From the Department of Medicine, Case Western Reserve
University School of Medicine, Cleveland, Ohio 44106,
Holland
Labs, American Red Cross, Rockville, Maryland 20855, and
** Center for Nutrition and Toxicology, Karolinska Institute at Huddinge
University Hospital, S-14186 Huddinge, Sweden
Received for publication, May 9, 2000, and in revised form, August 23, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The very low density lipoprotein receptor
(VLDL-R) binds and internalizes several ligands, including very low
density lipoprotein (VLDL), urokinase-type plasminogen
activator:plasminogen activator inhibitor type 1 complexes, lipoprotein
lipase, and the 39-kDa receptor-associated protein that copurifies with
the low density lipoprotein receptor-related
protein/ The very low density lipoprotein receptor
(VLDL-R)1 is a member of the
low density lipoprotein receptor (LDL-R) family, which encompasses
several structurally related proteins such as the LDL receptor-related
protein/ Differences in the tissue distribution of the LDL and VLDL receptors
also suggest non-overlapping physiologic functions. Although the LDL-R
is highly expressed in liver and promotes LDL uptake and clearance
(21), the VLDL-R is most abundant in heart and skeletal muscle (22),
suggesting involvement in the uptake of triglyceride-rich lipoproteins
in tissues dependent on fatty acid metabolism (2, 7, 8). However, the
broad ligand specificity of the VLDL-R as well as the lack of
coordinate regulation between the VLDL-R and lipoprotein lipase
suggests that this receptor may also function in other processes
distinct from lipid metabolism (23). In this context, the VLDL-R
regulates cell surface urokinase receptor expression and urokinase
receptor-dependent cellular migration (24).
We previously reported that the VLDL-R is expressed by human arterial
and venous endothelial and smooth muscle cells in vitro and
in vivo (11, 25) as well as by macrophage-derived foam cells
within atherosclerotic plaques (25). Nakazato et al. (26) detected VLDL-R mRNA in smooth muscle cells and macrophages within atherosclerotic aorta from cholesterol-fed NZW and Watanabe hereditary hyperlipidemic rabbits (26). The VLDL-R is also present in the endothelium of capillaries, small arterioles, and coronary arteries of
mouse and bovine origin (27). These studies suggest a potential role
for the VLDL receptor in (patho)physiological vascular processes such
as atherosclerosis.
Unlike that of the LDL-R, expression of the VLDL-R is not
diminished by exogenous cholesterol (21, 28, 29). In
choriocarcinoma cells, VLDL-R mRNA levels are regulated by insulin,
8-bromo-cAMP, and clofibrate (30), whereas in HL-60 cells,
1,25-dihydroxy vitamin D3 stimulates receptor expression (31). In
vivo, thyroid hormone (32) and granulocyte-macrophage colony
stimulating factor (33) stimulate the expression of VLDL-R mRNA in
skeletal muscle, whereas estradiol stimulates VLDL-R expression in
myocardium (34). However, changes in mRNA levels may not reflect
the expression of a functional VLDL-R protein and whether the
expression or activity of the VLDL-R may be regulated through
post-transcriptional mechanisms has not been assessed. Here we report
that the ligand binding affinity of the VLDL-R is diminished after
protein kinase C (PK-C)-dependent phosphorylation.
Materials--
Tissue culture medium and reagents were from
Mediatech (Herndon, VA). Fetal bovine serum was from Hyclone
(Logan, Utah). The 39-kDa RAP, which co-purifies with the LDL
receptor-related protein/ Cell Culture--
THP-1 and HEK 293 cells were obtained from the
American Type Culture Collection. Human dermal microvascular
endothelial cells and human aortic smooth muscle cells were cultured as
described (39, 40). All primary cell cultures used in these studies were of passage 4 or lower.
Measurement of VLDL Receptor Expression by Ligand or
Immunoblotting--
Expression of functional VLDL-R was assessed by
ligand blotting using RAP or u-PA:PAI-1 complexes as ligands (16, 19). The latter were prepared by incubating two-chain urokinase with active
PAI-1 for 30 min at a 1:4 molar ratio. Complex formation was assessed
by immunoblotting using anti-u-PA and anti-PAI-1 antibodies, which
demonstrated that >80% of the u-PA was incorporated into a u-PA:PAI-1 complex.
For ligand blots, detergent extracts were prepared from control and
PMA-treated cells in a buffer containing 25 mM Hepes, pH
7.4, 1% Triton X-100, 0.05% Tween 20, 1 mM
phenylmethylsulfonyl fluoride, 0.02 mg/ml leupeptin, and 50 mM
D-phenylanalyl-L-propyl-L-arginine chloromethyl ketone). Cellular proteins (100 µg/lane) were separated by 7.5% SDS-PAGE, then transferred to PVDF membranes, which were blocked by incubation in Tris-buffered saline (0.15 M Tris,
0.1 M NaCl, pH 7.4) containing 5% nonfat milk and 0.1%
Tween 20. Membranes were then incubated with either 1.0 nM
RAP or 2 µg/ml u-PA:PAI-1 complex in the same buffer containing 5 mM CaCl2. After washing, membranes were
incubated with 5 µg/ml anti-RAP or anti-PAI-1 antibodies, and bound
antibody was detected using peroxidase-conjugated goat anti-rabbit or
rabbit anti-mouse IgG. Blots were developed by incubation with Super
Signal chemiluminescence reagent.
In selected experiments, the effect of VLDL-R dephosphorylation on
ligand binding was assessed. Briefly, extracts of cells that had been
exposed to 1.5 × 10
For immunoblots, cell extracts were prepared, separated by SDS-PAGE,
and transferred to PVDF before incubation with rabbit anti-human VLDL-R
antibodies. Membranes were then washed, incubated with
peroxidase-conjugated goat anti-rabbit IgG, and developed using chemiluminescence.
Measurement of VLDL Receptor mRNA--
VLDL receptor
mRNA was quantitated by Northern blotting. Total RNA was isolated
using Trizol (Life Technologies, Inc.) and separated on a 1%
denaturing agarose gel. RNA was transferred to nylon membranes in 20×
SSC (1× SSC contains 0.15 M NaCl, 0.015 M
sodium citrate, pH 7.0). The membrane was prehybridized at 42 °C in
a solution containing 50% formamide, 5× SSC, 5× Denhardt's solution, 1% SDS, and 100 µg/ml denatured salmon sperm DNA, and VLDL-R mRNA was detected by overnight hybridization at 42 °C
with a full-length VLDL-R cDNA (36) labeled with 32P
(Ready to Go labeling kit, Amersham Pharmacia Biotech). To ensure equal
loading of RNA in all lanes, blots were stripped and reprobed with a
32P-labeled polymerase chain reaction product corresponding
to nucleotides 384-684 of actin cDNA (42).
Binding of VLDL-R Ligands to Control and PMA-treated
Cells--
To assess whether PK-C activation altered the expression of
VLDL-R on intact cells, cell surface proteins were labeled using sulfosuccinimidyl 6-(biotinamido) hexanoate (NHS-LC biotin) (43) before
and after exposure of cells to 1.5 × 10
Two approaches were used to investigate the effect of PK-C activation
on the binding of VLDL-R ligands to cells. First, the binding of
125I-RAP to control THP-1 cells or cells incubated for 60 min in the presence of 1.5 × 10
In a second series of experiments, the binding of
1,1'-dioctadecyl-3,3',3,3'-tetramethylindocarbocyanine (DiI)-labeled
VLDL to control and PMA-treated THP 1 cells was analyzed using flow cytometry. Human VLDL was purified and labeled with DiI as described (45;46). THP-1 cells (2 × 106 cells/ml) were washed
and incubated for 2 h at 4 °C with or without 25 µg/ml
DiI-labeled VLDL. Cells were then washed again, fixed in 2%
paraformaldehyde, and analyzed using a FACScan flow cytometer (Becton-Dickinson; excitation wavelength, 575 nm; emission wavelength, 488 nm) (47). Binding of DiI-VLDL was quantitated by subtracting the
mean fluorescence intensity of control cells (autofluorescence) from
that of cells incubated with DiI-VLDL. Binding specificity was
confirmed by assessing the ability of a 100-fold excess of unlabeled
VLDL or RAP to block the binding of DiI-VLDL to cells.
Assessment of the Role of PK-C in Regulation of Receptor Ligand
Binding Affinity by PMA--
The role of PK-C in regulation of VLDL-R
affinity was assessed by determining the ability of the PK-C inhibitors
GF109203X and calphostin C to block PMA-mediated inhibition of VLDL-R
ligand binding using both ligand blotting and whole cell binding approaches.
