(Received for publication, May 4, 1995; and in revised form, July 13, 1995)
From the
The endocytic -macroglobulin receptor/low
density lipoprotein receptor-related protein (
MR/LRP)
binds several classes of extracellular ligands at independent sites. In
addition,
MR/LRP can bind multiple copies of the
39-40-kDa receptor-associated protein (RAP). Both amino-terminal
and carboxyl-terminal fragments of RAP exhibit affinity, and the
fragments apparently bind to different sites on the receptor. RAP
completely inhibits the binding of all presently known extracellular
ligands, whereas several ligands such as
-macroglobulin and tissue-type plasminogen activator
are poor inhibitors of RAP binding. Since RAP is largely an
intracellular molecule that normally does not occupy
MR/LRP at the cell surface, we hypothesized that an
established extracellular ligand might bind to those sites on the
receptor capable of binding the RAP fragments. We found complete
cross-competition between carboxyl-terminal RAP fragments and fragments
of lipoprotein lipase containing the recently identified binding domain
for
MR/LRP (Nykjær, A., Nielsen, M., Lookene,
A., Meyer, N., Røigaard, H., Etzerodt, M., Beisiegel, U.,
Olivecrona, G., and Gliemann, J.(1994) J. Biol. Chem. 269,
31747-31755). Moreover, the lipoprotein lipase fragment
completely inhibited the binding of several
MR/LRP
ligands in a pattern similar to that of carboxyl-terminal RAP
fragments. On the other hand, the amino-terminal RAP fragment was a
poor competitor of binding of the lipoprotein lipase fragment, whereas
it competed effectively with pro-uPA for binding to the receptor. The
results provide evidence that lipoprotein lipase binds to the site on
MR/LRP also available for binding of the
carboxyl-terminal domain of RAP and suggest that pro-uPA may bind to or
overlap the site available for the amino-terminal domain of RAP.
The -macroglobulin receptor/low density
lipoprotein receptor-related protein (
MR/LRP) (
)is a
600-kDa endocytic receptor that is translated as
a single polypeptide and cleaved in the Golgi network into an 85-kDa
membrane-spanning chain and a large extracellular chain that remains
attached to the 85-kDa chain via noncovalent linkages. The
extracellular chain contains multiple copies of two types of
cysteine-rich repeats, the growth factor repeats and the
complement-type repeats, that provide binding sites for several
structurally unrelated ligands. The complement-type repeats are
clustered in four regions containing two, eight, 10, and 11 repeats.
The ligands include the receptor-binding form of
-macroglobulin (
M*), apolipoprotein
E-containing lipoproteins, lactoferrin, tissue-type plasminogen
activator (tPA), pro-urokinase (pro-uPA), complexes of the type-1
plasminogen activator inhibitor (PAI-1) and tPA as well as uPA, and
lipoprotein lipase (LpL), both free and associated with lipoproteins
(for reviews, see (1, 2, 3, 4) ).
Several ligands do not compete with one another for binding to
MR/LRP. For example,
M* does not
compete with tPA or lactoferrin for binding to
MR/LRP(5, 6) , and
M* is a poor inhibitor of binding of apolipoprotein
E-containing lipoproteins (6, 7, 8) .
However, lactoferrin is able to inhibit the binding of apolipoprotein
E-containing lipoproteins to
MR/LRP, suggesting that
these ligands may bind to the same site(6) .
The
39-40-kDa receptor-associated protein (RAP) was first identified
as a component copurifying with MR/LRP prepared from
human placenta(9, 10, 11) . Subsequent
cloning and sequencing (12) revealed that it is the human
homologue of mouse heparin binding protein 44 (13) and of rat
Heymann nephritis target protein(14) . RAP was first shown to
inhibit the binding of
M* to the
receptor(15) , and several subsequent studies have demonstrated
that RAP, surprisingly, is capable of inhibiting the binding and uptake
of all known ligands to
MR/LRP in vitro and in vivo(1, 2, 3, 4, 16) . On
the other hand, most ligands have been reported to compete poorly for
binding of RAP to
MR/LRP. For instance, tPA and
M* at high concentrations cause little inhibition of
binding of labeled RAP to the receptor(2, 5) . This
lack of reciprocal cross-inhibition is presumably due to the complex
mode of binding of RAP to the receptor.
RAP binds with high affinity
to MR/LRP, and the capacity of the receptor has been
reported to be two (17) or up to six (18) RAP
molecules per receptor molecule. Previous studies have identified
strong binding of labeled RAP to fragments of
MR/LRP
containing the cluster of eight complement-type
repeats(19, 20) . In addition, most of the above cited
extracellular ligands are also bound in this region. Binding of RAP,
although weak, has also been observed to a domain containing the
cluster of 11 complement-type repeats(20) . It is uncertain
whether the stronger binding to the former domain is simply due to a
higher affinity or to multiple binding sites within that domain. In
addition to
MR/LRP, recent results have shown that RAP
also binds to the other known members of the LDL receptor family, i.e. gp330/megalin(21, 22) , VLDL
receptor(23, 24) , and LDL receptor(25) .
