The Low Density Lipoprotein Receptor-related Protein Complexes
with Cell Surface Heparan Sulfate Proteoglycans to Regulate
Proteoglycan-mediated Lipoprotein Catabolism*
Larissa C.
Wilsie and
Robert A.
Orlando
From the Department of Biochemistry and Molecular Biology,
University of New Mexico, Health Sciences Center, Albuquerque, New
Mexico 87131-0001
Received for publication, August 27, 2002, and in revised form, February 19, 2003
 |
ABSTRACT |
It has been proposed that clearance of
cholesterol-enriched very low density lipoprotein (VLDL) particles
occurs through a multistep process beginning with their initial binding
to cell-surface heparan sulfate proteoglycans (HSPG), followed by their
uptake into cells by a receptor-mediated process that utilizes members of the low density lipoprotein receptor (LDLR) family, including the
low density lipoprotein receptor-related protein (LRP). We have further
explored the relationship between HSPG binding of VLDL and its
subsequent internalization by focusing on the LRP pathway using a cell
line deficient in LDLR. In this study, we show that LRP and HSPG are
part of a co-immunoprecipitable complex at the cell surface
demonstrating a novel association for these two cell surface receptors.
Cell surface binding assays show that this complex can be disrupted by
an LRP-specific ligand binding antagonist, which in turn leads to
increased VLDL binding and degradation. The increase in VLDL binding
results from an increase in the availability of HSPG sites as treatment
with heparinase or competitors of glycosaminoglycan chain addition
eliminated the augmented binding. From these results we propose a model
whereby LRP regulates the availability of VLDL binding sites at the
cell surface by complexing with HSPG. Once HSPG dissociates from LRP, it is then able to bind and internalize VLDL independent of LRP endocytic activity. We conclude that HSPG and LRP together participate in VLDL clearance by means of a synergistic relationship.
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INTRODUCTION |
Remnant lipoproteins originating from lipolytic processing of
dietary chylomicrons and very low density lipoproteins
(VLDL)1 synthesized by the
liver are primary carriers in lipid transport and facilitate the
delivery of cholesterol and triglycerides to hepatic and extrahepatic
tissues. VLDL particles consist of apolipoprotein B (apoB) and
apolipoproteins CI, CII, and CIII (1) with esterified cholesterol and
triglycerides at their core, and an outer association of apolipoprotein
E (apoE). ApoCII is thought to activate lipoprotein lipase, necessary
for lipolytic processing (2-4). ApoB (5, 6) and apoE (7, 8) are
required for receptor-mediated clearance of VLDL particles.
Receptor-mediated clearance of circulating VLDL is a rapid and critical
step for maintenance of proper plasma lipid levels. The half-life of
injected VLDL is reported to be ~20 min in mice and rabbits (9).
Elevated VLDL levels, as seen in type III hyperlipoproteinemia (10,
11), lead to elevated plasma triglyceride levels making VLDL highly
atherogenic (5). Moreover, excessive VLDL uptake by macrophages results
in cholesterol accumulation and foam cell formation (12, 13) increasing
the risk for coronary heart disease and stroke.
Clearance of VLDL is thought to occur through a multistep process. VLDL
initially binds to cell surfaces through interactions with heparan
sulfate proteoglycans (HSPG) (14), and this binding is mediated by
apoE, which contains a high affinity heparan sulfate binding site (15,
16). Accordingly, apoE knockout mice display severe
hypercholesterolemia with a marked elevation of circulating remnants
(17, 18). Although it is established that HSPGs are the primary binding
sites for VLDL at the cell surface, their path of uptake into cells is
less clear and can occur through several independent receptor-mediated
events (19, 20). Studies have shown that the LDL receptor participates
in VLDL clearance in vivo (8, 21), however, other receptors
also contribute to its uptake, including the LDL receptor-related
protein (LRP) (22, 23) and HSPG (12, 14, 24, 25). LRP is a large cell
surface multiligand-binding protein and member of the LDL receptor
family (26). LRP is expressed predominantly in the liver but can also
be found in many diverse cell types (27). Impairment of LRP function
in vivo has been shown to result in an increase in
circulating levels of cholesterol and triglycerides (28, 29). Also,
aged rats were shown to have decreased LRP expression, which is
accompanied by a delayed clearance of VLDL remnants (30). HSPGs are
also able to directly internalize bound lipoproteins and mediate their
catabolism, although their internalization rates are slow when compared
with those of the LDL receptor family members (31, 32). Syndecan-1,
which is expressed on the peri-sinusoidal membrane of hepatocytes, is
one such example (33). Syndecan-1 is of the transmembrane class of
proteoglycans (34, 35) and has been shown to independently mediate the
binding, internalization, and lysosomal delivery of lipoproteins with
internalization kinetics of t1/2 ~ 1 h
(31).
