its ligand binding properties were further characterized. In vitro binding experiments documented that lipoprotein lipase (LpL), a key enzyme in lipoprotein catabolism, binds with high affinity to purified VLDL receptor. In addition, urokinase complexed with plasminogen activator-inhibitor type I (uPAbulletPAI-1) also bound to the purified VLDL receptor with high affinity. To assess the capacity of the VLDL receptor to mediate the cellular internalization of ligands, an adenoviral vector was used to introduce the VLDL receptor gene into a murine embryonic fibroblast cell line deficient in the VLDL receptor and the LDL receptor-related protein, another endocytic receptor known to bind LpL and uPAbulletPAI-1 complexes. Infected fibroblasts that express the VLDL receptor mediate the cellular internalization of I-labeled LpL and uPAbulletPAI-1 complexes, leading to their degradation. Non-infected fibroblasts or fibroblasts infected with the lacZ gene did not internalize these ligands. These studies confirm that the VLDL receptor binds to and mediates the catabolism of LpL and uPAbulletPAI-1 complexes. Thus, the VLDL receptor may play a unique role on the vascular endothelium in lipoprotein catabolism by regulating levels of LpL and in the regulation of fibrinolysis by facilitating the removal of urokinase complexed with its inhibitor.


INTRODUCTION

The low density lipoprotein (LDL) (^1)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

Human VLDL receptor was purified from detergent extracts of human embryonic kidney 293 cells infected with the adenovirus containing the human VLDL receptor cDNA (Ad-VLDL receptor) (see below). 25 150-mm plates of infected cells were extracted in 10 ml of ice-cold 50 mM HEPES, pH 7.4, 0.5 M NaCl, 0.05% Tween 20, 1% Triton X-100, containing the following proteinase inhibitors: 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, 5 µg/ml PPACK, and 2 µg/ml pepstatin (extraction buffer). The cell extract was sheared with a 21-gauge needle and then centrifuged at 14,000 rpm for 10 min. The VLDL receptor was purified over RAP-Sepharose as described by Battey et al.(15) . An aliquot of each fraction was subjected to SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting to identify the VLDL receptor-containing fractions. Receptor concentrations were determined by the assay of Bradford (23) using bovine serum albumin as a standard. Human RAP was expressed in bacteria as a fusion protein with glutathione S-transferase (GST) and purified free of GST as described by Williams et al.(19) . Bovine milk LpL was isolated as described(24) . The carboxyl-terminal domain of human LpL (LPLC) was produced as a fusion protein with GST in bacteria as described(25) . Human pro-urokinase (pro-uPA), and two chain uPA were provided by Dr. Jack Henkin (Abbott Laboratories, Abbott Park, IL). Active recombinant PAI-1 was purchased from Molecular Innovations (Wayne, MI). uPA was complexed with PAI-1 by mixing at a 1:1 molar ratio in 0.15 M Tris, 0.15 M NaCl, pH 8.0, at room temperature for 30 min. Human alpha(2)M was isolated from plasma and activated with methylamine (alpha(2)M:Me) as described (19) . Bovine serum albumin fraction V was purchased from Sigma. All proteins (RAP, LPL, uPA, pro-uPA, and alpha(2)M:Me) were iodinated with I by IODO-GEN (Amersham) as described previously (25) with specific activities ranging from 3 to 17 µCi/µg. I-uPAbulletPAI-1 complexes were prepared by reacting I-labeled uPA with PAI-1 at a 1:1 molar ratio.

Cell Culture

Human aortic endothelial cells, human umbilical vein endothelial cells, and human aortic smooth muscle cells were grown in plates previously coated with fibronectin (1 µg/ml). The cells were grown in Medium 199 (Biowhittaker, Walkersville, MD) containing 10% fetal bovine serum, 2 mML-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 5 units/ml heparin, and 10 ng/ml fibroblast growth factor-1. The LRP-deficient cell line, PEA13, and mouse embryonic fibroblasts were generously provided by Dr. J. Herz (Dallas, TX) and were grown as described (26) in Dulbecco's modified Eagle's medium from Cellgrow (Washington, D. C.) containing 10% bovine calf serum (Hyclone Laboratories, Logan, UT) and 2 mML-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin.

