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(Received for publication, May 10,
1995; and in revised form, June 16, 1995) From the
The ability of glycoprotein 330/low density lipoprotein
receptor-related protein-2 (LRP-2) to function as a lipoprotein
receptor was investigated using cultured mouse F9 teratocarcinoma
cells. Treatment with retinoic acid and dibutyryl cyclic AMP, which
induces F9 cells to differentiate into endoderm-like cells, produced a
50-fold increase in the expression of LRP-2. Levels of the other
members of the low density lipoprotein (LDL) receptor (LDLR) family,
including LDLR, the very low density lipoprotein receptor, and LRP-1,
were reduced. When LDL catabolism was examined in these cells, it was
found that the treated cells endocytosed and degraded at 10-fold higher
levels than untreated cells. The increased LDL uptake coincided with
increased LRP-2 activity of the treated cells, as measured by uptake of
both
Lipoprotein receptors comprise a family of proteins that are
structurally related to the low density lipoprotein (LDL) ( To investigate the cellular function of LRP-2, we
have identified several LRP-2-expressing cell lines (Stefansson et
al., 1995). One of these cell lines is F9 teratocarcinoma cells,
which when treated with retinoic acid (RA) and dibutyryl cyclic AMP
(Bt
Figure 1:
Immunoblot analysis of the expression
of members of the LDLR family in extracts of F9 cells treated with
RA/Bt
Figure 2:
Assay of cellular internalization and
degradation of LDLR family member ligands or antibodies by F9 cells
treated with RA/Bt
The two other LDLR family members that were detected in extracts of
F9 cells (untreated and RA/Bt In
contrast to the observation that LDLR levels did not increase in
response to RA/Bt
Figure 3:
Binding of LRP-2 and LDL in solid-phase
binding assays. In A,
The major structural
component of LDL is apoB100. This apolipoprotein is known to mediate
the binding of LDL to LDLR (Milne et al., 1983). To evaluate
the role of apoB100 in LDL binding to LRP-2, a monoclonal anti-apoB100
IgG (mAb 4G3) was used as a blocking agent in the solid-phase
Figure 4:
LRP-2 antibody inhibits the increased
cellular uptake of LDL that occurs in F9 cells treated with
RA/Bt
The
effect of monoclonal anti-apoB100 IgG (mAb 4G3) on cellular uptake and
degradation of LDL was also examined. As shown in Fig. 5, mAb
4G3 blocked the endocytosis and degradation of
Figure 5:
Monoclonal apoB100 antibody inhibits the
increased cellular uptake and degradation of LDL that occurs in F9
cells treated with RA/Bt
This study establishes for the first time that LRP-2 is a LDL
receptor capable of mediating LDL endocytosis and lysosomal
degradation. This conclusion is supported by in vitro binding
assays showing high affinity binding of LRP-2 and LDL and cell assays
showing that the uptake and degradation of radiolabeled LDL in F9 cells
are inhibited by LRP-2 antibodies. In addition, the LRP-2 interaction
with LDL was inhibitable with apoB100 antibody in both solid-phase and
cellular assays, thereby indicating that apoB100 is the component of
LDL recognized by LRP-2. RAP was shown to be a potent inhibitor of LDL
binding to LRP-2, having a lower K The major question
raised by these findings is the in vivo relevance of
LRP-2-mediated uptake of LDL. The fact that LRP-2 is apparently
expressed only in extravascular sites (Kounnas et al., 1994b)
seems to preclude its role in the clearance of LDL directly from blood
in the adult. However, LRP-2 is expressed by embryonic trophectoderm
and on parietal and visceral endoderm (Buc-Caron et al., 1987;
Gueth-Hallonet et al., 1994). During early placental
formation, trophectoderm differentiates into trophoblast giant cells,
which surround the conceptus and make contact with decidual tissue and
maternal blood (Cross et al., 1994). Parietal endoderm forms a
layer underlying the trophoblast giant cells and mediates nutrient
exchange from the trophoblast giant cells to the yolk.