To attempt to clarify this complex mechanism of lipoprotein clearance,
a multistep model has been proposed suggesting that remnant lipoprotein
catabolism involves three distinct steps (20, 36). This model is based
on a number of independent, corroborative studies (25, 37-41). First,
circulating lipoproteins are sequestered to the cell surface by binding
to HSPG. Second, the "captured" remnants undergo further lipolytic
processing by hepatic lipase (42, 43) and lipoprotein lipase (38, 44).
And third, internalization of the remnants is mediated through combined
activities of the LDL receptor, LRP, and cell surface HSPG. Whether
these three receptors interact to facilitate lipoprotein clearance is
not known.
In the present study, we have begun to examine the relationship between
HSPG and LRP at the cell surface to identify if these receptors act
independently in lipoprotein clearance or if they coordinate their
activities in a synergistic manner. Using a cell line deficient in LDL
receptor expression, we have identified that HSPG and LRP are present
in a co-immunoprecipitable complex at the cell surface. Moreover,
incubation of cells with an antagonist of LRP ligand-binding activity
increases the availability of HSPG for VLDL binding resulting in
increased VLDL catabolism. These results suggest that HSPG and LRP are
associated in a macromolecular complex at the cell surface and upon
dissociation increase the availability of free HSPG to augment VLDL
binding to cells.
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EXPERIMENTAL PROCEDURES |
Materials--
Human VLDL, purchased from Intracel, Inc.
(Frederick, MD), was obtained from a mixed pool of normal, non-fasting
donors. Human
2-macroglobulin (
2M),
heparin, heparinase I, and
4-methylumbelliferyl-
-D-xylopyranoside were purchased
from Sigma-Aldrich (St. Louis, MO). Protein A-agarose was obtained from
Bio-Rad (Hercules, CA) and
p-nitrophenyl-
-D-xylopyranoside was from
Calbiochem (La Jolla, CA). [35S]Methionine and
35SO4 were purchased from PerkinElmer Life
Sciences (Boston, MA). Precast SDS-PAGE gels were from BioWhittaker
(East Rutherford, NJ). RAP-GST fusion protein was purified as
previously described (45). Anti-LRP polyclonal antibody was raised
against an 18-amino acid peptide from the cytoplasmic tail of human LRP
(46). Tissue culture plastics were purchased from Corning (Corning,
NY). Buffers, salts, and detergents were obtained from either
Sigma-Aldrich or Calbiochem.
Cell Culture--
Mouse embryo fibroblasts deficient in LRP
expression (MEF-2) or deficient in LDL receptor expression (MEF-3) were
generous gifts from Dr. Joachim Herz, University of Texas Southwestern Medical Center, Dallas, TX. Cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal
calf serum (Irvine Scientific, Santa Ana, CA), 100 µg/ml streptomycin
sulfate, and 100 units/ml penicillin G. Cells were cultured at 37 °C
with 5% CO2 and passaged twice weekly.
Immunoblotting and Immunoprecipitation--
For immunoblotting,
cell lysates were prepared in 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 10 mM CHAPS, and cellular proteins were separated by SDS-PAGE
and electrotransferred to polyvinylidene difluoride membranes using a
wet tank transfer method (Bio-Rad, Hercules, CA). Membranes were
blocked with 20 mM Tris, pH 7.4, 150 mM NaCl
(TBS), 0.1% Tween-20, 5% calf serum for 15 min at 23 °C and
incubated with anti-LRP antisera (1:2000) in blocking buffer for 2 h at 23 °C. Membranes were washed three times (10 min each) with
TBS, 0.1% Tween-20, 1% calf serum, and bound antibodies were detected
with species-specific HRP-conjugated secondary antibodies followed by
chemiluminescence detection according to the manufacturer's
instructions (Pierce, Rockford, IL).
For immunoprecipitation, cells were incubated with 125 µCi/ml of
either [35S]methionine in complete culture media or
35SO4 in sulfate-free media for 16 h.
Cells were rinsed twice with TBS and solubilized with TBS containing
1% Triton X-100, 1% bovine serum albumin (BSA), and protease
inhibitor mix (2 µg/ml chymostatin, leupeptin, antipain, and
pepstatin) for 30 min at 4 °C. Detergent-insoluble material was
removed by centrifugation (12,000 × g, 5 min,
4 °C), and soluble proteins were incubated with anti-LRP antisera
(1:200) and protein A-agarose for 16 h at 4 °C.