The involvement of the PK-C Direct Assessment of VLDL Receptor Phosphorylation--
VLDL-R
phosphorylation was assessed by measuring incorporation of
32P into the receptor. THP-1 cells (1 × 107 cells/ml) were pre-incubated in phosphate-free RPMI
1640 for 30 min and then divided into 4 aliquots that were each labeled for 3 h with 0.5 mCi/ml [32P]orthophosphate. Sixty
minutes before the termination of labeling, either 1.5 × 10
To determine whether 32P was incorporated into serine,
threonine, or tyrosine residues, immunoprecipitates from
32P-labeled cells were separated using 7.5% SDS-PAGE, then
transferred to PVDF. Membranes were washed in transfer buffer (50 mM Tris, 192 mM glycine, 20% methanol, 0.1%
SDS, pH 8.3) and then incubated for 1 h at 30 °C in 1.0 M KOH, which causes selective chemical dephosphorylation of
phosphoserine residues (50). Immobilized VLDL-R were also analyzed by
immunoblotting using specific monoclonal anti-phosphoserine,
anti-phosphothreonine, and anti-phosphotyrosine antibodies. Comparative
alignment of the cytoplasmic domains of the VLDL-R and low density
lipoprotein receptors was performed using the alignment editor function
of the PepTools software package (BioTools, Inc., Alberta, CA) with a
consensus threshold of 75%.
Preparation of a Truncated VLDL-R Mutant--
To further assess
the role of phosphorylation within the VLDL-R cytoplasmic domain on
ligand binding activity, a truncated VLDL-R mutant was prepared.
Briefly, the 3.2-kilobase VLDL-R cDNA was digested with
AseI and BglI to yield 2438, 578, and 238 nucleotide fragments. The gel-purified 2438- and 578-nucleotide
fragments were recloned in pRC-CMV to create a construct
(pRC-CMV-VLDL-TR) in which the 238-nucleotide cDNA sequence
encoding the VLDL-R cytoplasmic domain C-terminal to Leu-790
(proximal to all cytoplasmic domain serine, threonine or tyrosine
residues) was deleted. Polymerase chain reaction of the coding region
of this construct (sense primer 5'-CTAGTCAACAACCTGAATGA-3', antisense
primer 5'-TACACAAGCTTGATCCACGC-3') yielded a 418-nucleotide product, in
comparison to a 656-nucleotide product from the wild-type construct.
The DNA sequence of the mutant was confirmed by automated sequencing.
HEK 293 cells were transfected with wild type and mutant constructs,
and positive clones selected by culture in 400 µg/ml G418. Ligand
precipitation of biotinylated cell surface proteins using RAP-Sepharose
confirmed that wild type and truncated VLDL-R were expressed in similar
amounts on the cell surface.
Effect of Elevated Glucose Concentrations on the Ligand Binding
Activity of the VLDL-R--
Elevated glucose concentrations activate
endothelial cell PK-C Functional VLDL Receptor Expression Is Diminished after Exposure of
Cells to PMA--
We initially wished to determine the effect of
monocyte differentiation on VLDL-R expression. As a model of monocyte
differentiation, THP-1 cells were treated with PMA, which induced them
to become adherent and acquire a macrophage-like phenotype (55).
Contrary to expectations, the expression of both the ~105- and
~130-kDa VLDL-R isoforms by the PMA-treated cells, measured using RAP
ligand blotting, was markedly diminished within 60 min of exposure to PMA (Fig. 1). Maximal responses occurred
at a PMA concentration of ~1 × 10
To determine whether these responses were unique to transformed cell
lines such as THP-1, we assessed the effects of PMA on the expression
of VLDL-R by primary cultures of human dermal microvascular endothelial
cells and human aortic smooth muscle cells. As observed with THP-1
cells, exposure to PMA completely blocked the ligand binding activity
of VLDL-R from these primary cell cultures as well.
To assess whether these responses were limited to the binding of RAP,
which is not a physiological extracellular VLDL-R ligand, we determined
the effect of PMA on the binding of u-PA:PAI-1 complexes to the VLDL-R.
PMA caused a marked decrease in the binding of these complexes as well
(Fig. 2).
The Effects of PMA Reflect a Decrease in Affinity of the VLDL-R for
Its Ligands--
Exposure of THP-1 cells to PMA for 2 h did not
affect their content of VLDL-R mRNA (Fig.
3, lanes 1 and 2).
Similarly, immunoblotting studies revealed identical amounts of VLDL-R
protein within extracts of PMA-treated and control cells (Figs.
2B and 4, upper panel), although markedly
less RAP (Fig. 4, lower panel)
or u-PA:PAI-1 complex (Fig. 2A) bound to VLDL-R in extracts
from the PMA-treated cells. The observations that PMA did not affect
the amount of VLDL-R within cell extracts, as determined by
immunoblotting, but blocked ligand binding to the receptors suggested
that it decreased ligand binding affinity.
PMA Inhibits VLDL-R Ligand Binding on Intact Cells--
Two
approaches were undertaken to assess the effects of PMA on ligand
binding by VLDL receptors on intact cells. First, we compared the
binding of 125I-RAP to control and PMA-treated THP-1 cells.
125I-RAP bound specifically to ~31,400 ± 1,038 (S.E.) sites on control cells, with a Kd of
~1.8 ± 0.3 nM, and to ~ 40,110 ± 12,700 (S.E.) sites on PMA-treated cells, with a Kd of ~22.9 ± 4.5 nM (Fig.
5A). Protamine sulfate did not
affect the binding of 125I-RAP to either control or
PMA-treated cells, suggesting that the observed binding was mediated by
the VLDL receptor rather than glycosaminoglycans (56). Consistent with
the similar Bmax values determined for the
binding of RAP to control and treated cells, PMA did not affect the
amount of biotinylated VLDL-R that was immuno- or ligand-precipitated
from cell surface-biotinylated THP-1 cells.
We also examined the effect of PMA on the binding of human VLDL to
THP-1 cells. DiI-labeled VLDL (25 µg/ml) bound specifically to
control THP-1 cells, as demonstrated by the ability of a 100-fold molar
excess of unlabeled VLDL (Fig. 5B) or RAP (not shown) to block the binding of DiI-VLDL to cells by >90%. Exposure of THP-1 cells to PMA decreased the binding of DiI-VLDL by a similar extent as
unlabeled VLDL (467.2 to 17.0 arbitrary fluorescence units), with this
effect blocked by the PK-C inhibitor GF109203X (Fig. 5C).
Taken together, these studies demonstrate that exposure of THP-1 cells
to PMA leads to inhibition of the binding of VLDL-R ligands without
altering cell surface VLDL-R expression.
The Effects of PMA Depend upon PK-C Activation--
Since PMA
activates other protein kinases in addition to PK-C, we further
assessed the role of PK-C in the inhibition of VLDL-R ligand binding in
response to PMA. In contrast to the protein kinase A inhibitor H89 and
the protein-tyrosine kinase inhibitor genistein (not shown), the PK-C
inhibitor GF109203X (57) blocked the PMA-induced inhibition of
u-PA:PAI-1 complex (Fig. 2A, lane 3), RAP (Fig.
4, upper panel, lane 4), and VLDL binding (Fig. 5C) to the VLDL-R. Similar results were observed with
calphostin C, another specific PK-C inhibitor. Neither GF109203X (Fig.
3) nor calphostin C affected the levels of VLDL-R mRNA or protein (Fig. 2B, lane 3; Fig. 4, lower panel,
lane 4).
We next assessed the specific involvement of the PK-C
To further assess the role of PK-C
In contrast to cells transfected with the VLDL-R alone, co-transfected
cells demonstrated a marked increase in the amount of PKC- The VLDL Receptor Is Phosphorylated on Serine Residues--
The
results described above suggested that the VLDL-R was phosphorylated in
a PK-C-dependent manner, leading to diminished ligand
binding affinity. Therefore, we determined whether metabolically labeled THP-1 cells incorporated [32P]orthophosphate into
the receptor. In THP-1 cells, 32P was incorporated into
both the ~105- and ~130-kDa isoforms of the VLDL-R, with
approximately 3-fold more 32P incorporated after exposure
to PMA. Inclusion of GF109203X or LY379196 during metabolic labeling or
treatment of the immunoprecipitated VLDL-R with protein phosphatase 2B
reduced receptor phosphorylation to base-line levels (Fig.
8A). In control studies
performed in parallel, PMA did not affect the levels of
[35S]methionine-labeled VLDL-R (Fig. 8C),
suggesting that it did not influence receptor synthesis or degradation.