RA/Bt It is conceivable that LRP-2 acts as part
of a back-up system to LDLR for the uptake of cholesterol-rich LDL. In
animals genetically deficient for LDLR, developmental abnormalities are
not evident (Goldstein and Brown, 1983). It has been speculated that
increased de novo synthesis of cholesterol may compensate for
the absence of cholesterol derived via LDLR-mediated uptake of LDL
(Dietschy et al., 1983). However, de novo synthesis
of cholesterol cannot compensate for cellular requirements for
lipid-soluble vitamins, such as vitamin E, that are associated with
LDL. In humans and mice that are genetically deficient for apoB and
hence deficient in apoB-containing lipoproteins, neurological
abnormalities are apparent (Homanics et al., 1993; Kane and
Havel, 1989). Vitamin deficiency has been speculated to be a
contributing factor in the abnormal neurological phenotype associated
with genetic deficiency of apoB (Homanics et al., 1993; Farese et al., 1995). LRP-2-mediated uptake of LDL may therefore
serve as a mechanism to acquire lipid-soluble vitamins. The
identification of the apoB component of LDL as a ligand for LRP-2 adds
to a growing list of ligands that bind to this receptor. In addition to
LDL, the current list of LRP-2 ligands includes lipoprotein lipase and
the lipoprotein lipase-VLDL complex (Willnow et al., 1992;
Kounnas et al., 1993), apoE-enriched
Volume 270,
Number 33,
Issue of August 18, pp. 19417-19421, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
I-labeled monoclonal LRP-2 antibody and the LRP-2
ligand prourokinase. The ability of LDL to bind to LRP-2 was
demonstrated by solid-phase binding assays. This binding was
inhibitable by LRP-2 antibodies, receptor-associated protein (the
antagonist of ligand binding for all members of the LDLR family), or
antibodies to apoB100, the major apolipoprotein component of LDL. In
cell assays, LRP-2 antibodies blocked the elevated
I-LDL
internalization and degradation observed in the retinoic acid/dibutyryl
cyclic AMP-treated F9 cells. A low level of LDL endocytosis existed
that was likely mediated by LDLR since it could not be inhibited by
LRP-2 antibodies, but was inhibited by excess LDL, receptor-associated
protein, or apoB100 antibody. The results indicate that LRP-2 can
function to mediate cellular endocytosis of LDL, leading to its
degradation. LRP-2 represents the second member of the LDLR family
identified as functioning in the catabolism of LDL.
)receptor (LDLR). In addition to LDLR, the family includes
the very low density lipoprotein receptor (VLDLR) (Takahashi et
al., 1992; Gafvels et al., 1993), the LDLR-related
protein (LRP-1) (Herz et al., 1988; Jensen et al.,
1989; Strickland et al., 1991), and glycoprotein 330/LRP-2 (
)(Raychowdhury et al., 1989; Saito et
al., 1994; Korenberg et al., 1994). The roles of VLDLR,
LDLR, and LRP-1 as mediators of lipoprotein endocytosis have been
established through numerous studies using cultured cells and animal
models (for reference, see Gianturco and Bradley(1987), Yamamoto et
al. (1993), and Krieger and Herz(1994)). In contrast, the role of
LRP-2 in lipoprotein metabolism has largely been inferred from in
vitro binding data showing that it binds apoE-enriched 
-VLDL,
lipoprotein lipase-enriched VLDL (Willnow et al., 1992;
Kounnas et al., 1993), and apolipoprotein J (Kounnas et
al., 1995).cAMP), differentiate to endoderm-like epithelial cells
that express 50-fold higher levels of LRP-2 than untreated cells.
The levels of the other members of the LDLR family are reduced by the
treatment. During characterization of the expression and activity of
LDLR family members in this cell system, we discovered a novel function
of LRP-2, namely that it mediates endocytosis of LDL.
Proteins
LRP-2 was purified from pig kidney by
RAP-Sepharose affinity chromatography as described previously (Kounnas et al., 1994a). Human RAP was expressed in bacteria as a
fusion protein with glutathione S-transferase and purified
free of glutathione S-transferase as described by Williams et al.(1992). Human prourokinase was provided by Dr. Jack
Henkin (Abbott Laboratories, Abbott Park, IL). Human
-macroglobulin was purified according to Barrett(1981)
and complexed to trypsin, and the complex was purified as described by
Ashcom et al.(1990). LDL (d = 1.02-1.05
g/ml) and VLDL (S 100-400) were
isolated as described from normolipodemic human subjects who had the
most common apoE phenotype (E3/3) (Havel et al., 1955;
Chappell, 1988). Lipoproteins were labeled with
[
I]iodine (Amersham Corp.) to specific
activities of 1000-2000 cpm/ng by the iodine monochloride method
(Bilheimer et al., 1972) and used within 24 h. All other
proteins were iodinated using IODO-GEN (Pierce).