Immunoprecipitates were washed two times with radioimmune precipitation
assay buffer (10 mM Tris-HCl, pH 7.2, 150 mM
NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS) followed by two
times with TBS containing 0.1% Tween-20. Antibody-bound proteins were
solubilized in Laemmli sample buffer supplemented with 4%
-mercaptoethanol and heated at 95 °C for 5 min. Proteins were
resolved by SDS-PAGE, stained with 0.1% Coomassie Blue-R in
water:methanol:acetic acid (50:40:10, v/v), destained, and incubated
for 30 min in Amplify (Amersham Biosciences, Piscataway, NJ). Gels were
then dried and exposed to Kodak X-AR film at
80 °C.
Activation of
2-Macroglobulin--
2M was activated
for receptor binding by incubating with an equal volume of 0.4 M methylamine in 0.1 M Tris-HCl, pH 8, for 2 h at room temperature. Unbound methylamine was then removed by
passage over a desalting column (PD-10, Amersham Biosciences).
Radioiodination--
VLDL and
2M were
radioiodinated with Na125I (PerkinElmer Life Sciences)
using IODO-BEADs (Pierce) as previously described (47). Specific
activities were routinely between 3000 and 4000 cpm/ng of protein.
Using the Bligh-Dyer lipid extraction procedure, we determined that
~10% of the 125I label was incorporated into lipid of
the VLDL particle while the remaining 90% of label was found
coupled to protein.
Cell Surface 4 °C Binding Assay--
Cells were grown on
tissue culture plates precoated with 1% gelatin and used when
confluent. In some cases, cells were cultured for 72 h in 3 mM 4-methylumbelliferyl-
-D-xylopyranoside or
p-nitrophenyl-
-D-xylopyranoside or incubated
for 4 h at 37 °C with heparinase I (1 Sigma unit/ml) prior to performing the binding assay. Cells were rinsed twice with 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM
CaCl2 (buffer A), followed by incubation for 3 h with
125I-VLDL or 125I-
2M (2 µg/ml)
at 4 °C in the presence or absence of the indicated potential
competitors. 125I-Labeled ligands were diluted into buffer
A containing 1% BSA and chilled to 4 °C before adding to cells.
Unbound 125I-ligand was removed by rinsing cells three
times with cold buffer A after which cells with bound ligand were
solubilized with 0.1 N NaOH. Solubilized proteins were
added to EcoLume (ICN Biomedicals, Costa Mesa, CA) and subjected to
scintillation counting (73% efficiency for iodine 125). Results were
normalized to total cellular protein (BCA Protein Assay, Pierce,
Rockford, IL). Specificity was determined as the difference between
total binding (without competition) and nonspecific binding
(non-competable) (48). The actual amount of ligand bound to cells was
calculated as cpm divided by the specific activity of
125I-labeled ligand. All data points represent averages of
duplicates or triplicates with standard errors of <5%. Experiments
were repeated a minimum of three times. The paired t test
was used to determine statistical significance.
Solid-phase Assay--
Wells of 96-well plates contained either
confluent MEF-3 cells or were pre-coated with RAP-GST or BSA by
incubating for 16 h at 4 °C with 20 µg/ml protein diluted
into phosphate-buffered saline. Nonspecific sites were blocked by
incubating at 4 °C for 2 h with TBS containing 3% BSA.
125I-VLDL was diluted to 2 µg/ml in TBS, 3% BSA and
added to wells for 2 h at 4 °C in the presence or absence of
unlabeled VLDL (50 µg/ml). Wells were then rinsed with TBS, and
proteins were solubilized with 0.1 N NaOH and subjected to
scintillation counting as described above.
37 °C Ligand Degradation Assay--
Cells were incubated at
37 °C/5% CO2 with 2 µg/ml 125I-VLDL or
125I-
2M diluted into Dulbecco's modified
Eagle's medium containing 1% BSA in the presence or absence of the
indicated potential competitor. At the indicated times, media was
removed and processed for trichloroacetic acid precipitation (49).
Trichloroacetic acid-soluble material was added to EcoLume and
subjected to scintillation counting. Degradation was calculated as
trichloroacetic acid-soluble cpm divided by the specific activity of
the radioiodinated ligand.
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RESULTS |
Because we have chosen to examine the relationship between HSPG
and LRP toward VLDL clearance, we have selected a cell line (mouse
embryo fibroblasts, MEF-3) that is devoid of LDL receptor expression to
remove its contribution toward VLDL binding and clearance. MEF-3 cells
were originally established from LDL receptor knockout mice (50). To
confirm LRP expression in these cells, detergent lysates were prepared
and subjected to immunoblot analysis using polyclonal antibodies (pAb)
specific for the cytoplasmic tail of LRP (46). In MEF-3 cells, anti-LRP
antibodies clearly reacted with the light chain of LRP (~95 kDa),
which contains the receptor's cytoplasmic tail sequence (Fig.