Immunoblotting of immunoprecipitated VLDL-R using anti-VLDL-R
antibodies confirmed that similar amounts of VLDL-R were
immunoprecipitated from control and PMA-treated cells (Fig.
8D).
Incubation of PVDF-immobilized 32P-labeled VLDL-R with 1 M KOH, which chemically dephosphorylates phosphoserine
residues (50), released 32P from the receptor (Fig.
8B). The specificity of this approach was confirmed by the
observation that immobilized phospho-MAP kinase
(tyrosine-phosphorylated, from vascular endothelial cell growth
factor-stimulated human dermal microvascular endothelial cells) was not
dephosphorylated when subjected to identical treatment (not shown).
Moreover, VLDL-R immunoprecipitated from extracts of PMA-treated THP-1
cells were recognized by anti-phosphoserine but not
anti-phosphothreonine or anti-phosphotyrosine antibodies.
Direct Evidence for Modulation of VLDL Receptor Ligand Binding
Affinity by Phosphorylation--
Since the studies above demonstrated
that the VLDL-R was phosphorylated in intact cells and that receptor
phosphorylation diminished ligand binding affinity, we determined
whether the affinity of the receptor for RAP was restored after
enzymatic dephosphorylation. Treatment of extracts from PMA-treated
THP-1 cells (which do not bind RAP) with potato acid phosphatase
restored the binding of RAP to immobilized VLDL-R in a
concentration-dependent manner (Fig.
9, lanes 3-5). An identical
effect was observed after treatment of the phosphorylated receptor with
protein phosphatase 2B.
Additional evidence for a critical role for phosphorylation within the
VLDL-R cytoplasmic domain in regulation of receptor ligand binding
affinity was derived from study of a VLDL-R mutant lacking the
cytoplasmic domain of the receptor. This mutant receptor was expressed
in similar amount as the wild-type receptor (primarily as the
~105-kDa isoform) on the surface of transfected HEK 293 cells, as
determined by analysis of immunoprecipitated VLDL-R from cell
surface-biotinylated cells. However, unlike the wild-type receptor,
binding of RAP by the truncated receptor was unaffected after treatment
of cells with PMA (Fig. 10). Taken
together with the preceding studies, these experiments support the
hypothesis that phosphorylation of serine residues within the
cytoplasmic domain of the receptor plays an important role in
regulating ligand binding affinity.
Activation of PK-C by Elevated Glucose Concentrations Leads to
Inhibition of VLDL-R Ligand Binding Activity--
Elevated glucose
concentrations contribute to the pathogenesis of vascular disease in
diabetes mellitus (52, 63). Culture of microvascular endothelial
cells in the presence of high glucose or galactose stimulates the
activation of PK-C by inducing de novo synthesis of
diacylglycerol (51, 64). Treatment of diabetic rats with a specific
PK-C
To determine whether the ligand binding activity of the VLDL receptor
was affected by high glucose, microvascular endothelial cells were
incubated in the presence of various concentrations of glucose,
galactose, or mannitol. Exposure of cells to 16.5 mM
D-glucose or galactose caused a potent and rapid reduction in the binding of RAP by VLDL-R (Fig.
11, upper panel), whereas the level of VLDL-R detected by immunoblotting was unaffected (Fig. 11,
lower panel). The effects of glucose and galactose were blocked by GF109203X and LY379196 (Fig. 11, upper panel,
lanes 3 and 4 and 6 and 7,
respectively), demonstrating an important role for PK-C, particularly
the These studies demonstrate that PK-C activation leads to
phosphorylation of the VLDL-R and inhibition of its ligand binding activity in a number of cell types, including monocyte-derived THP-1
cells, human endothelial and vascular smooth muscle cells, and HEK 293 cells transfected with VLDL-R cDNA. PK-C-dependent phosphorylation of the VLDL-R leads to diminished binding not only of
RAP but of other VLDL-R ligands such as u-PA:PAI-1 complexes and VLDL.
The (patho)physiological relevance of this process is supported by the
observation that glucose concentrations similar to those that induce
PK-C activation in vitro and in vivo in
experimental diabetes cause VLDL-R phosphorylation and loss of ligand
binding activity in endothelial cells. Moreover, VLDL-R isolated from myocardium obtained from patients with advanced cardiomyopathies, a
condition associated with PK-C VLDL-R phosphorylation was associated with a rapid loss of ligand
binding activity, although levels of VLDL-R mRNA and protein and
the expression of the VLDL-R on the cell surface were unaffected. Taken
together, these observations suggest that phosphorylation results in
diminished affinity of the receptor for its ligands. This conclusion is
supported by direct radioligand binding studies in which
125I-RAP was found to bind to a similar number of sites on
control and PMA-treated THP-1 cells, although with ~13-fold lower
affinity to the latter. Furthermore, although the affinity of DiI-VLDL binding to THP-1 cells was not determined directly, the complete disappearance of binding after exposure of these cells to PMA suggests
that VLDL-R phosphorylation greatly diminishes the affinity of the
receptor for apoE-containing ligands as well.
Several observations support the conclusion that phosphorylation of
cytoplasmic domain serine residue(s) regulates the ligand binding
activity of the VLDL-R through a process involving PK-C. First, the
PMA-induced loss of ligand binding activity was prevented by the PK-C
inhibitors GF109203X (57), calphostin C (66), and LY379196 but not by
inhibitors of PK-A or tyrosine kinases. Second, enzymatic
dephosphorylation of the VLDL-R restored its ligand binding activity.
Third, 32P was directly incorporated into the VLDL-R, with
incorporation stimulated by PMA. Fourth, chemical dephosphorylation of
the VLDL-R, as well as immunoblotting with specific monoclonal
antibodies, were consistent with the phosphorylation of serine but not
threonine or tyrosine residues within the VLDL-R in response to PK-C
activation. Finally, the ligand binding activity of a mutant VLDL-R
lacking the cytoplasmic domain was unaffected by PMA.
The effects of phosphorylation on VLDL receptor activity likely reflect
a generalized mechanism through which phosphorylation within a receptor
cytoplasmic domain regulates its function by altering ligand binding
affinity. For example, phosphorylation of serine and threonine residues
within the cytoplasmic domains of the insulin (67, 68) and epidermal
growth factor receptors (69-75) diminishes their affinity for insulin
and epidermal growth factor, respectively. With respect to the
epidermal growth factor receptor, these effects mimic those involved in
regulation of the receptor by heterologous ligands (76) such as
platelet-derived growth factor (77) and those induced through a
negative feedback pathway triggered by binding of epidermal growth
factor itself (78).
Regulation of the activity of LDL receptor family members through
phosphorylation is a novel concept of which few examples exist. Though
Kishimoto et al. (79) observe that the LDL receptor could be
phosphorylated on Ser833 (79) by an LDL receptor kinase
purified from bovine adrenal cortex (80), these effects occurred only
in a cell-free system, and incorporation of
[32P]orthophosphate into the LDL receptors of intact
cells could not be demonstrated (80). More recently, however, Bu
et al. (81) demonstrate that the LDL receptor-related
protein/ Which serine residue(s) within the cytoplasmic domain of the VLDL-R are
involved in regulation of ligand binding affinity is uncertain,
although preliminary studies suggest the involvement of
Ser-829.3 Interestingly,
although the cytoplasmic domains of the VLDL and LDL receptors are
highly homologous (49% matching residues), only 2 of the 6 serines
within this region of the VLDL-R align with any of the 4 serines in the
corresponding domain of the LDL-R, perhaps explaining the different
susceptibility of these receptors to phosphorylation in intact cells.
In addition to hyperglycemia, PK-C activation may be induced by several
cytokines that affect the function of endothelial and smooth muscle
cells and monocytes (84-86). Mechanical stimuli (87, 88) and hypoxia
(89) also activate PK-C. The pathophysiologic consequences of excessive
PK-C Our studies must be considered in light of the fact that homozygous
disruption of the VLDL-R gene is not associated with major phenotypic
abnormalities (92). Although these results demonstrate that the VLDL
receptor does not play a critical role in lipoprotein clearance in
mice, they do not exclude a role for this receptor in normal and/or
pathophysiological processes. The ability of the VLDL-R to mediate
lipoprotein uptake in vivo has been demonstrated by gene
transfer studies (93, 94), and the receptor may be involved in other
processes in addition to lipoprotein metabolism. Hence, a complementary
interpretation of the VLDL receptor knockout studies, based on the
findings presented here, is that in at least some tissues the receptor
may be phosphorylated and in a relatively inactive state. If so, then
genetic deletion of the receptor might have relatively minor
consequences. Further evaluation of this hypothesis will require
in vivo examination of the function of VLDL-R lacking
specific serine residues involved in regulation of receptor affinity.
