(Received for publication, April 14, 1995; and in revised form, August 10, 1995)
From the
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 (uPAPAI-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
uPA
PAI-1 complexes. Infected fibroblasts that express the VLDL
receptor mediate the cellular internalization of
I-labeled LpL and uPA
PAI-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 uPA
PAI-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.
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).
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 uPA
PAI-1 in the
absence or presence of 1 µM RAP for 1 h at 25 °C. The
bound uPA
PAI-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).
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
uPA
PAI-1 complexes, and K
is the
dissociation constant. Since the free uPA
PAI-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
uPA
PAI-1 is approximately equal to the total uPA
PAI-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.
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).
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 nM
I-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.
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.
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
uPA
PAI-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 uPA
PAI-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 uPA
PAI-1 complexes. The
binding of uPA
PAI-1 complexes to the immobilized VLDL receptor
was visualized using an anti-uPA IgG. The results demonstrate that
uPA
PAI-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 uPA
PAI-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-uPA
PAI-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-uPA
PAI-1 complexes, PEA13 fibroblasts that
express the VLDL receptor following infection with Ad-VLDLR are
effective in internalizing and degrading
I-uPA
PAI-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-uPA
PAI-1 complexes (data not shown). Together,
these data provide compelling evidence that the VLDL receptor mediates
the cellular uptake of uPA
PAI-1 complexes leading to their
degradation.
Figure 5:
Internalization of I-labeled
uPA
PAI-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 nM
I-labeled uPA
PAI-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-uPA
PAI-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
uPA
PAI-1 complex.
Figure 6:
Cellular internalization of I-labeled pro-uPA, uPA
PAI-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 uPA
PAI-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).
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 nM
I-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-uPA
PAI-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) uPA
PAI-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
uPA
PAI-1 levels on the endothelial cell surface. It is apparent
that additional RAP-insensitive mechanisms also exist for the cellular
uptake of uPA
PAI-1 complexes.
Figure 9:
Effect of RAP on uptake and
internalization of I-uPA
PAI-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 nM
I-labeled uPA
PAI-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.
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 uPA
PAI-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
uPA
PAI-1 complexes leading to their degradation. Thus, the VLDL
receptor, like LRP (51) and gp330 (52) , binds to
uPA
PAI-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 uPA
PAI-1
levels. However, it is apparent that other RAP-insensitive mechanisms
exist on the vascular endothelium that contribute to the
internalization of uPA
PAI-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
uPA
PAI-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
uPA
PAI-1 levels on the vascular endothelium.