1, lane 2). MEF-2 cells, which
are devoid of LRP expression (51), show no reactivity with anti-LRP
demonstrating the specificity of this antibody (Fig. 1, lane
3).

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Fig. 1.
LRP is expressed in LDL receptor-deficient
mouse embryo fibroblasts (MEF-3). Cell lysates were prepared using
CHAPS detergent and proteins (20 µg/lane) were separated by SDS-PAGE
and immunoblotted with anti-LRP pAb. MEF-3 cells demonstrate LRP
expression (lane 2). The light chain of LRP (~95 kDa) is
the same molecular mass as that seen in normal rat liver
(lane 1). MEF-2 cells are LRP-deficient and serve as a
negative control for anti-LRP pAb (lane 3).
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Because VLDL is known to bind to HSPG on the cell surface prior to its
internalization, we examined the relationship between LRP and this
primary binding event by first determining if LRP is associated with
HSPG at the cell surface. MEF-3 cells were incubated with
[35S]methionine to label proteins or
35SO4 to label proteoglycans. Cell lysates were
prepared using a mild, non-denaturing detergent (1% Triton X-100) and
immunoprecipitated with pAb to LRP. With [35S]methionine
labeling, the heavy chain of LRP can be seen at 515 kDa (Fig.
2A). In cells labeled with
35SO4, a very large (>600 kDa) sulfated
molecule is found to co-immunoprecipitate with LRP. To determine if
this large sulfated molecule is an HSPG, immunoprecipitates were
prepared from 35SO4-labeled cells using
anti-LRP antibodies, and antibody-bound proteins were treated with or
without heparinase. Confirming our results obtained in Fig.
2A, anti-LRP antibodies co-immunoprecipitate a large
sulfated molecule from 35SO4-labeled cells
(Fig. 2B). When this material is treated with heparinase,
little or none of the high molecular weight sulfated molecule remains
thus identifying it as an HSPG. These data indicate that LRP is
associated with HSPG in a co-immunoprecipitable complex at the cell
surface in MEF-3 cells.

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Fig. 2.
HSPG co-immunoprecipitates with LRP.
A, cells were metabolically labeled for 16 h with
either [35S]methionine or 35SO4.
Cell lysates were prepared and immunoprecipitated with anti-LRP
polyclonal antibody. LRP is readily detected following
[35S]methionine labeling. In cells labeled with
35SO4, a high molecular weight, sulfated
molecule is seen to co-immunoprecipitate with LRP. B,
immunoprecipitates prepared from cells labeled with
35SO4 using anti-LRP antibodies were treated
with or without heparinase I (1 unit/ml). Heparinase I treatment
significantly reduced the amount of the large sulfated molecule
associated with LRP thus identifying it as an HSPG.
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We next examined the binding properties of VLDL on MEF-3 cells. Cells
were incubated at 4 °C (to prevent internalization, but permit
lateral movement of protein in the plasma membrane) with
125I-VLDL alone or together with unlabeled VLDL, heparin,
RAP, or mix of RAP and heparin. Unlabeled VLDL effectively competed for the binding of 125I-VLDL demonstrating the specificity of
its binding to the surface of MEF-3 cells (Fig.
3A). Heparin also effectively
competed for 125I-VLDL binding confirming that the initial
binding of VLDL to cells occurs through HSPG.

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Fig. 3.
RAP increases the availability of binding
sites for VLDL. Cells were incubated at 4 °C with
125I-VLDL (2 µg/ml) (A) or
125I- 2M (2 µg/ml) (B) in the
presence or absence of the indicated unlabeled ligands (VLDL, 50 µg/ml; heparin, 500 µg/ml; RAP, 50 µg/ml; 2M, 40 µg/ml). A, incubation of cells with RAP significantly
increased the binding of 125I-VLDL. Competition for this
binding by heparin, VLDL, or a mix of RAP and heparin suggests that the
increased 125I-VLDL binding due to RAP incubation is a
result of increased availability of cell surface HSPG. B,
RAP, 2M, or a combination of RAP and heparin compete for
the binding of 125I- 2M to cells
demonstrating that our preparation of RAP is an effective
ligand-binding antagonist for LRP. As expected, heparin alone has no
effect on 125I- 2M binding to cells. The
asterisk indicates a statistically significant
difference (p < 0.05).
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To determine if, in addition to HSPG, 125I-VLDL also bound
directly to LRP, we challenged its binding with the receptor-associated protein (RAP), because RAP is known to compete for the binding of all
ligands to LRP (52, 53), including VLDL (54, 55). Surprisingly,
co-incubation of 125I-VLDL with RAP resulted in a
significant increase in VLDL binding (Fig. 3A). Incubation
of 125I-VLDL together with RAP and heparin reduced its
binding to background levels suggesting that the increase in VLDL
binding by co-incubation with RAP is due to increased availability of
HSPG binding sites. To confirm that our preparation of RAP is active as
a ligand binding antagonist for LRP, we incubated MEF-3 cells with
125I-
2M in the presence or absence of RAP.