2-macroglobulin receptor. Although several
agonists regulate VLDL-R mRNA and/or protein expression,
post-transcriptional regulation of receptor activity has not been
described. Here, we report that the ligand binding activity of the
VLDL-R in THP-1 monocytic cells, endothelial cells, smooth muscle
cells, and VLDL-R-transfected HEK 293 cells is diminished after
treatment with phorbol 12-myristate 13-acetate. This response was
blocked by inhibitors of protein kinase C (PK-C), including a specific
inhibitor of the PK-C
II isoform, and was associated with
phosphorylation of serine residues in the cytoplasmic domain of the
receptor. Culture of endothelial cells in the presence of high glucose
concentrations, which stimulate diacylglycerol synthesis and PK-C
II
activation, also induced a PK-C-dependent loss of VLDL-R
ligand binding activity. Taken together, these studies demonstrate that
the ligand binding activity of the VLDL-R is regulated by
PK-C-dependent phosphorylation and that hyperglycemia may
diminish VLDL-R activity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin receptor, gp330 (megalin), and
apoE receptor 2 (1-4) among others. Members of this family are
characterized by a cytoplasmic domain NPXY sequence that
mediates ligand internalization through clathrin-coated pits (5) and an
extracellular domain comprised of cysteine-rich complement-type and
epidermal growth factor precursor-like repeats that regulate ligand
binding specificity (2). Except for the presence of an additional
complement-type repeat in its extracellular domain, the domain
structure of the VLDL-R is identical to that of the LDL-R (6-8).
Despite this homology, however, the ligand binding specificity of these
receptors differs. Though both receptors bind apoE-containing
lipoproteins (6, 7, 9, 10), only the LDL receptor binds lipoproteins
containing apoB-100 (6, 7), whereas the VLDL-R binds several additional
ligands such as lipoprotein lipase (9), (11) urokinase-type plasminogen activator:plasminogen activator inhibitor type 1 (u-PA:PAI-1) complexes
(11, 12), and Lp(a) (13). Finally, the 39-kDa receptor-associated
protein (RAP), which co-purifies with (14, 15) and binds with high
affinity to the LDL receptor-related protein/
2-macroglobulin receptor (16-18), binds tightly
to the VLDL-R (19) but with only low affinity to the LDL-R (19,
20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin receptor (14,
15) and inhibits ligand binding to this (16-18) as well as to the
VLDL-R (19), was expressed and purified as described (17). For selected
studies, RAP was conjugated to cyanogen bromide-activated Sepharose 4B
(8.9 mg/ml of packed beads) per the manufacturer's protocol (Amersham
Pharmacia Biotech) or labeled with 125I to a specific
activity of ~5.0 × 106 cpm/µg using iodobeads.
Affinity-purified anti-VLDL receptor antibodies, raised against a
peptide corresponding to the C-terminal 20 amino acids of the human
(35-37) mouse (38) and rabbit (6) VLDL-R, have been described (30). A
rabbit antibody raised against a polypeptide encompassing the 160 N-terminal amino acids of the VLDL-R (25) was used for detection of a
truncated VLDL-R mutant lacking the cytoplasmic domain.
Anti-phosphoserine (1C8) and anti-phosphothreonine (1E11) monoclonal
antibodies were from Biomol (Plymouth Meeting, PA),
anti-phosphotyrosine antibodies were from Upstate Biotechnology (Lake
Placid, NY), and anti-PK-C
II antibodies were from Santa Cruz
Biotechnology (Santa Cruz, CA). Two-chain u-PA, PAI-1, and anti-PAI-1
antibodies were from American Diagnostica, Inc. (Greenwich, CT).
Phorbol 12-myristate 13-acetate (PMA), protamine sulfate, streptavidin-peroxidase, and peroxidase-conjugated secondary antibodies were from Sigma. Genistein, potato acid phosphatase, recombinant (Escherichia coli) protein phosphatase 2B,
protease inhibitors, and the PK-C inhibitors GF109203X and calphostin C
were from Calbiochem. The PK-C
II-specific inhibitor LY379196 was a
kind gift from Dr. Kirk Ways, Eli Lilly (Indianapolis, IN).
Polyvinylidene fluoride (PVDF) membranes (Immobilon P) were from
Millipore (Bedford, MA), and Nytran membranes were from Schleicher & Scheull. Radiochemicals and Reflection® autoradiography film were from
PerkinElmer Life Sciences. Super Signal chemiluminescence
reagent and Iodobeads were from Pierce.
7 M PMA
were divided into four aliquots. Proteins within three of these were
incubated for 60 min with 0.48, 0.6 or 1.2 units/ml potato acid
phosphatase or protein phosphatase 2B, as described previously (41).
The fourth aliquot was incubated under identical conditions with an
equal volume of phosphate-buffered saline. Extracts were then analyzed
by RAP ligand blotting.
7 M PMA for 2 h. VLDL-R
were then ligand-precipitated from cell extracts using RAP-Sepharose
4B, separated using 7.5% SDS-PAGE, and transferred to PVDF.
Biotinylated proteins were detected by chemiluminescence after
incubation of membranes with streptavidin-peroxidase.
7
M PMA was assessed. 125I-RAP binding was
measured by incubating increasing concentrations of
125I-RAP with control or PMA-treated cells (2 × 106 cells/ml, 0.5 ml) for 2 h at 4 °C in the
absence or presence of a 100-fold excess of unlabeled RAP. Cells were
separated from unbound ligand by centrifugation (10,000 × g) through a 1-ml cushion of silicone oil (Fluid 500:Fluid
200, 7.5:1.5; Dow/Corning, Nye Lubricants, New Bedford, MA), and
cell-associated ligand was determined by counting the cell pellets in a
counter. Specific binding was defined as the difference between
total and nonspecific binding measured in the absence or presence of
excess unlabeled ligand, respectively. Saturation isotherms were
prepared from the binding data using non-linear regression and assuming
one-site binding (Prism; Graph Pad Software, San Diego, CA) and used to
determine the Kd and Bmax. In
selected experiments, the role of heparan sulfate proteoglycans in
125I-RAP binding was assessed by measuring binding in the
presence of 100 µg/ml of protamine sulfate (44) (Sigma).
II isoform in mediating the effects of
PMA was also assessed. First, we determined whether PK-C
II was
translocated to the membrane fraction after exposure of cells to PMA
(48). Next, the ability of the specific PK-C
II inhibitor LY379196
to block PMA-induced inhibition of VLDL-R ligand binding was measured.
Third, the expression of functional VLDL-R by HEK 293 cells transfected
with either VLDL-R cDNA alone, or co-transfected with VLDL-R
cDNA and a 2980-nucleotide PK-C
II cDNA (a gift from Dr.
George King, Joslin Diabetes Center, Boston, MA) was compared.
7 M PMA (final concentration),
1.5 × 10
7 M PMA and either
10 µM GF109203X or 30 nM LY379196, or an
equal volume of ethanol vehicle was added to the individual aliquots. Labeling was terminated by washing the cells in ice-cold
phosphate-buffered saline, and cell pellets were immediately extracted
in a buffer containing 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 50 mM
D-phenylanalyl-L-propyl-L-arginine chloromethyl ketone and 50 mM sodium fluoride, pH 7.4. Cell
extracts were incubated overnight with 30 µl of rabbit anti-VLDL-R
antibodies (0.66 mg/ml), and immune complexes were precipitated using
50 µl of protein A-Sepharose. Beads were collected by centrifugation and washed before elution of bound material in 50 µl of 1× Laemmli sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 10%
glycerol) (49). The precipitate from the cells exposed to PMA alone was
divided into two aliquots that were treated for 30 min at 30 °C with
either 200 units of recombinant protein phosphatase 2B or potato acid phosphatase or an equal volume of phosphate-buffered saline. Eluates were then separated in parallel lanes of a 7.5% SDS-PAGE gel that was
dried and exposed to Reflection autoradiographic film for 24 h at
80 °C. The incorporation of 32P into VLDL-R from each
aliquot of cells was determined by measuring the total radioactivity
incorporated into the ~130- and ~105-kDa VLDL-R bands using a
PhosphorImager (model 445SI; Molecular Dynamics, Sunnyvale, CA).
To assure that equal protein synthesis occurred under each of
these conditions, a parallel experiment was performed in which aliquots
of THP-1 cells were labeled under identical conditions using 0.2 mCi/ml
[35S]methionine.