The finding that RAP may function as a universal antagonist has led
to the hypothesis that it may modulate the function of LDL receptor
family members, particularly MR/LRP. However, whereas
MR/LRP is primarily confined to clathrin-coated pits
in the plasma membrane and within endosomal
vesicles(26, 27) , RAP is largely an intracellular
protein located in the endoplasmic reticulum and the Golgi
complex(27, 28, 29) . The function of RAP in
those compartments is presently unknown, although it has been
hypothesized that RAP may prevent binding of other ligands to the
multifunctional receptors before they reach the plasma
membrane(29, 30, 31) . Consistent with this,
RAP is only associated with
MR/LRP at the plasma
membrane to a small extent, if at all(27, 32) .
Furthermore, attempts to detect RAP in conditioned media of cell
incubations and in the blood have repeatedly been negative. Since
MR/LRP is normally unoccupied by RAP at the cell
surface, we considered the possibility that the RAP sites might bind
one of the established extracellular ligands. Thus, we decided to
investigate whether LpL might bind to those sites since we have
previously demonstrated that LpL, like RAP, binds to multiple sites on
the receptor(33) , and since it was recently reported that LpL
and the carboxyl-terminal folding domain of LpL inhibit binding of RAP
to
MR/LRP(34) . In the present analysis, we
made use of a peptide containing the receptor binding site of
LpL(35) . Since amino-terminal and carboxyl-terminal RAP
fragments can interact with separate sites on
MR/LRP(36) , it became important to analyze
the competition of the receptor-binding LpL fragment with both
amino-terminal and carboxyl-terminal RAP fragments(37) . We
find that the LpL fragment and the carboxyl-terminal RAP fragment
exhibit complete reciprocal cross-inhibition and that the LpL fragment
can inhibit the binding of several established ligands for
MR/LRP.
Fusion proteins of glutathione S-transferase with RAP or RAP fragments (GST-RAP constructs) were expressed and purified as described previously(37) . Briefly, polymerase chain reaction was used to amplify specific sequences of the RAP cDNA, which were ligated into the pGEX2T expression vector. Proteins expressed in E. coli were isolated and purified via affinity chromatography on glutathione-agarose. RAP-(1-114) and RAP-(115-319) were purified from the thrombin-treated GST constructs as described previously(37) . Detailed characterization of all constructs has been described(36, 37, 45) . The purity of all recombinant proteins was confirmed by SDS-polyacrylamide gel electrophoresis and staining with Coomassie Brilliant Blue.
In some
experiments, we used recombinant human RAP with the fusion tail
(HFX) cleaved off by factor X
(44) as
the labeled species. Separate experiments showed that the affinities
for binding of human RAP and rat RAP without fusion proteins to
purified
MR/LRP were indistinguishable. In addition,
the concentrations of rat RAP and GST-RAP required to cause
half-maximal inhibition of binding of
I-labeled human RAP
were similar, with EC
values ranging between 0.1 nM and 0.4 nM.
Figure 1:
Competition by LpL and
the receptor-binding fragment of LpL for binding of RAP to immobilized
MR/LRP. A, purified
MR/LRP
(about 100 fmol/well) was immobilized in microtiter wells, and
incubations (100 µl) were performed for 16 h at 4 °C with about
10 pM
I-labeled human RAP (40,000 cpm/ml) and
varying concentrations of dimeric bovine LpL
(
--
) or the fragment of human LpL containing
amino acids 378-448 (LpL-(378-448))
(
--
). Fifty percent of the added
I-RAP was bound to the immobilized receptor in the
absence of unlabeled competitor, and this maximal binding was set at
100%. The blank value (<1% bound tracer) determined in the presence
of 10 mM EDTA was subtracted from all other values. B, bovine LpL (about 10 fmol/well) was immobilized in
microtiter wells and incubated (16 h, 4 °C) with about 10 pM
I-
MR/LRP and varying concentrations
of LpL-(378-448). Fifty-five percent of the added
I-
MR/LRP was bound to the immobilized
LpL in the absence of unlabeled competitor and was set at 100%. The
blank value (<1% bound tracer) was subtracted from all other values.
The results show the mean values of triplicates ± 1
S.D.
Figure 2:
Competition by the receptor-binding LpL
fragment for binding and degradation of RAP in cells. Upper
panel, COS-1 cells (3.5 10
cells/well) were
incubated with about 20 pM
I-RAP for 16 h at 4
°C without additions, with 400 nM unlabeled RAP, or with 2
µM LpL-(347-448). The results show the cell-bound
I-RAP in percentage of the amount of tracer added to the
incubations. Lower panel, COS-1 cells (3.5
10
cells/ml) were incubated for 4 h at 37 °C with additions as
described in the upperpanel, followed by measurement
of the cell-bound (openbars), trichloroacetic
acid-precipitable (hatchedbars), and trichloroacetic
acid-soluble radioactivity (filledbars) in the
incubation medium. The results are the mean values of triplicate
incubations ± 1 S.D.