2M is known to be a specific ligand for LRP (56). Our
preparation of RAP proved to be an effective competitor for
125I-
2M binding to LRP (Fig. 3B).
Heparin, as expected, had no effect on
125I-
2M binding to LRP.
To determine if the effect of RAP on VLDL binding to MEF-3 cells is
concentration-dependent, cells were incubated at 4 °C with 125I-VLDL and varying concentrations of RAP. As shown
in Fig. 4, increasing amounts of RAP do
increase the availability of specific VLDL binding sites in a
concentration-dependent manner.

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Fig. 4.
The effect of RAP on VLDL binding to MEF-3
cells is concentration-dependent. MEF-3 cells were
incubated at 4 °C with 125I-VLDL (2 µg/ml) and the
indicated concentrations of RAP. Cells were also incubated in parallel
with RAP and heparin (500 µg/ml) together, and these values were
subtracted from total binding to obtain specific binding of
125I-VLDL to cells (shown). Increasing amounts of RAP
increased the availability of specific binding sites for VLDL on MEF-3
cells.
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The fact that heparin effectively competes for 125I-VLDL
binding to the increased sites made available by incubation with RAP suggests that these new sites are HSPG. To explore this possibility, we
treated MEF-3 cells with
4-methylumbelliferyl-
-D-xylopyranoside or
p-nitrophenyl-
-D-xylopyranoside, both of
which are known to be potent competitors of heparan sulfate
polysaccharide chain addition to proteoglycan core proteins (57, 58).
Cells treated with these reagents have reduced amounts of
glycosaminoglycan associated at the cell surface and have been shown to
bind reduced amounts of VLDL (59, 60). We incubated MEF-3 cells with
4-methylumbelliferyl-
-D-xylopyranoside (Fig.
5A) or
p-nitrophenyl-
-D-xylopyranoside (Fig.
5B) for 72 h, followed by incubation at 4 °C with
125I-VLDL in the presence or absence of RAP. As expected,
both 4-methylumbelliferyl-
-D-xylopyranoside and
p-nitrophenyl-
-D-xylopyranoside reduced
overall binding of 125I-VLDL to cells. In addition, both
reagents also significantly reduced VLDL binding to the additional
sites made available by RAP incubation indicating that these sites are
HSPG. To further confirm this observation, 125I-VLDL
binding study was performed on MEF-3 cells treated with heparinase
(Fig. 6). Following heparinase treatment,
the binding of 125I-VLDL to cells was markedly reduced as
expected. Moreover, the RAP-inducible binding sites were also
significantly reduced due to heparinase treatment. Together, these data
indicate that the additional VLDL binding sites made available by
incubation with RAP are HSPG.

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Fig. 5.
The RAP-inducible VLDL binding sites are
HSPG. MEF-3 cells were grown in the absence or presence of 3 mM 4-methylumbelliferyl- -D-xylopyranoside
(A) or 3 mM
p-nitrophenyl- -D-xylopyranoside
(B). After 72 h cells were incubated at 4 °C with
125I-VLDL (2 µg/ml) in the presence or absence of RAP (50 µg/ml). Cells were also incubated in parallel with RAP and heparin
(500 µg/ml) together to determine nonspecific binding of
125I-VLDL; this value has been subtracted from the reported
values. Treatment of MEF-3 cells with either
4-methylumbelliferyl- -D-xylopyranoside or
p-nitrophenyl- -D-xylopyranoside significantly
reduced 125I-VLDL binding. Moreover, these treatments also
reduced 125I-VLDL binding to cells incubated with RAP
indicating that the RAP-inducible binding sites are HSPG. The
asterisk indicates a statistically significant difference
(p < 0.05).
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Fig. 6.
Heparinase treatment of MEF-3 cells reduced
the number of RAP-induced binding sites for VLDL. MEF-3 cells were
incubated with or without heparinase (1 unit/ml) for 4 h at
37 °C, followed by incubation at 4 °C with 125I-VLDL
(2 µg/ml) in the presence or absence of unlabeled VLDL (50 µg/ml),
RAP (50 µg/ml), heparin (500 µg/ml), or combination of RAP and
heparin. Heparinase treatment of cells reduced VLDL binding to levels
comparable to those obtained by competition with unlabeled VLDL and
heparin. Similarly, heparinase also reduced 125I-VLDL
binding to RAP-inducible sites to levels comparable to those obtained
by competition with a combination of RAP and heparin. The
asterisk indicates a statistically significant
difference (p < 0.05).