II in vitro and in vivo
(51, 52), and inhibition of hyperglycemia-induced PKC-
II activation
ameliorated the microvascular complications of diabetes in animal
models (53, 54). To determine whether hyperglycemia induced VLDL-R
phosphorylation, we incubated human dermal microvascular endothelial
cells for 6 h in the following: 1) control media containing 5.5 mM D-glucose, 2) high glucose medium containing
16.5 mM D-glucose, 3) high galactose medium, identical to high glucose medium except for substitution of galactose (16.5 mM) for glucose, and 4) high mannitol medium in which
the inert sugar, mannitol (16.5 mM), was substituted for
glucose or galactose. Osmolality of all medium was 311 mosmol/kg. Cell
extracts were then prepared and separated by 7.5% SDS-PAGE, and the
amount of VLDL-R protein and its ligand binding activity were assessed by immunoblotting and RAP ligand blotting, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7
M. VLDL-R expression was not altered by treatment of cells
with the inactive phorbol ester, 4
-phorbol 12,13-didecanoate.
View larger version (62K):
[in a new window]
Fig. 1.
Effect of PMA on the binding of RAP to
VLDL-R. THP-1 cells were cultured in the absence (lane
1) or presence (lanes 2-5) of 1.5 × 10 7 M PMA for the indicated
times. Detergent extracts were then prepared, and 100 µg of protein
from each extract analyzed using RAP ligand blotting, as described
under "Experimental Procedures." The ~105-kDa and ~130-kDa
bands represent the two isoforms of the VLDL receptor, whereas the
39-kDa band reflects endogenous RAP.
View larger version (43K):
[in a new window]
Fig. 2.
Effect of PMA on the binding of u-PA:PAI-1
complexes to VLDL-R. THP-1 cells were cultured for 2 h in the
absence (lane 1 of each blot) or presence (lane 2 of each blot) of 1.5 × 10 7 PMA, or PMA
and GF109203X (lane 3 of each blot). Detergent extracts from
cells incubated under each of these conditions were then divided into
equal aliquots and analyzed by ligand blotting using u-PA:PAI-1
complexes as the ligand (A) or immunoblotting, using
anti-VLDL-R antibodies (B).
View larger version (27K):
[in a new window]
Fig. 3.
Effect of PMA on the levels of VLDL-R
mRNA. THP-1 cells were incubated for 2 h in medium
containing either no additions (lane 1), 1.5 × 10 7 M PMA (lane 2),
1.0 µM calphostin C (lane 3), or 1.5 × 10
7 M PMA and 1.0 µM calphostin C (lane 4). Total cellular RNA
was then isolated, and 10 µg of RNA from cells treated under each of
these conditions were analyzed by Northern blotting for VLDL-R
(upper panel) or actin mRNA (lower
panel).
View larger version (49K):
[in a new window]
Fig. 4.
Effect of PMA on expression of functional
VLDL-R and VLDL-R protein. THP-1 cells were incubated for 2 h
in medium containing no additions (lane 1 of each blot),
1.5 × 10 7 M PMA (lane
2 of each blot), 10.0 µM GF109203X (lane
3 of each blot), or 1.5 × 10
7
M PMA and 10.0 µM GF109203X (lane
4 of each blot). Cell extracts were then prepared, and equal
amounts of protein from each were separated by 7.5% SDS-PAGE and
analyzed by RAP ligand blotting (upper panel) or
immunoblotting using anti-human VLDL-R antibodies (lower
panel).
View larger version (24K):
[in a new window]
Fig. 5.
Effect of PMA on binding of VLDL-R ligands to
intact cells. A, the specific binding of
125I-RAP to THP-1 cells preincubated for 2 h in the
absence ( ) or presence (----) of 1.5 × 10
7 M PMA was measured as
described under "Experimental Procedures." Error bars
represent S.E. of triplicate points from a single experiment
representative of three so performed. B,
specificity of DiI-VLDL binding to THP-1 cells. Histograms represent
control THP-1 cells not exposed to PMA or DiI-VLDL (red) and
THP-1 cells incubated with DiI-VLDL in the absence (green)
or presence (black) of a 100-fold excess of unlabeled VLDL.
C, effect of PMA on binding of DiI-VLDL to THP-1 cells.
Histograms represent control cells not exposed to PMA or DiI-VLDL
(red), cells preincubated for 2 h in the absence
(green) or presence (black) of 1.5 × 10
7 M PMA before incubation with
25 µg/ml DiI-VLDL, or cells preincubated for 2 h in the presence
of 1.5 × 10
7 M PMA and 10 µM GF109203X (blue) before incubation with
DiI-VLDL.
II isoform in
the decreased VLDL-R ligand binding occurring upon PK-C activation.
PK-C
II is strongly expressed in failing human heart (58), a tissue
in which we have observed that the VLDL-R is phosphorylated (59). Upon
exposure of THP-1 cells to PMA, PK-C
II was rapidly translocated
from the cytosolic to the cell membrane fraction (Fig.
6A). Moreover, as observed
with GF109203X, a specific PK-C
II inhibitor, LY379196, blocked the
PMA-induced decrease in RAP binding (Fig. 6B).
View larger version (49K):
[in a new window]
Fig. 6.
Involvement of PK-C
II in regulation of VLDL-R ligand binding
affinity. A, subcellular localization of PK-C
II
(~62 kDa) before and after exposure of THP-1 cells to 1.5 × 10
7 M PMA for 60 min. Cytosolic
(upper panel) and membrane (lower panel)
fractions were isolated before and at increasing times after exposure
of cells to PMA. Proteins from these fractions were separated by 10%
SDS-PAGE, transferred to PVDF, and detected by immunoblotting using
PK-C
II-specific antibodies. B, inhibition of the
PK-C-associated decrease in VLDL-R ligand binding by the PK-C
II-specific inhibitor LY379196. THP-1 cells were incubated for
2 h in the absence or presence of 1.5 × 10
7 M PMA or in the presence of
PMA and either GF109203X or LY379196. Cell extracts were then prepared
and analyzed by RAP ligand blotting.
II, HEK 293 cells were
transfected with VLDL-R cDNA alone or co-transfected with VLDL-R and PK-C
II cDNA. As previously reported for bovine aortic
endothelial cells (60) and mammary carcinoma cells (61, 62),
VLDL-R-transfected HEK 293 cells expressed primarily the ~105-kDa
isoform of the receptor lacking exon 16, which binds ligands
identically to the larger ~130-kDa isoform (2, 62).
II in the
membrane fraction (not shown). Moreover, ligand blot analysis revealed
dramatically reduced binding of RAP to immobilized VLDL-R from the
co-transfected cells, despite similar amounts of VLDL-R protein (Fig.
7, lane 1 versus
lane 5). Moreover, although the PK-C inhibitors GF109203X
and LY379196 blocked the inhibitory effects of PMA on the ligand
binding activity of the VLDL-R in singly transfected cells, these
inhibitors not only blocked the effect of PMA but markedly stimulated
VLDL-R ligand binding activity (compared with unstimulated cells) in
the co-transfected cells (compare lanes 5 versus lanes
7 and 8). These results are consistent with the
increased base-line activity of PK-C
II in the co-transfected
cells.
View larger version (30K):
[in a new window]
Fig. 7.
Transfection of HEK 293 cells with VLDL-R and
PKC- II cDNA. HEK 293 cells were
transfected with a full-length VLDL-R cDNA as described under
"Experimental Procedures." Successfully transfected clones were
maintained in 400 µg/ml geneticin or co-transfected with a
full-length PK-C
II cDNA. After further selection, the ligand
binding activity of VLDL-R from VLDL-R or VLDL-R/PK-C
II-transfected
cell lines was analyzed by ligand blotting (A) and the
amount of immunologically detectable receptor quantitated by
immunoblotting (B). Compared with VLDL-R from the singly
transfected cells, VLDL-R from the co-transfected cells bound less RAP
(lane 1 versus lane 5). PMA diminished
the ligand binding activity of VLDL-R from single and cotransfectants
(lanes 2 and 6). The PK-C inhibitors GF109203X or
LY379196 blocked the inhibition of VLDL-R ligand binding activity
caused by PMA (lanes 3 and 4), whereas these
inhibitors not only blocked the effects of PMA but markedly stimulated
the VLDL-R ligand binding activity of the cotransfectants (lanes
7 and 8 versus lane 5).
View larger version (67K):
[in a new window]
Fig. 8.