Antibodies
IgG was isolated from rabbit polyclonal
anti-LRP-2 serum (rb239 or rb784) (Kounnas et al., 1994b) and
anti-LRP-1 serum (rb777 and rb810) (Strickland et al., 1991)
by affinity chromatography on protein G-Sepharose followed by affinity
selection on either LRP-2- or LRP-1-Sepharose (1-2 mg/ml of
resin). IgG was isolated from the rabbit polyclonal antiserum raised
against a synthetic peptide corresponding to the last 11 residues of
the LRP-1 cytoplasmic domain (rb704) (Kounnas et al., 1992) by
protein G-Sepharose chromatography. The mouse anti-LRP-2 monoclonal
antibody 1H2 was provided by Dr. Robert McCluskey
(Harvard/Massachusetts General Hospital, Boston). The mouse anti-LRP-1
monoclonal antibody 5A6, which is directed against the 85-kDa light
chain of the receptor, has been previously described (Strickland et
al., 1990). IgGs from 1H2 and 5A6 ascitic fluids were purified by
protein G-Sepharose chromatography. The monoclonal antibody 4G3 to the
ligand-binding domain of apolipoprotein B100 (Milne et al.,
1983) and the mouse anti-apoE monoclonal antibody 1D7 (Weisgraber et al., 1983) were provided by Drs. Ross Milne and Yves Marcel
(Clinical Research Institute of Montreal). Rabbit anti-LDLR serum was
provided by Dr. Joachim Herz (Russell et al., 1984). Rabbit
antiserum to a synthetic peptide corresponding to the carboxyl terminus
of human and rabbit VLDLRs has been described previously (Battey et
al., 1994).Cells
Mouse embryonic F9 teratocarcinoma cells
(ATCC CCL 185) were obtained from American Type Culture Collection and
grown on plates (Corning, Corning, NY) coated with 0.1% gelatin in
Dulbecco's modified Eagle's media (DMEM; Life Technologies,
Inc.) supplemented with 10% bovine calf serum (Hyclone Laboratories,
Logan, UT), penicillin, and streptomycin. Subconfluent cultures were
treated with 0.1 µM RA (Calbiochem; diluted from a 0.1 M stock in dimethyl sulfoxide) and 0.2 µM BtcAMP (Sigma; diluted from a 0.1 M stock in
dimethyl sulfoxide) for periods of up to 7 days without a change of
medium.Immunoblotting
Detergent extracts of the cells
were prepared by solubilizing cells in 1% Triton X-100, 0.5% Tween 20,
0.5 M NaCl, 50 mM Hepes, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA. Equal amounts
of protein from the extracts were loaded onto 4-12%
polyacrylamide gradient gels in the presence of SDS. Following
electrophoresis, the proteins were transferred onto nitrocellulose
membranes, blocked with 3% non-fat milk and phosphate-buffered saline,
and probed with antibodies to LDLR family members or RAP. Detection of
bound antibody was achieved by autoradiography using the Renaissance
chemiluminescence kit (DuPont NEN). Autoradiographs were analyzed by
densitometry using a Lynx imaging system (Applied Imaging Corp.).Solid-phase Binding Assays
I-Labeled
LDL (0.9 µg/ml) was incubated in microtiter wells coated with LRP-2
or BSA (3 µg/ml coating concentration) in the presence of
increasing concentrations of unlabeled competitor (RAP, monoclonal IgG
4G3, or polyclonal IgG to either LRP-1 or LRP-2) as described by
Williams et al.(1992). The computer program Ligand (Munson and
Rodbard, 1980) was used to analyze the competition data and to
determine dissociation constants (K
) for
receptor-ligand interactions.
Assay of Cellular Internalization and Degradation of
Ligands
To evaluate ligand internalization and degradation in F9
cells treated with RA/BtcAMP, cells from successive days of
treatment were released by trypsin/EDTA and reseeded onto
gelatin-coated wells (0.5-1.5 10
cells/wells,
24-well plates) in serum-containing DMEM with RA and
BtcAMP. Prior to the addition of radiolabeled ligand, the
cells were cultured for 18 h at 37 °C and 5% CO. Ligand
internalization and degradation assays were performed as described
previously (Kounnas et al., 1993; Stefansson et al.,
1995). Briefly, F9 monolayers were washed twice with serum-free DMEM
and incubated in serum-free DMEM containing 1.5% BSA and Nutridoma
serum substitute (DMEM/BSA/SS). Antagonists of receptor ligand binding,
RAP (800 nM), or polyclonal LRP-2 antibodies (250 µg/ml)
in DMEM/BSA/SS were incubated with the cells for 30 min prior to the
addition of radiolabeled ligand. Radiolabeled ligands (prourokinase (5
nM); -macroglobulin-trypsin (1 nM);
mouse IgG 1H2, 5A6, and control IgG (3 nM); and LDL (2
µg/ml)) in DMEM/BSA/SS were incubated with the cells for
3.5-5 h at 37 °C and 5% CO. Determination of the
amount of radiolabeled ligand internalized and degraded was done as
described previously (Kounnas et al., 1993; Stefansson et
al., 1995). The amount of I-LDL specifically
internalized and degraded was determined by subtracting the amount of
radioactivity internalized or degraded in the presence of a 20-fold
molar excess of unlabeled ligand from the amount of radioactivity that
was internalized or degraded in the absence of excess unlabeled LDL. Of
the total
I-LDL internalized after 3.5 h, typically 70%
could be blocked by excess unlabeled LDL. Similarly, of the total
cell-mediated
I-LDL degradation observed after 3.5 h,
typically 80% could be blocked by excess unlabeled LDL.