Adenovirus Infection of PEA-13 Cells

The production of recombinant replication-deficient adenoviral vectors was prepared as described(27) . Briefly, the vectors were constructed from an adenovirus type 5 (Ad5) mutant, which lacks most of viral sequence regions E1a and E1b and a portion of E3. By homologous recombination techniques, either Escherichia coli lacZ marker cDNA or the human VLDL receptor cDNA(9) , driven by the human cytomegalovirus 3`-promoter region, was inserted into the viral genome. Viral stocks were propagated in human embryonic kidney 293 cells, and the titer was estimated by spectrophotometric density and/or plaque assays. To determine the optimal concentration of virus for infection, PEA13 cells were grown in 150-mm dishes and infected with various concentrations of Ad-VLDLR and Ad-lacZ. After 48 h, the cells were solubilized with extraction buffer, and the extract was subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted to measure VLDL receptor protein expression. Optimal expression occurred at 60,000 particles/cell. Ad-lacZ infections were performed using the same concentration of virus particles.

Cell Internalization and Degradation Assays

Human umbilical vein endothelial cells were plated in 6-well dishes at 2.4 times 10^5 cells/well 24 h prior to the assay. For PEA13 cells infected with Ad-VLDLR and Ad-lacZ, the cells were first infected in 150-mm dishes. After incubating for 24 h, the cells were transferred to 12-well dishes (Costar, Cambridge MA) at 1.5 times 10^5 cells/well. Cellular internalization and degradation assays were performed 24 h following the replating (48 h following infection). Cellular internalization and degradation assays were performed as described (28) with 1-5 nMI-labeled ligands. Nonspecific uptake and degradation was measured using 100-200-fold molar excess unlabeled ligands and accounted for 18% of the total degradation. In the case of I-labeled LpL, 100 µg/ml heparin was used to determine nonspecific uptake and degradation and accounted for 20% of the total degradation. RAP blocking experiments were performed utilizing 1 µM recombinant RAP.

Antibodies

Rabbit polyclonal antibodies were prepared against a synthetic peptide corresponding to the human VLDL receptor carboxyl terminus as described by Battey et al.(15) , and the antibody was affinity purified on peptide-Sepharose as described by Wittmaack et al.(29) . Polyclonal antibodies to human LRP (Rb777) were developed as described (30) and purified by affinity chromatography on LRP-Sepharose followed by chromatography on RAP-Sepharose to remove any anti-RAP reactivity. Antibodies against human RAP (Rb80) were affinity purified on RAP-Sepharose as described (30) . Monoclonal antibody to uPA was generously provided by Jack Henkin (Abbot Laboratories).

Preparation of Cell Extracts

Cell extracts were prepared from cells grown in either 100- or 150-mm dishes, washed twice in 5 ml of isotonic phosphate-buffered saline, and then solubilized in 300 µl of extraction buffer and used directly for immunoblotting and RAP-ligand blotting experiments. In immunoblot experiments of human umbilical vein endothelial cells, six 150-mm dishes of cells were solubilized in 1.8 ml of extraction buffer and centrifuged, and the supernatant was then applied to a RAP-Sepharose column. The column was washed and eluted as described(15) , and an aliquot from the eluted fraction was subjected to immunoblot analysis. Protein concentrations were determined by the method of Bradford (23) using BSA as a standard.

Immunoblotting and Ligand Blotting

Cell extracts were subjected to SDS-PAGE on gradient gels (4-12% Tris-glycine gels from Novex, San Diego, CA) under non-reducing conditions and electrophoretically transferred to nitrocellulose membranes. Filters were blocked with 3% non-fat milk in 50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM CaCl(2) for 1 h at 25 °C (blocking buffer). For immunoblotting experiments, the membranes were then incubated with an affinity-purified polyclonal antibody at 1 µg/ml in blocking buffer plus 0.05% Tween 20 (incubation buffer) for 1 h at 25 °C and then washed three times with incubation buffer. The membranes were then incubated with a goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad) in incubation buffer for 1 h at 25 °C and washed as described above. Antibody binding was visualized by using the Renaissance chemiluminescence kit (DuPont NEN).