Incorporation of 32P into the
VLDL-R. A, THP-1 cells were metabolically labeled by
incubation for 3 h in phosphate-free medium containing 0.5 mCi/ml
of [32P]orthophosphate. During the last 60 min of the
labeling period, either ethanol control (lane 1), 1.5 × 10 7 M PMA (lane 2),
1.5 × 10
7 M PMA and 10 µM GF109203X (lane 3), or 1.5 × 10
7 M PMA and 30 nM
LY379196 (lane 4) were added to the cell suspensions. An
aliquot from the cells exposed to PMA was also treated with recombinant
protein phosphatase 2B after labeling (lane 5). Detergent
extracts were prepared, and VLDL receptors were immunoprecipitated
using anti-VLDL-R antibodies and protein A-Sepharose. Precipitated
VLDL-R were eluted with 50 µl of 1× Laemmli buffer, and proteins
within equal volumes of eluate were separated using 7.5% SDS-PAGE,
transferred to PVDF, and detected using autoradiography. B,
the PVDF membranes used to prepare the autoradiogram in panel
A were incubated in 1 M KOH for 1 h, and
autoradiograms were repeated. C, THP-1 cells were
metabolically labeled with [35S]methionine under
identical conditions as in panel A, and VLDL-R were then
immunoprecipitated and analyzed as in panel A. D,
immunoblot analysis, using anti-VLDL-R antibodies, of the
immunoprecipitated VLDL-R depicted in panels A and
B. U, units.
View larger version (49K):
[in a new window]
Fig. 9.
Effect of VLDL-R dephosphorylation on RAP
binding activity. Detergent extracts were prepared from either
untreated THP-1 cells (lane 1) or cells exposed to 1.5 × 10 7 M PMA for 2 h
(lane 2). Extracts from the PMA-treated cells, containing
100 µg of total protein, were incubated with either
phosphate-buffered saline (lane 2) or increasing amounts of
potato acid phosphatase at 30 °C for 60 min (lanes 3-5)
and analyzed by RAP ligand blotting. Similar results were obtained
using recombinant protein phosphatase 2B.
View larger version (30K):
[in a new window]
Fig. 10.
PK-C independence of a truncated VLDL-R
mutant. Stable transfectants expressing wild-type VLDL-R
(panel A) or a truncated VLDL-R mutant lacking the
cytoplasmic domain (panel B) were prepared in HEK 293 cells.
Each of the transfectants were incubated in the absence (lane
1) or presence (lane 2) of 1.5 × 10 7 M PMA or PMA and either 10 µM GF109203X (lane 3) or LY379196 (lane
4) for 2 h. Detergent extracts were then prepared and
analyzed by RAP ligand blotting. PMA diminished the ligand binding
activity of the wild-type receptor (panel A, lanes
1 versus 2), with its effects prevented by
PK-C (
II) inhibition (lanes 3 and 4), but did
not affect that of the truncated receptor (panel B).
II inhibitor inhibits the development of vascular dysfunction
that leads to retinal, renal, and neurologic disease (53, 54, 65).
II isoform, in this response. An identical concentration of
mannitol, which does not induce diacylglycerol synthesis or PK-C
II
activation in endothelial cells (51), did not affect the binding of RAP
by VLDL-R.
View larger version (35K):
[in a new window]
Fig. 11.
Effect of hyperglycemia on VLDL-R ligand
binding activity; PK-C dependence. Human dermal microvascular
endothelial cells were cultured for 6 h in control medium
(lane 1) or medium containing elevated concentrations of
glucose, galactose, or mannitol in the absence or presence of GF109203X
or LY379196 (lanes 2-8). Detergent extracts were then
prepared, and VLDL-R ligand binding activity was assessed using RAP
ligand blotting (upper panel). Elevated concentrations of
glucose (lane 2) or galactose (lane 5) inhibited
VLDL-R ligand binding activity, with these effects blocked by GF109203X
(lanes 3 and 6) or LY379196 (lanes 4 and 7). Elevated concentrations of mannitol (lane
8) did not affect VLDL-R ligand binding activity. The amount of
VLDL-R protein, as determined by immunoblotting using anti-VLDL-R
antibodies (lower panel), was unaffected by any of these
treatments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II activation in the heart (58), are
also phosphorylated, whereas receptors from normal human myocardium are
not.2
2-macroglobulin receptor is phosphorylated in
neuronal cells in response to nerve growth factor, leading to enhanced
expression and endocytic activity. Our studies suggest that the
cytoplasmic domain of the VLDL-R plays an important role in regulating
receptor function, a hypothesis consistent with the complete sequence
conservation of this domain in the murine (38, 82), rabbit (6), and
human (35-37) receptors. Trommsdorf et al. (83) also
demonstrate a critical role for the cytoplasmic domains of the VLDL-R
and apoE receptor 2 in binding of the cytoplasmic adaptor protein
mammalian Disabled (mDab1), an interaction critical for appropriate
migration and layering of neurons during development of the cerebellum
and cerebral cortex. Whether this process may be affected by VLDL-R
phosphorylation has not been assessed.
II activation in myocardium, as occurs in diabetes (63, 65) and
congestive cardiomyopathies (58), is supported by transgenic mouse
models in which myocardial PKC
II overexpression leads to a fatal
postnatal cardiomyopathy (90). Although our studies demonstrate the
involvement of PK-C
II in phosphorylation of the VLDL-R, however,
they do not exclude the possibility that other PK-C isoforms such as
PK-C
may also mediate this activity (91).
![]() |
FOOTNOTES |
---|
* This work was supported by National Institute of Health Grants HL50827 (to K. R. M.) and HL50787 (to D. K. S.) and a Research Award from the American Diabetes Association (to K. R. M.)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.
§ A recipient of a Scientist Development Grant from the American Heart Association.
¶ A recipient of a postdoctoral fellowship from the Ohio Valley affiliate of the American Heart Association.
To whom correspondence should be addressed: Hematology-Oncology
Division, BRB 329, Case Western Reserve University School of Medicine,
10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-368-6606; Fax:
216-368-1166; E-mail: kxm71@po.cwru.edu.
Published, JBC Papers in Press, September 28, 2000, DOI 10.1074/jbc.M003953200
2 R. Sakthivel, manuscript in preparation.
3 R. Sakthivel and K. McCrae, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
VLDL, very low
density lipoprotein receptor;
LDL-R, low density lipoprotein
associated;
PMA, phorbol 12-myristate 13-acetate;
RAP, LDL
receptor-associated protein/2-macroglobulin
receptor-associated protein;
PK-C, protein kinase C;
u-PA, two-chain
urokinase-type plasminogen activator;
PAI-1, plasminogen activator
inhibitor type 1;
PVDF, polyvinylidene difluoride;
PAGE, polyacrylamide
gel electrophoresis;
DiI, 1,1'-dioctadecyl-3,3',3,3'-tetramethylindocarbocyanine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Herz, J., and Willnow, T. E. (1994) Functions of the LDL Receptor Gene Family , pp. 14-19, New York Academy of Sciences, New York |
2. | Hussain, M. M., Strickland, D. K., and Bakillah, A. (1999) Annu. Rev. Nutrition 19, 141-172[CrossRef][Medline] [Order article via Infotrieve] |
3. | Willnow, T. E. (1999) J. Mol. Med. 77, 306-315[CrossRef][Medline] [Order article via Infotrieve] |
4. | Jingami, H., and Yamamoto, T. (1995) Curr. Opin. Lipidol. 6, 104-108[Medline] [Order article via Infotrieve] |
5. |
Kibbey, R.,
Rizo, J.,
Gierasch, L.,
and Anderson, R.
(1998)
J. Cell Biol.
142,
59-67 |
6. | Takahashi, S., Kawarabayasi, Y., Nakai, T., Sakai, J., and Yamamato, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9252-9256[Abstract] |
7. | Yamamoto, T., Takahashi, S., Sakai, J., and Kawarabayasi, Y. (1993) Trends Cardiovasc. Med. 3, 144-148 |
8. | Nimpf, J., and Schneider, W. J. (1998) Atherosclerosis 141, 191-202[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Takahashi, S.,
Suzuki, J.,
Kohno, M.,
Oida, K.,
Tamai, T.,
Miyabo, S.,
Yamamoto, T.,
and Nakai, T.
(1995)
J. Biol. Chem.
270,
15747-15754 |
10. | Takahashi, S., Oida, K., Ookubo, M., Suzuki, J., Kohno, M., Murase, T., Yamamoto, T., and Nakai, T. (1996) FEBS Lett. 386, 197-200[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Argraves, K. M.,
Battey, F. D.,
MacCalman, C. D.,
McCrae, K. R.,
Gåfvels, M.,
Chappell, D. A.,
Strauss, J. F., III,
and Strickland, D. K.