LRP-2 Expression by F9 Cells Is Enhanced by Retinoic
Acid and Dibutyryl Cyclic AMP Treatment
We had previously found
that F9 cells expressed both LRP-1 and LRP-2 (Stefansson et
al., 1995). Knowing that F9 cells can be induced to differentiate
into an absorptive endoderm-like cell type by treatment with RA and
BtcAMP (Sherman and Miller, 1978), the effects on the
expression of LDLR family members were examined. Immunoblot analysis
was performed on detergent extracts of cells from successive days of
treatment. As shown in Fig. 1, over the 7-day course of the
differentiation, LRP-2 levels increased and after 7 days were
50-fold higher compared with untreated cells. In contrast, LRP-1
levels decreased
10-fold during differentiation. The level of
39-kDa RAP increased
5-fold after 7 days of treatment, which is in
agreement with findings reported by Furukawa et al.(1990). The
results indicate that LRP-2 and RAP levels increase coordinately during
RA/Bt
cAMP-induced F9 cell differentiation, while levels of
the other members of the LDLR family decrease.
cAMP. Labels on the right of each panel indicate the
LDLR family member (or RAP) that is immunologically stained. The M
values of the stained bands shown are as
follows: 600,000 for LRP-2, 85,000 for the LRP-1 light chain, 110,000
for LDLR, 130,000 and 120,000 for the two forms of VLDLR, and 39,000
for RAP. In each panel, lane0 corresponds to
detergent extracts made from untreated F9 cells. Lanes 1-7 correspond to extracts made from F9 cells on successive days of
RA/BtcAMP treatment.
Functional Activity of Individual LDLR Family Members in
Differentiated F9 Cells
To determine whether changes in receptor
levels observed during the course of differentiation correlated with
changes in receptor activity displayed by the cells, endocytosis of
receptor-specific antibodies or ligands was examined. Internalization
of radioiodinated monoclonal antibodies to LRP-1 and LRP-2 was used as
a means to measure the levels of endocytosis mediated by each receptor.
As shown in Fig. 2A, the amount of I-LRP-2 antibody (mAb 1H2) internalized by cells
increased over the course of the RA/Bt
cAMP-induced
differentiation. The pattern of increase in the uptake of I-LRP-2 antibody over the course of treatment paralleled
the increase in LRP-2 protein levels as shown in Fig. 1. In
contrast, the level of
I-LRP-1 antibody (mAb 5A6) that
was internalized by the cells over the course of treatment was not
significantly different from that of a control IgG of the same isotype.
The lack of
I-LRP-1 antibody (mAb 5A6) internalization by
F9 cells was not due to failure of the antibody to recognize mouse
LRP-1 since a different mouse teratocarcinoma cell line (SCC-PSA1
cells; ATCC CRL 1535) specifically internalized the antibody (data not
shown). Despite the fact that the monoclonal LRP-2 antibody was
internalized, it was not degraded (Fig. 2B), which is
in agreement with previous observations (Stefansson et al.,
1995). The basis for this may be that the antibody does not dissociate
from the receptor in the low pH environment of the endosomes, but is
recycled back to the surface with the receptor, as has been described
for antibodies to LRP-1 (Herz et al., 1990). Our results
suggest that there is little LRP-1 expressed on the surface of the F9
cells. Consistent with this conclusion was the observation that the F9
cells failed to endocytose or degrade
-I-macroglobulin-trypsin complexes, a
LRP-1-specific ligand, during the course of RA/Bt
cAMP
treatment (Fig. 2, C and D). Internalization
and degradation of I-prourokinase, a ligand for both
LRP-2 (Stefansson et al., 1995) and LRP-1 (Kounnas et
al., 1993), increased over the course of treatment (Fig. 2, C and D). Because F9 cells display little LRP-1
activity, this suggests that the increasing level of endocytosis and
degradation of
I-prourokinase is mediated by LRP-2.
cAMP. A and B show the
cellular internalization (A) and degradation (B) of I-LRP-2 antibody 1H2 (▪),
I-LRP-1
antibody 5A6 (▴), or a control mouse IgG (
) by F9 cells at
the indicated days of treatment with Bt
cAMP (DBC). C and D show the cellular internalization (C) and degradation (D) of I-prourokinase (Pro-uPA; ▪),
I-prourokinase plus RAP (
), and
-I-macroglobulin
(
M)-trypsin (
) by F9 cells at the indicated days
of treatment. E and F show the cellular
internalization (E) and degradation (F) of I-VLDL (
) and I-VLDL plus RAP (
)
by F9 cells at the indicated days of treatment. G and H show the cellular internalization (G) and degradation (H) of
I-LDL (
) and I-LDL
plus RAP (
) by F9 cells at the indicated days of treatment. The
data presented are representative of three experiments, each performed
in duplicate. Each plotted value represents the average of duplicate
determinations with the range indicated by the bars. DBC, Bt
cAMP.
