(Received for publication, June 7, 1994; and in revised form, September 26, 1994)
From the
The 39-kDa receptor-associated protein (RAP) is co-synthesized
and co-purifies with the low density lipoprotein receptor-related
protein (LRP)/-macroglobulin receptor and is thought
to modulate ligand binding to LRP. In addition to binding LRP, RAP
binds two other members of the low density lipoprotein (LDL) receptor
family, gp330 and very low density lipoprotein (VLDL) receptors. Here,
we show that RAP binds to LDL receptors as well. In normal human
foreskin fibroblasts, RAP inhibited LDL receptor-mediated binding and
catabolism of LDL and VLDL with S
20-60 or
100-400. RAP inhibited
I-labeled LDL and
S
100-400 lipoprotein binding at 4 °C
with K
values of 60 and 45 nM,
respectively. The effective concentrations for 50% inhibition
(EC
) of cellular degradation of 2.0 nM
I-labeled LDL, 4.7 nM
I-labeled S
20-60, and
3.6 nM
I-labeled S
100-400 particles were 40, 70, and 51 nM,
respectively. Treatment of cells with lovastatin to induce LDL
receptors increased cellular binding, internalization, and degradation
of RAP by 2.3-, 1.7-, and 2.6-fold, respectively. In solid-phase
assays, RAP bound to partially purified LDL receptors in a
dose-dependent manner. The dissociation constant (K
) of RAP binding to LDL receptors in
the solid-phase assay was 250 nM, which is higher than that
for LRP, gp330, or VLDL receptors in similar assays by a factor of 14
to 350. Also, RAP inhibited
I-labeled LDL and
S
100-400 VLDL binding to LDL receptors in
solid-phase assays with K
values of 140
and 130 nM, respectively. Because LDL bind via apolipoprotein
(apo) B100 whereas VLDL bind via apoE, our results show that RAP
inhibits LDL receptor interactions with both apoB100 and apoE. These
studies establish that RAP is capable of binding to LDL receptors and
modulating cellular catabolism of LDL and VLDL by this pathway.
The -macroglobulin receptor-associated protein
(RAP) (
)is a 39-kDa polypeptide that co-purifies with the
-macroglobulin (
M) receptor/low
density lipoprotein receptor-related protein (LRP)(1) . A
fraction of intracellular RAP associates with LRP immediately after its
synthesis and is transferred to the cell surface in a complex with LRP;
the remainder remains intracellular(2, 3) . RAP is not
secreted into the extracellular fluid and is not found in plasma. RAP
also co-purifies with gp330, another member of the LDL receptor
family(4, 5) . Mature human RAP consists of 323 amino
acids and has 73% identity with the rat Heymann nephritis antigen and
77% identity with a mouse heparin-binding protein called
HBP-44(2) . The carboxyl-terminal domain of RAP also has 26%
sequence identity with a region of apolipoprotein (apo) E containing
the LDL receptor binding domain(2) .
The physiological role
of RAP is not yet clear. It is well established that RAP inhibits the
interactions of LRP with all of its known ligands including
M-proteinase complexes(6, 7) ,
apoE-enriched
-migrating very low density lipoproteins
(
-VLDL)(6) , lipoprotein lipase(8, 9) ,
complexes of tissue-type and urokinase plasminogen activators with
their
inhibitor(10, 11, 12, 13, 14) ,
and Pseudomonas exotoxin A(15) . In addition,
fibroblasts genetically engineered to overexpress RAP also overexpress
LRP. (
)Thus, it is believed that RAP modulates LRP-ligand
interactions and may function in the intracellular transport of LRP as
well. Kounnas et al. (3) have shown that RAP also
binds gp330 with a high affinity and may modulate interactions of gp330
with its ligands(3, 16) .
Because RAP binds to LRP (17, 18) and gp330(3, 19) , both members of the LDL receptor family, we speculated that RAP might also bind LDL receptors themselves. Here, we demonstrate that RAP binds to LDL receptors both in normal human foreskin fibroblasts and solid-phase binding assays. Our findings suggest that RAP can modulate LDL receptor-mediated binding and catabolism of normal LDL and VLDL.