(1995)
J. Biol. Chem.
270,
26550-26557 |
12. |
Heegaard, C. W.,
Simonson, A. C. W.,
Oka, K.,
Kjöller, L.,
Christensen, A.,
Madsen, B.,
Ellgaard, L.,
Chan, L.,
and Andreasen, P. A.
(1995)
J. Biol. Chem.
270,
20855-20861 |
13. |
Argraves, K. M.,
Kozarsky, K. F.,
Fallon, J. T.,
Harpel, P. C.,
and Strickland, D. K.
(1997)
J. Clin. Invest.
100,
2170-2181 |
14. | Kristensen, T., Moestrup, S. K., Gliemann, J., Bendtsen, L., Sand, O., and Sottrup-Jensen, L. (1992) FEBS Lett. 276, 151-155[CrossRef] |
15. |
Strickland, D. K.,
Ashcom, J. D.,
Williams, S.,
Burgess, W. H.,
Migliorini, M.,
and Argraves, W. S.
(1990)
J. Biol. Chem.
265,
17401-17404 |
16. |
Herz, J.,
Goldstein, J. L.,
Strickland, D. K.,
Ho, Y. K.,
and Brown, M. S.
(1991)
J. Biol. Chem.
266,
21232-21238 |
17. |
Williams, S. E.,
Ashcom, J. D.,
Argraves, W. S.,
and Strickland, D. K.
(1992)
J. Biol. Chem.
267,
9035-9040 |
18. |
Bu, G.,
and Rennke, S.
(1996)
J. Biol. Chem.
271,
22218-22224 |
19. |
Battey, F. D.,
Gåfvels, M. E.,
Fitzgerald, D. J.,
Argraves, W. S.,
Chappell, D. A.,
Strauss, J. F. I.,
and Strickland, D. K.
(1994)
J. Biol. Chem.
269,
23268-23273 |
20. |
Medh, J. D.,
Fry, G. L.,
Bowen, S. L.,
Pladet, M. W.,
Strickland, D. K.,
and Chappell, D. A.
(1995)
J. Biol. Chem.
270,
536-540 |
21. | Brown, M. S., and Goldstein, J. L. (1986) Science 23, 34-47 |
22. | Webb, J. C., Patel, D. D., Jones, M. D., Knight, B. L., and Soutar, A. K. (1994) Hum. Mol. Genet. 3, 531-537[Abstract] |
23. | Niemeier, A., Gåfvels, M., Heeren, J., Meyer, N., Angelin, B., and Beisiegel, U. (1996) J. Lipid Res. 37, 1733-1742[Abstract] |
24. |
Webb, D. J.,
Nguyen, D. H.,
Sankovic, M.,
and Gonias, S. L.
(1999)
J. Biol. Chem.
274,
7412-7420 |
25. | Multhaupt, H. A. B., Gåfvels, M. E., Kariko, K., Jin, H., Arenas-Elliott, C., Goldman, B. I., Strauss, J. F. I., Angelin, B., Warhol, M. J., and McCrae, K. R. (1996) Am. J. Pathol. 148, 1985-1997[Abstract] |
26. | Nakazato, K., Ishibashi, T., Shindo, J., Shiomi, M., and Maruyama, Y. (1996) Am. J. Pathol. 149, 1831-1838[Abstract] |
27. |
Wyne, K. L.,
Pathak, R. K.,
Seabra, M. C.,
and Hobbs, H. H.
(1996)
Arterioscler. Thromb. Vasc. Biol.
16,
407-415 |
28. | Russell, D. W., Yamamoto, T., Schneider, W. J., Slaughter, C. J., Brown, M. S., and Goldstein, J. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7501-7505[Abstract] |
29. | Suzuki, J., Takahashi, S., Oida, K., Shimada, A., Kohno, M., Tamai, T., Miyabo, S., Yamamoto, T., and Nakai, T. (1995) Biochem. Biophys. Res. Commun. 206, 835-842[CrossRef][Medline] [Order article via Infotrieve] |
30. | Wittmaack, F. M., Gåfvels, M. E., Bronner, M., Matsuo, H., McCrae, K., Tomaszewski, J., Robinson, S. L., Strickland, D. K., and Strauss, J. F. I. (1995) Endocrinol. 136, 340-348[Abstract] |
31. | Kohno, M., Takahashi, S., Oida, K., Suzuki, J., Tamai, T., Yamamoto, T., and Nakai, T. (1997) Atherosclerosis 133, 45-49[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Jokinen, E. V.,
Landschultz, K. T.,
Wyne, K. L.,
Ho, Y. K.,
Frykman, P. K.,
and Hobbs, H. H.
(1994)
J. Biol. Chem.
269,
26411-26418 |
33. | Ishibashi, T., Yokoyama, K., Shindo, J., Hamazaki, Y., Endo, Y., Sata, T., Takahashi, S., Kawarabayasi, Y., Shiomi, M., Yamamoto, T., and Maruyama, Y. (1994) Arterioscler. Thromb. 14, 1534-1541[Abstract] |
34. | Masuzaki, H., Jingami, H., Yamamoto, T., and Nakao, K. (1994) FEBS Lett. 347, 211-214[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Sakai, J.,
Hoshino, A.,
Takahashi, S.,
Miura, Y.,
Hirofumi, I.,
Hiroyuki, S.,
Kawarabayasi, Y.,
and Yamamoto, T.
(1994)
J. Biol. Chem.
269,
2173-2182 |
36. | Gåfvels, M. E., Caird, M., Britt, D., Jackson, C. L., Patterson, D., and Strauss, J. F. I. (1993) Somatic Cell Mol. Genet. 19, 557-569[Medline] [Order article via Infotrieve] |
37. | Oka, K., Tzung, K. W., Sullivan, M., Lindsay, E., Baldini, A., and Chan, L. (1994) Genomics 20, 298-300[CrossRef][Medline] [Order article via Infotrieve] |
38. | Oka, K., Ishimura-Oka, K., Chu, M.-J., Sullivan, M., Krushkal, J., Li, W.-H., and Chan, L. (1994) Eur. J Biochem. 224, 975-982[Abstract] |
39. | McCrae, K. R., DeMichele, A., Samuels, P., Roth, D., Kuo, A., Meng, Q., Rauch, J., and Cines, D. B. (1991) Br. J. Haematol. 79, 595-605[Medline] [Order article via Infotrieve] |
40. | Christ, G., Seiffert, D., Hufnagl, P., Gessl, A., Wojta, J., and Binder, B. R. (1993) Blood 81, 11277-11284 |
41. | Bingham, E. W., Farrell, H. M., Jr., and Dahl, K. J. (1976) Biochim. Biophys. Acta 429, 448-460[Medline] [Order article via Infotrieve] |
42. | Ponte, P., Ng, S. Y., Engel, J., Gunning, P., and Kedes, L. (1984) Nucleic Acids Res. 12, 1687-1696[Abstract] |
43. |
Yanase, K.,
Smith, R. M.,
Puccetti, A.,
Jarett, L.,
and Madaio, H. P.
(1997)
J. Clin. Invest.
100,
25-31 |
44. |
Warshawsky, I.,
Herz, J.,
Broze, G. J., Jr.,
and Schwartz, A. L.
(1996)
J. Biol. Chem.
271,
25873-25879 |
45. | Rudling, M. (1992) J. Lipid Res. 33, 493-501[Abstract] |
46. | Redgrave, T. G., Roberts, D. C., and West, C. E. (1975) Anal. Biochem. 65, 42-49[Medline] [Order article via Infotrieve] |
47. | Ando, M., Gåfvels, M., Bergström, J., Lindholm, B., and Lundkvist, I. (1997) Kidney Int. 51, 785-792[Medline] [Order article via Infotrieve] |
48. |
Castagna, M.,
Takai, Y.,
Kalbuchi, K.,
Sano, K.,
Kikkawa, U.,
and Nishizawa, O.