cAMP-treated) are LDLR and
VLDLR. To examine the activity of these receptors in the F9 cells, we
used the VLDLR- and LDLR-specific lipoprotein ligands, VLDL and LDL,
respectively. Although VLDL binds to LDLR, the capacity of LDLR to
mediate their catabolism is considerably less than that of LDL
(Chappell et al., 1993). I-VLDL was found to be
internalized and degraded by F9 cells during the course of
RA/Bt
cAMP treatment; however, there was no change in the
amount taken up and degraded by the cells (Fig. 2, E and F). RAP was found to completely block the
internalization and degradation of I-VLDL, as has been
reported previously (Battey et al., 1994; Medh et
al., 1995). These findings were consistent with immunoblot
analysis of detergent extracts of the F9 cells (Fig. 1C), which indicated that the level of VLDLR did
not increase throughout the course of treatment. Furthermore, VLDL has
been shown not to interact with LRP-2 unless it is enriched with
lipoprotein lipase (Kounnas et al., 1993). Therefore, the
increased levels of LRP-2 in the RA/Bt
cAMP-treated cells
would not be expected to promote an increase in VLDL uptake.cAMP treatment, there was a 10-fold
increase in the amount I-LDL that was internalized and
degraded by the cells over the course of RA/Bt
cAMP
treatment (Fig. 2, G and H). The similarity
between the pattern of increase in I-LDL uptake over the
course of treatment and that of the uptake of
I-LRP-2
antibody and the LRP-2 ligand
I-prourokinase suggests
that LRP-2 might be mediating the endocytosis of LDL.
LRP-2 Binds to LDL in Solid-phase Binding
Assays
To evaluate the ability of LRP-2 to bind directly to LDL, in vitro binding assays were performed using purified LRP-2.
As shown in Fig. 3A, I-LRP-2 bound to
microtiter wells coated with LDL, but not to BSA-coated wells. This
binding could be inhibited by unlabeled LRP-2. Based on a fit of these
data, a dissociation constant (K
) of 45
nM (n = 4) was derived. In a like manner,
I-LDL bound to microtiter wells coated with LRP-2, and
binding could be inhibited by increasing doses of unlabeled LDL (Fig. 3B). By fitting these data, assuming a molecular
mass of 0.513
10
Da for LDL (Chappell et
al., 1993), a K of 50 nM (n = 2) was determined. RAP, the antagonist of LRP-2
ligand binding, inhibited the binding of
I-LDL to LRP-2 (Fig. 3C), with an inhibition constant (K
) of 3.3 nM (n = 3). The binding of
I-LDL to LRP-2 could
also be blocked by polyclonal LRP-2 antibodies (Fig. 3D), but not by polyclonal LRP-1 antibodies (data
not shown). The results indicate that LRP-2 can bind LDL particles. By
comparison to the LDLR-LDL interaction, the LDL-LRP-2 affinity is
10-fold lower (Medh et al., 1995).
I-LRP-2 (0.1 nM)
and various concentrations of unlabeled LRP-2 were incubated with wells
coated with LDL (
) or BSA (). In B,
I-LDL (1.6 nM) and various concentrations of
unlabeled LDL were incubated with wells coated with LRP-2 (▪) or
BSA (
). In C,
I-LDL and various
concentrations of RAP were incubated with wells coated with LRP-2
(▪) or BSA (
). In D,
I-LDL and various
concentrations of polyclonal LRP-2 antibody (rb239) were incubated with
wells coated with LRP-2 (▪) or BSA (
). In E,
I-LDL and various concentrations of either apoB100
antibody (mAb 4G3) (▪) or apoE antibody (mAb 1D7) (
) were
incubated with wells coated with LRP-2 (▪) or BSA (
). The
curves represent the best fit of the data to a single class of sites.
The data presented in A-E are representative of two,
three, five, three, and two experiments, respectively, with each
performed in duplicate. Each plotted value represents the average of
duplicate determinations with the range indicated by the bars.
I-LDL-LRP-2 binding assays. The antibody 4G3 binds to the
receptor-binding region of apoB100 and can block interaction with LDLR
(Milne and Marcel, 1982; Milne et al., 1983). As shown in Fig. 3E, mAb 4G3 inhibited the binding of
I-LDL to LRP-2 coated on microtiter wells. Neither a
control IgG of the same isotype as mAb 4G3 nor the apoE antibody 1D7
(known to block apoE-mediated binding to LDLR (Weisgraber et
al., 1983)) had inhibitory effects on the binding. The results
indicate that apoB100 serves as the ligand that mediates interaction of
LDL with LRP-2.