In some experiments, bound ligands were detected by an enzyme-linked immunoabsorbent assay. Wells were coated with 0-10 µg of RAP for 5 h at 4 °C, blocked with 1% BSA for 2 h at 4 °C, washed, and incubated for 16 h at 4 °C with 100 µl of buffer containing 1 µg/µl DE52 eluant. The wells were washed again and incubated for 2 h at 4 °C with 100 µl of buffer containing 30 µg/ml IgG-C7. Bound IgG-C7 was quantitated by incubating the wells for 2 h at 4 °C with 100 µl of a 1:10,000 dilution of alkaline phosphatase-conjugated rabbit anti-mouse IgG (Sigma). Then, wells were washed and 100 µl of substrate (1 mg/ml p-nitrophenylphosphate, Sigma) was added. After 20 min at 25 °C, absorbance at 405 nm was measured. Standard curves prepared simultaneously by coating wells with known amounts of IgG-C7 were linear (correlation coefficients >0.98).
Figure 1:
RAP
inhibits cell-surface binding of I-labeled LDL and
S
100-400 to human fibroblasts at 4 °C.
Fibroblasts were treated with lipoprotein-deficient serum and
lovastatin to up-regulate their LDL receptor number as described under
``Experimental Procedures.'' Cells were then incubated for 3
h at 4 °C in media containing 3 nM of either
I-labeled LDL (A) or
I-labeled
S
100-400 VLDL (B) in the presence
of increasing concentrations of RAP (closedcircles)
or LDL (opentriangles). After washing as described,
bound radioactivity was dissociated by incubating cells for 30 min at 4
°C with buffer containing 10 mg/ml polyphosphate. The moles of
bound ligand were calculated from the radioactivity released. The curves represent the best fit of the data to a single class of
sites using K
values of 2.4 and 2.0
nM for
I-labeled LDL and
I-labeled
S
100-400 particles,
respectively.
Figure 2:
RAP competes for I
lipoprotein degradation by human fibroblasts. Cells were incubated for
5 h at 37 °C with media containing 2.0 nM
I-labeled LDL (opencircles), 4.7
nM
I-labeled S
20-60
VLDL (closedcircles), or 3.6 nM
I-labeled S
100-400 VLDL (opentriangles) in the presence of various
concentrations of RAP. Degradation was measured as described under
``Experimental Procedures.''
Figure 3:
Lovastatin treatment increases I-labeled RAP catabolism by fibroblasts. Cells were
treated either with lipoprotein-deficient serum and lovastatin (closedcircles) as described under
``Experimental Procedures'' or maintained in
lipoprotein-containing media (opencircles). They
were then incubated for 5 h at 37 °C in the presence of various
concentrations of
I-labeled RAP. Degradation was
calculated as described under ``Experimental Procedures.''
The data are corrected for cellular protein content in each
well.
Fig. 4A shows that
increasing amounts of I-labeled RAP bound to increasing
amounts of LDL receptors immobilized to plastic wells coated with
IgG-C7. In three separate experiments, specific binding of
I-labeled RAP directly correlated with the amount of
immobilized receptor. To verify these results, we also measured binding
of unlabeled RAP using the
I-labeled monoclonal anti-RAP
antibody, IgG-7F1 (Fig. 4A). Like
I-labeled LDL binding,
I-labeled RAP
binding to LDL receptors was EDTA sensitive (data not shown). However,
1 mM EDTA was sufficient to totally inhibit
I-labeled LDL binding, whereas 10 mM EDTA was
required to completely eliminate
I-labeled RAP binding.
Figure 4:
RAP
binding to immobilized LDL receptors in a solid-phase assay. Microtiter
wells were first coated with IgG-C7 and blocked with BSA as described
under ``Experimental Procedures'' and then incubated with
various concentrations of DE52 eluant of the whole cell extract for 16
h at 4 °C. The wells were then incubated for 3 h at 4 °C with
buffer containing (A) either 13 nMI-labeled RAP (closedcircles) or
260 nM unlabeled RAP (opentriangles) or (B) 9.8 nM
I-labeled LDL (closedcircles) or 3.9 nM
I-labeled
M* (opentriangles). Unlabeled RAP
was detected by incubating with 3.3 nM
I-labeled
7F1 for 3 h at 4 °C. Nonspecific binding to BSA-coated wells was
subtracted from total binding.
We also measured I-labeled
M* binding
to immobilized LDL receptors (Fig. 4B). Whereas
I-labeled LDL bound in a dose-dependent manner,
I-labeled
M* failed to bind proteins
immobilized by anti-LDL receptor antibodies. These data exclude a
significant component of LRP binding in these assays. The ligand
binding capacity of partially purified receptors varied among different
preparations and decreased with storage at -70 °C. Thus, it
is not appropriate to compare the stoichiometry of
I-labeled RAP binding in Fig. 4A with
that of
I-labeled LDL binding in Fig. 4B.