(1982)
J. Biol. Chem.
257,
7847-7851 |
49. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
50. | Kamps, M. P., and Sefton, B. M. (1989) Anal. Biochem. 176, 22-27[Medline] [Order article via Infotrieve] |
51. | Xia, P., Inoguchi, T., Kern, T. S., Engerman, R. L., Oates, P. J., and King, G. L. (1994) Diabetes 43, 1122-1129[Abstract] |
52. | King, G. L., Kunisaki, M., Nishio, Y., Inoguchi, T., Shiba, T., and Xia, P. (1996) Diabetes 45 Suppl. 3, 105-108[Abstract] |
53. | Ishii, H., Jirousek, M. R., Koya, D., Takagi, C., Xia, P., Clermont, A., Bursell, S. E., Kern, T. S., Ballas, L. M., Heath, W. F., Stramm, L. E., Feener, E. P., and King, G. L. (1996) Science 272, 728-731[Abstract] |
54. | Nakamura, J., Kato, K., Hamada, Y., Nakayama, M., Chaya, S., Nakashima, E., Naruse, K., Kasuya, Y., Mizubayashi, R., Miwa, K., Yasuda, Y., Kamiya, H., Ienaga, K., Sakakibara, F., Koh, N., and Hotta, N. (1999) Diabetes 48, 2090-2094[Abstract] |
55. | Mehta, K., and Lopez-Berestein, G. (1986) Cancer Res. 46, 1388-1394[Abstract] |
56. | Iadonato, S. P., Bu, G., Maksymovitch, E., and Schwartz, A. L. (1993) Biochem. J. 296, 867-875[Medline] [Order article via Infotrieve] |
57. |
Toullec, D.,
Pianetti, P.,
Coste, H.,
Bellevergue, P.,
Grand-Perret, T.,
Ajakane, M.,
Baudet, V.,
Boissin, P.,
Boursier, E.,
Loriolle, F.,
Duhamel, L.,
Charon, D.,
and Kirilovsky, J.
(1991)
J. Biol. Chem.
266,
15771-15781 |
58. |
Bowling, N.,
Walsh, R. A.,
Guojie, S.,
Estridge, T.,
Sandusky, G.,
Fouts, R. L.,
Mintze, K.,
Pickard, T.,
Roden, R.,
Bristow, M. R.,
Sabbah, H. N.,
Mizrahi, J. L.,
Gromo, G.,
King, G. L.,
and Vlahos, C. J.
(2000)
Circulation
99,
384-391 |
59. | Sakthivel, R., Strickland, D. K., Margulies, K., Gåfvels, M., and McCrae, K. R. (1998) Vascular Biology '98 April 15-18, p. 32, Abstr. 132, American Heart Association, San Francisco, CA |
60. |
Magrane, J.,
Reina, M.,
Pagan, R.,
Luna, A.,
Casaroli-Marano, R. P.,
Angelin, B.,
Gåfvels, M.,
and Vilaro, S.
(1998)
J. Lipid Res.
39,
2172-2181 |
61. | Simonson, A. C., Heegaard, C. W., Rasmussen, L. K., Ellgaard, L., Kjöller, L., Christensen, A., Etzerodt, M., and Andreasen, P. A. (1994) FEBS Lett. 354, 279-283[CrossRef][Medline] [Order article via Infotrieve] |
62. | Martensen, P. M., Oka, K., Christensen, L., Rettenberger, P. M., Petersen, H. H., Christensen, A., Chan, L., Heegard, C. W., and Andreasen, P. (1997) Eur. J. Biochem. 248, 583-591[Abstract] |
63. | Inoguchi, T., Battan, R., Handler, E., Sportsman, J. R., Heath, W., and King, G. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11059-11063[Abstract] |
64. | Lee, T.-S., Saltsman, K. A., Ohashi, H., and King, G. L. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5141-5145[Abstract] |
65. | King, G. L., and Koya, D. (1998) Diabetes 47, 859-866[Abstract] |
66. | Kobayashi, E., Nakano, H., Morimoto, M., and Tamaoki, T. (1989) Biochem. Biophys. Res. Commun. 159, 548-553[Medline] [Order article via Infotrieve] |
67. | Jacobs, S., Sahyoun, N. E., Saltiel, A. R., and Cuatrecasas, P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6211-6213[Abstract] |
68. |
Grunberger, G.,
and Gorden, P.
(1982)
Am. J. Physiol.
243,
E319-E324 |
69. |
Iwashita, S.,
and Fox, C. F.
(1984)
J. Biol. Chem.
259,
2559-2567 |
70. | Shoyab, M., De Larco, J., and Todaro, G. J. (1979) Nature 279, 387-391 |
71. | Lee, L. S., and Weinstein, E. B. (1978) Science 202, 313-315[Medline] [Order article via Infotrieve] |
72. |
McCaffrey, P. G.,
Friedman, B.,
and Rosner, M. R.
(1984)
J. Biol. Chem.
259,
12502-12507 |
73. | Hunter, T., Ling, N., and Cooper, J. A. (1985) Nature 311, 480-483 |
74. | Lin, C. R., Chen, W. S., Lazar, C. S., Carpenter, C. D., Gill, G. N., Evans, R. M., and Rosenfeld, M. G. (1986) Cell 44, 839-848[Medline] [Order article via Infotrieve] |
75. | Beguinot, L., Hanover, J. A., Ito, S., Richert, N. D., Willingham, M. C., and Pastan, I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2774-2778[Abstract] |
76. | Brown, K. D., Blay, J., Irvine, R. F., Heslop, J. P., and Berridge, M. J. (1984) Biochem. Biophys. Res. Commun. 123, 377-384[Medline] [Order article via Infotrieve] |
77. | Wrann, M., Fox, C. F., and Ross, R. (1980) Science 210, 1363-1364[Medline] [Order article via Infotrieve] |
78. | Whiteley, B., and Glaser, L. (1986) J. Cell Biol. 103, 1355-1362[Abstract] |
79. |
Kishimoto, A.,
Brown, M. S.,
Slaughter, C. A.,
and Goldstein, J. L.
(1987)
J. Biol. Chem.
262,
1344-1351 |
80. |
Kishimoto, A.,
Goldstein, J. L.,
and Brown, M. S.
(1987)
J. Biol. Chem.
262,
9367-9373 |
81. |
Bu, G.,
Sun, Y.,
Schwartz, A. L.,
and Holtzman, D. M.
(1998)
J. Biol. Chem.
273,
13359-13365 |
82. | Gåfvels, M. E., Paavola, L. G., Boyd, C. O., Nolan, P. M., Wittmaack, F., Chawla, A., Lazar, M. A., Bucan, M., Angelin, B., and Strauss, J. F. I. (1994) Endocrinol. 135, 387-394[Abstract] |
83. | Trommsdorf, M., Gotthardt, M., Hiesberger, T., Shelton, J., Stockinger, W., Nimpf, J., Hammer, R. E., Richardson, J. A., and Herz, J. (1999) Cell 97, 689-701[Medline] [Order article via Infotrieve] |
84. | Deisher, T. A., Haddix, T. L., Montgomery, K. F., Pohlman, T. H., Kauchansky, K., and Harlan, J. M. (1993) FEBS Lett. 331, 285-290[CrossRef][Medline] [Order article via Infotrieve] |
85. | Magnuson, D. K., Maier, R. V., and Pohlman, T. H. (1989) Surgery 106, 216-223[Medline] [Order article via Infotrieve] |
86. |
Xia, P.,
Aiello, L. P.,
Ishii, H.,
Jiang, Z. Y.,
Park, D. J.,
Robinson, G. S.,
Takagi, H.,
Newsome, W. P.,
Jirousek, M. R.,
and King, G. L.
(1996)
J. Clin. Invest.
98,
2018-2026 |
87. |
Tseng, H.,
Petersen, T. E.,
and Berk, B. C.
(1995)
Circ. Res.
77,
869-878 |
88. |
Takahashi, M.,
and Berk, B. C.
(1996)
J. Clin. Invest.
98,
2623-2631 |
89. |
Yan, S.-F.,
Lu, J.,
Zou, Y. S.,
Kisiel, W.,
Mackman, N.,
Leitges, M.,
Steinberg, S.,
Pinsky, D.,
and Stern, D.
(2000)
J. Biol. Chem.
275,
11921-11928 |
90. |
Wakasaki, H.,
Koya, D.,
Scheun, F. J.,
Jirousek, M. R.,
Ways, D. K.,
Hoit, B. D.,
Walsh, R. A.,
and King, G. L.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
9320-9325 |
91. | Park, J. Y., Takahara, N., Gabriele, A., Chou, E., Naruse, K., Suzuma, K., Yamauchi, T., Ha, S. W., Meier, M., Rhodes, C. J., and King, G. L. (2000) Diabetes 49, 1239-1248[Abstract] |
92. | Frykman, P. K., Brown, M. S., Yamamoto, T., Goldstein, J. L., and Herz, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8453-8457[Abstract] |
93. | Kozarsky, K. F., Jooss, K., Donahee, M., Strauss, J. F. I., and Wilson, J. M. (1996) Nat. Genet. 13, 54-62[Medline] [Order article via Infotrieve] |
94. |
Kobayashi, K.,
Oka, K.,
Forte, T.,
Ishida, B.,
Teng, B.,
Ishimura-Oka, K.,
Nakumata, M.,
and Chan, L.
(1996)
J. Biol. Chem.
271,
6852-6860 |