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 uPAbulletPAI-1 complexes, nitrocellulose membranes were incubated with 50 nM uPAbulletPAI-1 in the absence or presence of 1 µM RAP for 1 h at 25 °C. The bound uPAbulletPAI-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

The binding of uPAbulletPAI-1 complexes to microtiter wells coated with the purified VLDL receptor was performed essentially as described earlier(15) . Wells of microtiter plates (Dynatech Immulon 2, Dynatech Laboratories Inc., Chantilly, VA) were coated with 100 µl of 20 µg/ml affinity-purified anti-VLDL receptor IgG in 0.1 M sodium bicarbonate, pH 9.0, overnight at 4 °C. The wells were then blocked with 3% BSA in 50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM CaCl(2) for 1 h at 25 °C. The VLDL receptor, purified as described above, was captured on the antibody-coated microtiter wells overnight at 4 °C, and following washing, various concentrations of uPAbulletPAI-1 were added to the wells and also to control wells coated with just BSA. After an overnight incubation at 4 °C, the wells were washed and incubated with a mouse monoclonal antibody to uPA (1 µg/ml) for 1 h at 25 °C. Following washing, the IgG that was bound was detected with a goat anti-mouse IgG conjugated to horseradish peroxidase using the substrate 3,3`5,5`-tetramethylbenzidine (Kirkegaard & Perry, Gaithersburg, MD). Data were analyzed by nonlinear regression analysis using ,

where A is the absorbance at 650 nm, A(max) is the absorbance value at saturation, A(min) is the background absorbance in the absence of ligand, [L] is the molar concentration of free uPAbulletPAI-1 complexes, and K(D) is the dissociation constant. Since the free uPAbulletPAI-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 uPAbulletPAI-1 is approximately equal to the total uPAbulletPAI-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(2), 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

To investigate the role of the VLDL receptor in mediating the cellular uptake of ligands, a suitable cell line that expresses relatively large amounts of the VLDL receptor and relatively small amounts of other LDL receptor family members was required. For this purpose, PEA13 fibroblasts were employed. PEA13 fibroblasts are genetically deficient in LRP(26) . In addition, they do not express gp330 or VLDL receptor. To express the VLDL receptor in these cells an adenovirus vector, Ad-VLDLR, which contains the human VLDL receptor cDNA under the regulation of a cytomegalovirus promoter, was used. As a control, PEA13 fibroblasts were infected with an adenovirus vector, Ad-lacZ, containing the lacZ cDNA instead of the VLDL receptor cDNA. To measure production of the VLDL receptor in cells infected with Ad-VLDLR, cell extracts were immunoblotted with an anti-VLDL receptor IgG. The results of a typical experiment are shown in Fig. 1and demonstrate that cells infected with Ad-VLDLR expressed the VLDL receptor (Fig. 1A, left panel). In contrast, no immunoreactive material can be detected in parental PEA13 fibroblasts or in PEA13 fibroblasts infected with Ad-lacZ. The reaction with antibody was judged specific, since no immunoreactivity was noted when the anti-VLDL receptor IgG was omitted from the experiment (Fig. 1A, right panel). The minor band noted at approximately 105 kDa likely represents the VLDL receptor precursor, while the band noted at approximately 260 kDa is presumed to represent a dimer and has been observed previously(15) .


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

The tissue distribution of the VLDL receptor is quite similar to that of LpL(12) , and thus LpL has been proposed to play an important role in conjunction with the VLDL receptor in the catabolism of lipoproteins(8) . Consequently, studies to examine whether the VLDL receptor mediates the catabolism of LpL were initiated. The ability of the VLDL receptor to mediate the cellular internalization of I-LpL was examined using PEA13 cells infected with Ad-VLDLR. The results of these experiments are shown in Fig. 2. PEA13 fibroblasts that express the VLDL receptor effectively internalize (Fig. 2A) and degrade (Fig. 2B) I-LpL. Both of these processes are blocked when RAP is included in the culture media. PEA13 fibroblasts infected with Ad-lacZ internalized and degraded a small amount of I-LpL; these processes were not blocked by RAP. Parental PEA13 fibroblasts were identical to fibroblasts infected with Ad-lacZ (data not shown). These experiments confirm that the VLDL receptor, like LRP (31) , mediates the cellular internalization of LpL leading to its degradation.