DE52 eluants prepared from fibroblasts lacking LDL receptors bound RAP
at 50-60% lower levels than did eluants from normal fibroblasts
under identical assay conditions (data not shown), indicating that LDL
receptors were responsible for a majority of RAP binding.
To further confirm the interaction of RAP with LDL receptors in solid-phase assays, we coated plastic wells with increasing amounts of RAP and allowed LDL receptors in the DE52 eluant to bind (Fig. 5). Bound LDL receptors were detected using IgG-C7, a monoclonal antibody against the LDL receptor. Receptor binding was directly proportional to the amount of RAP immobilized. Negligible amounts of IgG-C7 bound in the absence of DE52 eluant, providing evidence for the specificity of binding.
Figure 5: LDL receptor binding to immobilized RAP in a solid-phase assay. Microtiter wells were coated with various amounts of RAP, blocked, and incubated in the presence (closedcircles) or absence (opentriangles) of DE52 eluant as described. Bound LDL receptors were quantitated using the anti-LDL receptor antibody IgG-C7.
We determined the K for RAP binding to
LDL receptors in the solid-phase assay. Immobilized LDL receptors were
incubated with increasing concentrations of RAP, and the amount of RAP
bound was determined using
I-labeled 7F1, an anti-RAP
monoclonal antibody. Fig. 6A shows the data obtained in
a representative experiment. The best fit of this data to a single-site
model predicted a K
of 250 nM for RAP
binding to LDL receptors. Fig. 6B shows inhibition of
I-labeled RAP binding to LDL receptors in the solid-phase
assay by unlabeled RAP (K
= 290
nM).
Figure 6:
Affinity of RAP binding to LDL receptors
in a solid-phase assay. Microtiter wells coated with IgG-C7 were used
to immobilize LDL receptors from DE52 eluants. The wells were then
incubated for 3 h at 4 °C with buffer containing either (A) increasing concentrations of unlabeled RAP followed by
another incubation with 3.3 nMI-labeled 7F1 as
in Fig. 4A or (B) 13 nM
I-labeled RAP in the presence of various
concentrations of unlabeled RAP. Curve fitting to a single-site model
is shown.
Ligand blotting to DE52 eluants was attempted but required micromolar concentrations of RAP to visualize LDL receptors. Under these conditions, background binding was much increased as was binding to several unidentified proteins in both normal fibroblasts and those lacking LDL receptors (data not shown).
Figure 7:
RAP competes for I
lipoprotein binding to LDL receptors in a solid-phase assay. LDL
receptors from DE52 eluants were immobilized onto IgG-4A4-coated
microtiter wells. The wells were then incubated with buffer containing (A) 1 nM
I-labeled LDL or (B)
2.1 nM
I-labeled S
100-400 particles in the presence of various concentrations
of unlabeled RAP as described under ``Experimental
Procedures.'' Curve fitting to a single-site model is
shown.
These data establish that RAP inhibits both LDL and VLDL
binding and catabolism by normal skin fibroblasts. We confirmed the
interaction of RAP with LDL receptors by several independent methods
using solid-phase assays. RAP bound to LDL receptor-coated wells, and
LDL receptors bound to RAP-coated wells. Also, RAP inhibited
lipoprotein binding to partially purified LDL receptors and vice
versa. It is unlikely that interaction of RAP with other members
of the LDL receptor family confounds our findings. Gp330 and VLDL
receptors are not expressed in normal fibroblasts to a significant
degree, and VLDL receptors do not bind LDL, as was observed in our
assays. Although LRP is present in fibroblasts, previously published
data show that neither LRP nor gp330 binds normal lipoproteins in the
absence of exogenously added lipoprotein lipase or
apoE(8, 25, 33, 34, 35, 37) .
The absence of M* binding in the solid-phase assay
eliminates the possibility of a significant component of LRP-mediated
binding. Also, induction of LDL receptors with lovastatin induced
catabolism of RAP, which would not be expected with other members of
the LDL receptor family. Finally, the affinity of RAP for other members
of the LDL receptor family is substantially higher than that reported
here.