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Previous studies have shown that lipoprotein lipase (LPL) is able to
increase the binding of VLDL to cells (25, 36, 38, 40, 41) by binding
directly to both VLDL particles and HSPG and bridge their interaction.
To determine if RAP acts in a similar manner as LPL, we assessed if RAP
is able to directly interact with VLDL. To this end,
125I-VLDL was incubated in wells coated with RAP or BSA or
containing MEF-3 cells in the presence or absence of unlabeled VLDL.
Specific binding of 125I-VLDL to MEF-3 cells was seen as
expected, however, no specific binding of the ligand was measured to
RAP or BSA (Fig. 7) demonstrating that
RAP is unable to bind to VLDL and mediate its binding to cells.

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Fig. 7.
RAP does not bind to VLDL.
125I-VLDL (2 µg/ml) was incubated with RAP or BSA
immobilized in 96-well plates or with MEF-3 cells at 4 °C for 2 h in the presence or absence of unlabeled VLDL (50 µg/ml). Binding of
125I-VLDL to MEF-3 cells was competed by unlabeled VLDL
confirming specific binding of the ligand. No measurable specific
binding of 125I-VLDL to RAP or BSA was detected.
Differences in binding of 125I-VLDL to RAP and BSA are not
statistically significant (p < 0.05).
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After establishing that RAP is able to increase the availability of
HSPG for VLDL binding at the cell surface, we next determined the
effect of RAP incubation on VLDL internalization and degradation. To
this end, cells were incubated at 37 °C with 125I-VLDL
in the presence or absence of unlabeled VLDL, RAP, heparin, or RAP
together with heparin. After 16 h, culture media was subjected to
trichloroacetic acid precipitation and radioactivity in the trichloroacetic acid-soluble fraction, representing degraded ligand, was quantitated. 125I-VLDL uptake and degradation was
inhibited by both unlabeled VLDL and heparin demonstrating that
specific receptor-mediated internalization is dependent on cell surface
HSPG (Fig. 8A). Interestingly, RAP increased the intracellular degradation of VLDL, which is consistent with its ability to increase the number of VLDL binding sites as shown in Figs. 3-6. The RAP-induced increase in
125I-VLDL degradation is competable by heparin suggesting
that degradation occurs through an HSPG-mediated internalization
pathway. To confirm that our preparation of RAP maintains its
inhibitory properties for ligand binding to and uptake by LRP, we
incubated MEF-3 cells with 125I-
2M in the
presence or absence of RAP (Fig. 8B). RAP effectively blocked uptake and intracellular degradation of
125I-
2M verifying its strong ligand-binding
inhibitory property toward LRP. Heparin showed no competition for
125I-
2M binding as expected.

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Fig. 8.
RAP increases 125I-VLDL
degradation by MEF-3 cells. MEF-3 cells were incubated at 37 °C
with 125I-VLDL (2 µg/ml) (A) or
125I- 2M (2 µg/ml) (B) in the
presence or absence of unlabeled VLDL (100 µg/ml), unlabeled
2M (40 µg/ml), RAP (50 µg/ml), heparin (500 µg/ml), or a combination of RAP and heparin. After 16 h, culture
media were subjected to trichloroacetic acid precipitation, and soluble
radioactivity, representing degraded ligand, was quantitated.
A, unlabeled VLDL and heparin inhibited the degradation of
125I-VLDL. RAP increased 125I-VLDL degradation,
however, this increase was prevented by a combination of RAP and
heparin. B, unlabeled 2M and RAP blocked the
degradation of 125I- 2M confirming the
inhibitory properties of RAP toward ligand uptake by LRP. Heparin
showed little or no effect on 125I- 2M
degradation. C, MEF-3 cells were incubated with
125I-VLDL in the presence or absence of RAP at 37 °C for
5.5 or 29 h. Trichloroacetic acid-soluble radioactivity in the
media was quantitated. RAP competed for 125I-VLDL
degradation during shorter incubations (5.5 h), however, increased
125I-VLDL degradation during longer incubations (29 h). The
asterisk indicates a statistically significant difference
(p < 0.05).
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Other investigators have reported that RAP inhibits VLDL degradation by
cultured cells. In these studies, degradation assays were performed for
2-5 h (36, 40, 41, 61) as compared with 16 h in the current
study. To directly compare these studies to our current results, we
incubated MEF-3 cells with 125I-VLDL at 37 °C for 5.5 or
29 h, followed by trichloroacetic acid precipitation analysis of
the culture media. Consistent with prior studies, RAP effectively
competed for the degradation of 125I-VLDL after 5.5 h
of incubation. However, after 29 h of incubation RAP increased
125I-VLDL degradation by greater than 2-fold over cells
treated without RAP. These data indicate that 125I-VLDL
degradation is mediated by a RAP-sensitive, LRP-dependent pathway during shorter incubations and an LRP-independent pathway during longer incubations.