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 (bullet) were plated into wells (1.5 times 10^5 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. circle, Ad-lacZ + RAP; box, Ad-VLDLR + RAP.



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

Previous studies have determined that the carboxyl-terminal domain of LpL, termed LpLC, when expressed as a fusion protein with GST, binds to LRP(25) , and thus it was of interest to determine whether this region of LpL also interacts with the VLDL receptor. For these studies, an enzyme-linked immunosorbent assay was employed. This assay revealed that the purified VLDL receptor binds to microtiter wells coated with LpL but not to BSA-coated microtiter wells (Fig. 3A). The apparent K(D) of 1 nM is comparable to that measured for the binding of LRP to microtiter wells coated with LpL(25) . Fig. 3B demonstrates that the purified VLDL receptor also binds to GST-LpLC coated microtiter wells, and the apparent K(D) of 1.2 nM is close to that measured for the binding of LRP to GST-LpLC using a similar assay (25) .


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(2), 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 uPAbulletPAI-1 Complexes and Mediates Their Cellular Catabolism

It was of interest to determine if other LRP ligands, such as uPAbulletPAI-1 complexes, could bind to the VLDL receptor. Fig. 4A shows that uPAbulletPAI-1 complexes bind to purified VLDL receptor captured on microtiter wells coated with anti-VLDL receptor IgG. No binding of uPAbulletPAI-1 complexes to the microtiter wells coated with BSA was detected. An apparent K(D) of 15 nM for the binding of uPAbulletPAI-1 complexes to the VLDL receptor was estimated from fitting the data by nonlinear regression analysis to the equation described under ``Materials and Methods.'' This value is close to that obtained for the interaction of uPAbulletPAI-1 complexes with LRP using a similar assay (data not shown).


Figure 4: Binding of uPAbulletPAI-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 uPAbulletPAI-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(2), 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 uPAbulletPAI-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 uPAbulletPAI-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 uPAbulletPAI-1 complexes. The binding of uPAbulletPAI-1 complexes to the immobilized VLDL receptor was visualized using an anti-uPA IgG. The results demonstrate that uPAbulletPAI-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 uPAbulletPAI-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-uPAbulletPAI-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-uPAbulletPAI-1 complexes, PEA13 fibroblasts that express the VLDL receptor following infection with Ad-VLDLR are effective in internalizing and degrading I-uPAbulletPAI-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-uPAbulletPAI-1 complexes (data not shown). Together, these data provide compelling evidence that the VLDL receptor mediates the cellular uptake of uPAbulletPAI-1 complexes leading to their degradation.


Figure 5: Internalization of I-labeled uPAbulletPAI-1 complexes by murine PEA13 fibroblasts infected with Ad-VLDLR. PEA13 fibroblasts infected with Ad-VLDLR () or PEA13 fibroblasts infected with Ad-lacZ (bullet) were plated into wells (1.5 times 10^5 cells/well), and 2 nMI-labeled uPAbulletPAI-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. circle, Ad-lacZ + RAP; box, 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-uPAbulletPAI-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 uPAbulletPAI-1 complex.


Figure 6: Cellular internalization of I-labeled pro-uPA, uPAbulletPAI-1 complexes, and methylamine-activated alpha(2)M (alpha(2)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 times 10^5 cells/well). I-Labeled pro-uPA (2 nM) (panels A and D), I-labeled uPAbulletPAI-1 complex (2 nM) (panels B and E), and I-labeled alpha(2)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 alphaM