LRP-2 Mediates Endocytosis of LDL in F9 Cells
To
demonstrate that LRP-2 expressed by F9 cells functions to mediate LDL
endocytosis, we measured the endocytosis and degradation of I-LDL in the presence of polyclonal antibodies that had
previously been shown to block LRP-2 function (Stefansson et
al., 1995). As shown in Fig. 4, LRP-2 antibodies blocked
75% of the
I-LDL internalized by the cells treated
with RA/Bt
cAMP. The LRP-2 antibody was not able to block I-LDL internalization to the same extent as did excess
unlabeled LDL. However, RAP was able to block
I-LDL
uptake to a similar extent compared with excess unlabeled LDL. The
results indicate that while LRP-2 accounts for the increase in LDL
uptake observed during RA/Bt
cAMP treatment, there is a
basal level of I-LDL uptake that does not involve LRP-2.
The fact that this basal level of LDL uptake is RAP-sensitive suggests
that some LDLR family member, most likely LDLR, is involved.
cAMP. Shown are the amounts of I-LDL
internalized by F9 cells on successive days of treatment with
RA/Bt
cAMP (DBC) in the presence of LRP-2 antibody
(250 µg/ml; ▪), control IgG (250 µg/ml; ), RAP
(800 nM;
), or LDL (40 µg/ml;
) or in the
absence of competitor (
). Each plotted value represents the
average of duplicate determinations with the range indicated by the bars. The data presented are representative of two
experiments, each performed in duplicate.
I-LDL to a
similar extent compared with excess LDL and RAP. The apoE antibody 1D7
had little or no effect on these processes. The results indicate that
LDL uptake and degradation by F9 cells are apoB100-dependent. This,
along with the observed inhibitory effects of apoB100 antibody on in vitro LDL-LRP-2 binding (Fig. 3) and the fact that
LRP-2 antibodies block the increased
I-LDL uptake and
degradation in the treated cells (Fig. 4), indicates that LRP-2
interaction with apoB100 mediates the increased LDL clearance exhibited
by the treated cells.
cAMP. Shown are the amounts of I-LDL internalized (A) and degraded (B)
by normal F9 cells (open bars) and by F9 cells treated with
RA/Bt
cAMP (DBC) for 7 days (filled bars)
in the presence of RAP, LRP-2 antibody, LRP-1 antibody, and apoB100
antibody (mAb 4G3). All values depicted have been corrected by
subtraction of nonspecifically internalized or degraded LDL as
described under ``Materials and Methods.'' The data presented
are representative of five experiments, each performed in duplicate.
Each plotted value represents the average of duplicate determinations
with the range indicated by the bars.
(3.3
nM) than that reported for inhibition of LDL binding to LDLR
(140 nM (Medh et al., 1995)).
cAMP-differentiated F9 cells have been shown to have
characteristics consistent with parietal endoderm (Damjanov et
al., 1994). The observed LRP-2-mediated uptake of LDL by cultured
RA/BtcAMP-differentiated F9 cells may represent an
experimental model for the uptake of maternal LDL by embryonic
trophoblast and endodermal cells of the yolk sac placenta. In addition
to cells of the placenta, LRP-2 has been found to be expressed by a
number of other specialized epithelial cells including those of choroid
plexus, lung alveoli, and kidney proximal tubules (Kounnas et
al., 1994b; Zheng et al., 1994; Assmann et al.,
1986). Each of these epithelia is in contact with extravascular fluids
with LRP-2 localized on the apical surface of the cells, exposed to the
fluids. However, with the exception of cerebrospinal fluid of the adult
human, which contains low levels (0.77 mg/liter) of apoB (Carlsson et al., 1991), little or no information exists as to the LDL
content in these fluids.-VLDL (Willnow et al., 1992), apolipoprotein J (Kounnas et al.,
1995), prourokinase (Stefansson et al., 1995), plasminogen
activator inhibitor-1 (Stefansson et al., 1995), complexes of
tissue-type plasminogen activator or urokinase with plasminogen
activator inhibitor-1 (Willnow et al., 1992; Moestrup et
al., 1993; Stefansson et al., 1995), thrombospondin-1
(Godyna et al., 1995), lactoferrin (Willnow et al.,
1992), and RAP (Kounnas et al., 1992; Orlando et al.,
1992; Christensen et al., 1992). It is not obvious whether
there is a functional linkage among these ligands that accounts for
their having a common receptor. It seems that LRP-2, and also LRP-1,
can function in two major physiological arenas, lipoprotein metabolism
and proteinase regulation. It remains to be determined whether there
exists a link between these apparently distinct physiological processes
that could explain the evolution of a single class of receptors.
