(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 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.
Lipoprotein receptors comprise a family of proteins that are
structurally related to the low density lipoprotein (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).
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
(BtcAMP), 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.
Figure 1:
Immunoblot analysis of the expression
of members of the LDLR family in extracts of F9 cells treated with
RA/BtcAMP. 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/Bt
cAMP treatment.
Figure 2:
Assay of cellular internalization and
degradation of LDLR family member ligands or antibodies by F9 cells
treated with RA/BtcAMP. 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.
The two other LDLR family members that were detected in extracts of
F9 cells (untreated and RA/BtcAMP-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.
In
contrast to the observation that LDLR levels did not increase in
response to RA/BtcAMP 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.
Figure 3:
Binding of LRP-2 and LDL in solid-phase
binding assays. In A, 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.
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 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.
Figure 4:
LRP-2 antibody inhibits the increased
cellular uptake of LDL that occurs in F9 cells treated with
RA/BtcAMP. 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.
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 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.
Figure 5:
Monoclonal apoB100 antibody inhibits the
increased cellular uptake and degradation of LDL that occurs in F9
cells treated with RA/BtcAMP. 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.
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(3.3
nM) than that reported for inhibition of LDL binding to LDLR
(140 nM (Medh et al., 1995)).
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/BtcAMP-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/Bt
cAMP-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.
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 -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.