A recent study (32) reported that both native and activated alpha(2)M bind to the chicken VLDL receptor in ligand blotting experiments. Further, the investigators demonstrated that COS cells transfected with the chicken VLDL receptor cDNA were able to internalize activated alpha(2)M. Since COS cells are known to express large amounts of LRP(33) , it is not easy to demonstrate that the VLDL receptor is functional in internalizing activated alpha(2)M using these cells. Consequently, it was of interest to investigate if the human VLDL receptor could mediate the cellular uptake of activated alpha(2)M. The results demonstrate that PEA13 fibroblasts expressing the VLDL receptor following infection with Ad-VLDLR were unable to internalize (Fig. 6C) or degrade (Fig. 6F) I-labeled alpha(2)M activated by treatment with methylamine. In the same experiment, these cells were able to mediate the internalization of I-uPAbulletPAI-1 complexes (Fig. 6B), indicating that the VLDL receptor is indeed functional. In contrast, mouse embryo fibroblasts, which express LRP, were efficient in internalizing and degrading I-labeled activated alpha(2)M in a process that was inhibited by RAP (Fig. 6, C and F). These experiments indicate that the human VLDL receptor does not mediate the cellular uptake of activated alpha(2)M.

The VLDL Receptor Is Expressed in Vascular Cells

Previous studies have detected VLDL receptor mRNA in human umbilical endothelial cells by Northern blot analysis(29) . To determine if protein is expressed in these cells, RAP ligand blotting experiments were performed on cell extracts. Previous studies have shown that this technique is approximately 10-fold more sensitive than immunoblotting using an affinity-purified anti-VLDL receptor peptide IgG(15) . The results of RAP blotting studies shown in Fig. 7(left panel), demonstrate that human aortic endothelial cells, human umbilical vein endothelial cells, and human aortic smooth muscle cells all contain an 130-kDa polypeptide that comigrates with the VLDL receptor. A 105-kDa polypeptide was also detected in all three types of cells. This polypeptide could represent the VLDL receptor precursor or the variant form of the VLDL receptor lacking the O-linked sugar domain(10) . A large polypeptide, with a mobility identical to LRP was detected in the human aortic smooth muscle cells. Only trace amounts of this polypeptide were detected in endothelial cells. This molecule was identified as LRP by immunoblot analysis using an anti-LRP IgG (Fig. 7, middle panel). Human umbilical vein endothelial cells and human aortic endothelial cells do not appear to express much, if any, LRP (Fig. 7, middle panel). Cellular uptake experiments confirmed that I-labeled alpha(2)M-proteinase complexes are not internalized by human umbilical vein endothelial cells (data not shown), confirming that very little LRP is present in these cells. Taken together, these experiments suggest that endothelial cells express the VLDL receptor but negligible amounts of LRP.


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 times 10^5 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 (circle) and degradation (bullet) was determined as described under ``Materials and Methods.''



The internalization and degradation of I-uPAbulletPAI-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) uPAbulletPAI-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 uPAbulletPAI-1 levels on the endothelial cell surface. It is apparent that additional RAP-insensitive mechanisms also exist for the cellular uptake of uPAbulletPAI-1 complexes.


Figure 9: Effect of RAP on uptake and internalization of I-uPAbulletPAI-1 complexes by human umbilical vein endothelial cells. Human umbilical vein endothelial cells were plated in 6-well plates at 1.9 times 10^5 cells/well, and 5 nMI-labeled uPAbulletPAI-1 complexes were added to each well (bullet). 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, beta-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(D) = 0.7 nM) (15) but binds weakly to the LDL receptor (K(D) = 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 uPAbulletPAI-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 uPAbulletPAI-1 complexes. This conclusion is supported by in vitro binding studies, which document a high affinity interaction between uPAbulletPAI-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 uPAbulletPAI-1 complexes leading to their degradation. Thus, the VLDL receptor, like LRP (51) and gp330 (52) , binds to uPAbulletPAI-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 uPAbulletPAI-1 levels. However, it is apparent that other RAP-insensitive mechanisms exist on the vascular endothelium that contribute to the internalization of uPAbulletPAI-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 uPAbulletPAI-1 complexes. This might relate to a decreased affinity of these receptors for pro-uPA when compared with uPAbulletPAI-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 uPAbulletPAI-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 uPAbulletPAI-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.

(^1)
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

We thank Dr. Jeffery Winkles for providing the endothelial cells and human aortic smooth muscle cells utilized for some of these experiments. We also thank Evan Behre for preparing RAP and Sue Robinson for preparing the anti-VLDL receptor IgG. We also thank Dr. J. Herz for providing the PEA13 cells used in these studies and Dr. Jack Henkin for providing pro-uPA, uPA, and monoclonal antibodies against uPA.


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