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DISCUSSION |
In the present study we have examined the relationship between LRP
and HSPG at the cell surface and the functional consequences of this
relationship toward lipoprotein binding and internalization. First, we
have demonstrated that LRP and HSPG form a co-immunoprecipitable complex at the cell surface. Second, we have shown that incubation of
cells with the universal ligand-binding antagonist for LRP, namely RAP,
increases the number of VLDL binding sites at the cell surface. Third,
we have demonstrated that these newly available VLDL binding sites are
HSPG using two different competitors of glycosaminoglycan chain
addition and heparinase treatment. Fourth, we have shown that by
incubating cells with RAP we are able to increase the uptake and
degradation of VLDL through this LRP-independent, HSPG pathway. Based
on these data, we propose the following model for LRP-HSPG synergistic
activity at the cell surface (Fig. 9). LRP and HSPG are associated at the cell surface in a
co-immunoprecipitable complex, and the complexed HSPG is unavailable
for VLDL binding (Fig. 9A). Through incubation with RAP, the
LRP·HSPG complex dissociates, which increases the availability
of VLDL binding sites at the surface (Fig. 9B). This
activation of additional VLDL binding sites serves to augment the
sequestration of VLDL particles to the cell surface, ultimately
increasing the efficiency of particle clearance by cells. The
measurable increase in intracellular degradation of VLDL due to RAP
incubation is consistent with an increased clearance efficiency.
Moreover, the observed increase in VLDL degradation during longer
incubations with RAP also suggests that uptake occurs through a HSPG
internalization pathway, because RAP is known to block direct binding
of lipoproteins to LRP (41). Collectively, our data describe a
synergistic activity between LRP and HSPG for VLDL binding and
clearance and describe a novel function for LRP in regulating the
presentation of HSPG at the cell surface.

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|
Fig. 9.
RAP-induced dissociation of LRP from HSPG
increases the availability of VLDL cell surface binding sites.
A, LRP and HSPG associate at the cell surface to form a
co-immunoprecipitable complex. HSPG, when complexed with LRP, is unable
to bind VLDL particles. B, when LRP is activated to release
HSPG, as we have shown by RAP incubation, increased VLDL binding sites
become available. As our data shows (Fig. 8), this in turn translates
into greater efficiency of VLDL uptake and degradation.
|
|
The data presented here contrasts with previous studies identifying the
role of LRP and HSPG in lipoprotein clearance (22, 62). The
sequestration-internalization hypothesis (20, 36), based on a number of
comprehensive studies on lipoprotein clearance (25, 37-41), proposes
that circulating lipoproteins bind first to cell surface HSPG
(sequestration step), followed by receptor-mediated internalization
either by the LDL receptor or LRP. In this model, HSPGs serve a passive
role in lipoprotein clearance by simply mediating the localization of
lipoprotein particles to the cell surface. Particle uptake is proposed
to occur following ligand transfer from HSPG to the classic endocytic
receptors, the LDL receptor, and LRP. By contrast, our data describe a
mechanism whereby LRP regulates the availability of lipoprotein binding sites by associating with HSPG. Because LRP dissociates from HSPG to
uncover additional lipoprotein binding sites, the increased availability of HSPG at the cell surface in turn serves to increase VLDL clearance. Thus, our data propose a more active role for HSPG in
lipoprotein clearance, because we show they are able to mediate both
the binding and internalization of VLDL. Others have also demonstrated
that HSPGs are able to directly bind and internalize lipoprotein
particles, however, many of these studies have remained in the shadow
of those reporting particle clearance by the more classic endocytic
receptors. Our data strongly support an active, independent role for
HSPG in lipoprotein clearance and describe a novel role for LRP in
regulating the presentation of HSPG at the cell surface.
We also observed a shift in receptor utilization when measuring VLDL
internalization and degradation. Our data shows that shorter incubation
times with RAP (<5.5 h) reduced VLDL metabolism, as seen in previous
studies (36, 40, 41), suggesting that LRP may be responsible for rapid,
immediate VLDL internalization. By contrast, longer incubation times
with RAP (>16 h) increased VLDL degradation indicating that an
LRP-independent HSPG pathway contributes significantly to lipoprotein
clearance over more extended time periods.