)-macroglobulin receptor low density lipoprotein
receptor-related protein; LRP-2, glycoprotein 330/low density
lipoprotein receptor-related protein-2; RA, retinoic acid;
BtcAMP, dibutyryl cyclic AMP; RAP, receptor-associated
protein; DMEM, Dulbecco's modified Eagle's medium; BSA,
bovine serum albumin; mAb, monoclonal antibody.)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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  S. Dugue-Pujol, X. Rousset, D. Chateau, D. Pastier, C. Klein, J. Demeurie, C. Cywiner-Golenzer, M. Chabert, P. Verroust, J. Chambaz, et al. Apolipoprotein A-II is catabolized in the kidney as a function of its plasma concentration J. Lipid Res., October 1, 2007; 48(10): 2151 - 2161. [Abstract] [Full Text] [PDF]
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 B. J. Spencer and I. M. Verma From the Cover: Targeted delivery of proteins across the blood-brain barrier PNAS, May 1, 2007; 104(18): 7594 - 7599. [Abstract] [Full Text] [PDF]
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 E. Allen, K. B. Piontek, E. Garrett-Mayer, M. Garcia-Gonzalez, K. L. Gorelick, and G. G. Germino Loss of polycystin-1 or polycystin-2 results in dysregulated apolipoprotein expression in murine tissues via alterations in nuclear hormone receptors Hum. Mol. Genet., January 1, 2006; 15(1): 11 - 21. [Abstract] [Full Text] [PDF]
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S. Yoshida and Y. Wada Transfer of maternal cholesterol to embryo and fetus in pregnant mice J. Lipid Res., October 1, 2005; 46(10): 2168 - 2174. [Abstract] [Full Text] [PDF]
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 P. J. Verroust and E. I. Christensen Megalin and cubilin--the story of two multipurpose receptors unfolds Nephrol. Dial. Transplant., November 1, 2002; 17(11): 1867 - 1871. [Full Text] [PDF]
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 R. A. McCarthy, J. L. Barth, M. R. Chintalapudi, C. Knaak, and W. S. Argraves Megalin Functions as an Endocytic Sonic Hedgehog Receptor J. Biol. Chem., July 5, 2002; 277(28): 25660 - 25667. [Abstract] [Full Text] [PDF]
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J. A. McConihay, P. S. Horn, and L. A. Woollett Effect of maternal hypercholesterolemia on fetal sterol metabolism in the Golden Syrian hamster J. Lipid Res., July 1, 2001; 42(7): 1111 - 1119. [Abstract] [Full Text] [PDF]
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 E. I. Christensen and H. Birn Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule Am J Physiol Renal Physiol, April 1, 2001; 280(4): F562 - F573. [Abstract] [Full Text] [PDF]
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S. M. Hammad, J. L. Barth, C. Knaak, and W. S. Argraves Megalin Acts in Concert with Cubilin to Mediate Endocytosis of High Density Lipoproteins J. Biol. Chem., April 14, 2000; 275(16): 12003 - 12008. [Abstract] [Full Text] [PDF]
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 H. BIRN, H. VORUM, P. J. VERROUST, S. K. MOESTRUP, and E. I. CHRISTENSEN Receptor-Associated Protein Is Important for Normal Processing of Megalin in Kidney Proximal Tubules J. Am. Soc. Nephrol., February 1, 2000; 11(2): 191 - 202. [Abstract] [Full Text] [PDF]
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E. I. CHRISTENSEN and T. E. WILLNOW Essential Role of Megalin in Renal Proximal Tubule for Vitamin Homeostasis J. Am. Soc. Nephrol., October 1, 1999; 10(10): 2224 - 2236. [Full Text]
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S. M. Hammad, S. Stefansson, W. O. Twal, C. J. Drake, P. Fleming, A. Remaley, H. B. Brewer Jr., and W. S. Argraves Cubilin, the endocytic receptor for intrinsic factor-vitamin B12 complex, mediates high-density lipoprotein holoparticle endocytosis PNAS, August 31, 1999; 96(18): 10158 - 10163. [Abstract] [Full Text] [PDF]
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E. I. CHRISTENSEN, J. O. MOSKAUG, H. VORUM, C. JACOBSEN, T. E. GUNDERSEN, A. NYKJ&Aelig;R, R. BLOMHOFF, T. E. WILLNOW, and S. K. MOESTRUP Evidence for an Essential Role of Megalin in Transepithelial Transport of Retinol J. Am. Soc. Nephrol., April 1, 1999; 10(4): 685 - 695. [Abstract] [Full Text]