Because LDL and VLDL bind LDL receptors via apoB100 and apoE,
respectively, RAP may share binding sites for these ligands. Studies by
Russell et al.(38, 39) and others suggest
that repeats 3-7 of the LDL receptor are required for binding
apoB100, whereas repeat 5 is sufficient for binding apoE. RAP may
prevent lipoprotein binding by inducing a conformational change in the
LDL receptor. It is also possible that a single LDL receptor may bind
more than one RAP molecule by direct interaction with more than one
ligand-binding repeat. A definitive demonstration of the binding
stoichiometry has not been done. The affinity of RAP for LDL receptors
(250 nM) is less than that predicted from its ability to
inhibit lipoprotein binding to LDL receptors on cells (50
nM) and in solid-phase assays (
140 nM). We
currently have no explanation for these differences. However, they
suggest that the interaction of RAP with LDL receptors is stronger in
the presence of lipoproteins.
Both LDL receptors and LRP contribute
to catabolism of RAP by normal fibroblasts. At a concentration of RAP
greater than saturation for LRP but below saturation for LDL receptors,
a polyclonal antibody against LRP inhibited 40% of
I-labeled RAP degradation by normal fibroblasts. This
suggests the existence of an LRP-independent pathway. When LDL
receptors were up-regulated with lovastatin, catabolism of
I-labeled RAP was increased by
2.5-fold (Fig. 3), despite the fact that catabolism of LDL increased
>20-fold. These results suggest that both LDL receptors and LRP
contribute to the total cellular catabolism of RAP. Consistent with
this idea, RAP inhibited the catabolism of
I-labeled
lipoproteins, but the reverse is not true (data not shown). Although we
cannot completely explain this finding, it partially reflects the fact
that native lipoproteins cannot bind LRP and therefore would not
inhibit LRP-mediated catabolism of RAP. Another factor to consider is
that RAP is a heparin-binding protein (2) and conceivably could
interact with cell-surface proteoglycans, an interaction that may not
be blocked by lipoproteins.
RAP is the first new ligand for the LDL
receptor since the discovery that apoB100 and apoE bind to this
receptor(38, 40) . Previous studies establish that RAP
binds to two other members of the LDL receptor family, LRP (K = 18 nM) and gp330 (K
= 8
nM)(3, 17, 18, 19) .
Recently, Battey et al. (41) found that RAP also binds
to the VLDL receptor (K
= 0.7 nM).
The affinity of RAP for LDL receptors in solid-phase assays is lower by
a factor of 14-350 than that for LRP, gp330, or the VLDL receptor (41) and, in contrast to these receptors, is too low to permit
ligand blotting. The LDL receptor family members share several regions
of homology, particularly the ligand binding and growth factor-type
repeats(42) . It is known that RAP binds to a cluster of 8
complement-type repeats in LRP(43) , and it is likely that a
similar region in the LDL receptor is responsible for RAP binding.
Williams et. al(18) found that a single LRP may bind
two RAP molecules, supporting the notion that more than one
ligand-binding repeat interacts with RAP. RAP contains a region (amino
acids 203-321) with 26% sequence homology to apoE (2) that could form an amphipathic helix homologous to the
secondary structure of apoE known to be involved in apoE-LDL receptor
interactions(44, 45) . This region of RAP has been
shown to inhibit the interactions of LRP with its ligand (46) and may be responsible for interactions with other LDL
receptor family members.
The physiological significance of an
interaction between RAP and LDL receptors is currently unknown. Our
results suggest that RAP may modulate lipoprotein catabolism in
vivo. In a recent study, Mokuno et al.(47) show
that intravenous injection of glutathione-S-transferase-RAP
into rats reduces the clearance of human I-labeled LDL
from the blood. This is consistent with a regulatory role for RAP in
lipoprotein catabolism via LDL receptors. RAP is present both
intracellularly and on the cell surface but is not
secreted(2) . Conceivably, RAP could act as a chaperone to
assist proper LRP and LDL receptor folding and transport to the cell
surface. On the other hand, intracellular RAP may promote receptor
recycling and uncoupling from endocytosed ligands. RAP exists in
specialized intracellular compartments, in which the affinity for LDL
receptors may be dependent on its local concentration, proximity to
membranes, local pH, and calcium concentration. Clearly, more studies
are needed to address these possibilities. However, our results and
those of Mokuno et al. (47) demonstrate that RAP may
not be an appropriate experimental reagent to discriminate between LDL
receptor and LRP-mediated phenomena.