RAP is a well-characterized chaperone for members of the LDL receptor
gene family (52, 53) and is primarily found in the endoplasmic
reticulum (63, 64), retained there by its C-terminal His-Asn-Glu-Leu
sequence (65). As a chaperone, RAP has been shown to assist in the
folding and exocytic trafficking of LRP (66). RAP also has the unique
property of blocking the binding of all known ligands to LRP (52, 53),
which has proven invaluable for functional studies of LRP. Because of
its endoplasmic reticulum retention properties, little or no RAP can be
found at the cell surface. Therefore, we believe that RAP is mimicking
the effect of an LRP-specific extracellular ligand in dissociating LRP
from HSPG to make additional sites available for lipoprotein binding. Recent studies have shown that addition of lipoprotein lipase increases
lipoprotein particle binding to cell surface HSPG (25, 36, 38, 41, 67)
as well as increase their LRP-mediated clearance (41, 61). LPL is
thought to increase lipoprotein metabolism by bridging VLDL particle
binding to HSPG and LRP, because it is able to bind both cell surface
receptors (36, 38, 39). However, activation of LRP to release HSPG
sites likely occurs by a different mechanism than ligand bridging,
because we show here that RAP is unable to bind VLDL and thus unable to bridge VLDL binding to both HSPG and LRP. Alternatively, RAP may bind
HSPG and simply displace a protein already bound to cell surface HSPG
thereby making HPSG available to bind other soluble ligands. However,
we have only observed the binding of RAP to heparin following its
denaturation (45). We have interpreted this to indicate that the
heparin binding domain in RAP is cryptic in the native protein. Other
studies have confirmed this showing that radiolabeled RAP is unable to
bind to cell surface HSPG in fibroblasts (68).
The nature of the association between HSPG and LRP is currently
unknown. No member of the LDL receptor family has been reported to bind
heparin or heparan sulfate moieties, so the interaction likely does not
involve direct glycosaminoglycan interactions with LRP. Recently,
CASK/LIN-2 has been identified to interact with the cytoplasmic tail of
all four members of the syndecan HSPG family (69, 70) and is thought to
mediate syndecan coupling to the actin cytoskeleton through
interactions with protein 4.1 (70). LRP has also been reported to bind
cytosolic adaptors, such as FE65 and Disabled, to its cytoplasmic tail
(71). Therefore, it is possible that a bridging protein may bring
together HSPG and LRP through cytosolic interactions.
LRP was originally thought to be an independent endocytic receptor for
many soluble extracellular ligands. After binding these ligands at the
cell surface, LRP rapidly internalizes to deliver ligand to lysosomes
for degradation and recycles to the surface for additional rounds of
binding and internalization. However, we and others have recently
identified a greater complexity in LRP function. LRP is now known to
interact with other cell surface receptors and affect their activities.
For example, LRP associates with the amyloid precursor protein (72, 73)
and through this interaction increases proteolysis of the amyloid
precursor protein to generate A
peptide (74), the amyloidogenic
factor responsible for neurologic plaques in Alzheimer disease. LRP
is also known to form a co-immunoprecipitable complex with urokinase
receptor on the surface of human fibrosarcoma cells, and through this
interaction establishes a functional relationship with the plasminogen
activation system that affects the invasive phenotype of this cancer
line (75). These studies suggest that LRP activity is more complex than
originally thought and affects other cell surface receptor systems in
addition to its function in soluble ligand clearance. Our studies here
extend the functions of LRP to regulating HSPG availability at the cell
surface. Moreover, because integral HSPGs are known to shed from the
cell surface through proteolytic cleavage (76), LRP may also protect
them from proteolysis and prevent a loss of HSPGs that are critical to
cell function. In addition to lipoproteins, HSPGs are also known to
bind cytokines such as basic fibroblast growth factor (77) and
hepatocyte growth factor (78). Binding of these factors by HSPG affects
their binding to receptors and downstream signaling activities (77,
79). Although it remains to be determined if LRP regulates the
availability of HSPG sites for these growth factors, such a regulation
may suggest that the functional properties of LRP extend to a role in
modulating diverse cell signaling events.
 |
ACKNOWLEDGEMENT |
We are grateful for the generosity of Dr.
Joachim Herz from the University of Texas Southwestern Medical Center,
Dallas, TX, for supplying us with MEF-2 and MEF-3 cells.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant HL63291 (to R. A. O.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: MSC08-4670, 1 University of New Mexico, Albuquerque, NM 87131. Tel.: 505-272-5593; Fax: 505-272-3518; E-mail: rorlando@salud.unm.edu.
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M208786200
 |
ABBREVIATIONS |
The abbreviations used are:
VLDL, very low
density lipoprotein;
HSPG, heparan sulfate proteoglycan;
LDL, low
density lipoprotein;
LRP, lipoprotein receptor-related protein;
2M,
2-macroglobulin;
GST, glutathione
S-transferase;
MEF, mouse embryo fibroblast;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
BSA, bovine serum albumin;
pAb, polyclonal antibody;
RAP, receptor-associated protein.
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