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 A. Niemeier, T. Willnow, H. Dieplinger, C. Jacobsen, N. Meyer, J. Hilpert, and U. Beisiegel Identification of Megalin/gp330 as a Receptor for Lipoprotein(a) In Vitro Arterioscler. Thromb. Vasc. Biol., March 1, 1999; 19(3): 552 - 561. [Abstract] [Full Text] [PDF]
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S. Ranganathan, C. Knaak, C. R. Morales, and W. S. Argraves Identification of Low Density Lipoprotein Receptor-related Protein-2/megalin as an Endocytic Receptor for Seminal Vesicle Secretory Protein II J. Biol. Chem., February 26, 1999; 274(9): 5557 - 5563. [Abstract] [Full Text] [PDF]
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J. Hilpert, A. Nykjaer, C. Jacobsen, G. Wallukat, R. Nielsen, S. K. Moestrup, H. Haller, F. C. Luft, E. I. Christensen, and T. E. Willnow Megalin Antagonizes Activation of the Parathyroid Hormone Receptor J. Biol. Chem., February 26, 1999; 274(9): 5620 - 5625. [Abstract] [Full Text] [PDF]
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M. ZIAK, D. KERJASCHKI, M. G. FARQUHAR, and J. ROTH Identification of Megalin as the Sole Rat Kidney Sialoglycoprotein Containing Poly {alpha}2,8 Deaminoneuraminic Acid J. Am. Soc. Nephrol., February 1, 1999; 10(2): 203 - 209. [Abstract] [Full Text]
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 J Yochem, S Tuck, I Greenwald, and M Han A gp330/megalin-related protein is required in the major epidermis of Caenorhabditis elegans for completion of molting Development, January 2, 1999; 126(3): 597 - 606. [Abstract] [PDF]
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 M. Rudling, M. Gafvels, P. Parini, G. Gahrton, and B. Angelin Lipoprotein Receptors in Acute Myelogenous Leukemia : Failure to Detect Increased Low-Density Lipoprotein (LDL)Receptor Numbers in Cell Membranes despite Increased Cellular LDLDegradation Am. J. Pathol., December 1, 1998; 153(6): 1923 - 1935. [Abstract] [Full Text] [PDF]
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 M. K. Cooper, J. A. Porter, K. E. Young, and P. A. Beachy Teratogen-Mediated Inhibition of Target Tissue Response to Shh Signaling Science, June 5, 1998; 280(5369): 1603 - 1607. [Abstract] [Full Text]
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K. L. Wyne and L. A. Woollett Transport of maternal LDL and HDL to the fetal membranes and placenta of the Golden Syrian hamster is mediated by receptor-dependent and receptor-independent processes J. Lipid Res., March 1, 1998; 39(3): 518 - 530. [Abstract] [Full Text]
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S. M. Hammad, S. Ranganathan, E. Loukinova, W. O. Twal, and W. S. Argraves Interaction of Apolipoprotein J-Amyloid beta -Peptide Complex with Low Density Lipoprotein Receptor-related Protein-2/Megalin. A MECHANISM TO PREVENT PATHOLOGICAL ACCUMULATION OF AMYLOID beta -PEPTIDE J. Biol. Chem., July 25, 1997; 272(30): 18644 - 18649. [Abstract] [Full Text] [PDF]
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R. A. Orlando, M. Exner, R.-P. Czekay, H. Yamazaki, A. Saito, R. Ullrich, D. Kerjaschki, and M. G. Farquhar Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor-ligand interactions PNAS, March 18, 1997; 94(6): 2368 - 2373. [Abstract] [Full Text] [PDF]
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 P.A. Beachy, M.K. Cooper, K.E. Young, D.P. von Kessler, W.-J. Park, T.M. T. Hall, D.J. Leahy, and J.A. Porter Multiple Roles of Cholesterol in Hedgehog Protein Biogenesis and Signaling Cold Spring Harb Symp Quant Biol, January 1, 1997; 62(0): 191 - 204. [Abstract] [PDF]
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S. Swarnakar, J. Beers, D. K. Strickland, S. Azhar, and D. L. Williams The Apolipoprotein E-dependent Low Density Lipoprotein Cholesteryl Ester Selective Uptake Pathway in Murine Adrenocortical Cells Involves Chondroitin Sulfate Proteoglycans and an alpha 2-Macroglobulin Receptor J. Biol. Chem., June 8, 2001; 276(24): 21121 - 21128. [Abstract] [Full Text] [PDF]
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B. Garner, A. H. Merry, L. Royle, D. J. Harvey, P. M. Rudd, and J. Thillet Structural Elucidation of the N- and O-Glycans of Human Apolipoprotein(a). ROLE OF O-GLYCANS IN CONFERRING PROTEASE RESISTANCE J. Biol. Chem., June 15, 2001; 276(25): 22200 - 22208. [Abstract] [Full Text] [PDF]
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