Figure 3:
Competition by the LpL fragment for
binding of MR/LRP to amino- and carboxyl-terminal RAP
fragments. About 25 pmol of the indicated GST-RAP constructs were
slot-blotted onto polyvinylidene difluoride membranes and incubated for
16 h at 4 °C with about 30 pM
I-
MR/LRP (100,000 cpm/ml) in the absence
or presence of 1.4 µM LpL-(347-448), followed by
autoradiography. The binding of
I-
MR/LRP
to the various constructs was measured by laser scanning densitometry,
and the results of this and analogous experiments are summarized in Table 1. The weak residual reaction observed with
GST-RAP-(1-100) and with all GST-RAP constructs in the presence
of LpL-(347-448) was not different from that observed with
polyvinylidene difluoride membranes in the absence of GST-RAP
constructs.
To
answer this question, we first analyzed the abilities of the GST
constructs containing the amino-terminal and the carboxyl-terminal RAP
fragments, and their truncated variants, to compete with binding of I-labeled full-length RAP to immobilized
MR/LRP. As shown in Table 1, unlabeled
GST-RAP-(115-319) was nearly as effective a competitor as
unlabeled GST-RAP-(1-319). The truncated variants of the
carboxyl-terminal RAP fragment, e.g. RAP-(187-319), also
competed effectively, with EC
values of approximately 2
nM. On the other hand, the NH
-terminal fragment
and its truncated variants, which bound to the receptor approximately
as effectively as GST-RAP-(187-319), were poor competitors for
binding of
I-labeled full-length RAP, with EC
values of 200 nM or higher. Since the LpL fragment and
the carboxyl-terminal RAP fragment both competed effectively with
full-length RAP for binding to
MR/LRP, the possibility
arose that they might bind to the same site on the receptor.
To
further test this hypothesis, the amino- and carboxyl-terminal GST-RAP
fragments were I-labeled and incubated with
MR/LRP immobilized in microtiter wells. Table 2shows that the binding of
I-labeled
GST-RAP-(1-114) was effectively competed for by unlabeled
GST-RAP-(1-114) and, as expected, by the carboxyl-terminal
fragments. In fact, the inhibitory potencies of GST-RAP-(115-319)
and GST-RAP-(187-319) toward binding of
I-labeled
GST-RAP-(1-114) on the one hand, and
I-labeled
GST-RAP-(115-319) on the other, were of similar magnitudes. By
contrast, GST-RAP-(1-114) was a poor inhibitor of binding of the
I-labeled carboxyl-terminal RAP fragments. Thus, the
receptor-binding fragment of LpL showed a pattern similar to the
carboxyl-terminal RAP fragments in the sense that the inhibitory
potencies were similar toward binding of the labeled amino- and
carboxyl-terminal fragments. In additional experiments, the LpL
fragment was immobilized in microtiter plates and incubated with
I-labeled
MR/LRP with or without
GST-RAP-(115-319) or GST-RAP-(1-114), or the corresponding
RAP fragments produced by thrombin cleavage of the GST constructs. As
shown in Fig. 4, the inhibitory potencies of
GST-RAP-(115-319) and RAP-(115-319) were similar to that of
RAP, although the GST moiety appeared to cause a slight increase in the
inhibitory potency of the carboxyl-terminal domain. The potency of
GST-RAP-(1-114) was lower than that of GST-RAP-(115-319),
and RAP-(1-114) caused inhibitions only at very high
concentrations (i.e. its inhibitory potency was much lower
than that of RAP-(115-319)). We conclude from the combined data
that the receptor-binding domain of LpL and the carboxyl-terminal RAP
fragment cross-compete, indicating that they bind to the same or
strongly overlapping sites on
MR/LRP. In addition,
like the carboxyl-terminal RAP fragment(36) , the LpL fragment
is an effective competitor for binding of the amino-terminal RAP
fragment to the receptor, whereas the amino-terminal RAP fragment is a
poor competitor for binding of both the carboxyl-terminal RAP fragment
and the LpL fragment.
Figure 4:
Competition by carboxyl- and
amino-terminal RAP fragments for binding of MR/LRP to
the LpL fragment. LpL-(348-478) was immobilized in microtiter
wells (about 500 fmol/well) and incubated for 16 h at 4 °C with
about 30 pM
I-
MR/LRP (100,000
cpm/ml) in the absence or presence of the indicated concentrations of
unlabeled RAP (leftpanel), RAP-(115-319) (middlepanel), and RAP-(1-114) (rightpanel) or the GST-constructs of the RAP fragments (filled bars). The binding of
I-
MR/LRP in the absence of unlabeled
competitor (maximal binding) was set at 100% and ranged in individual
experiments from 11 to 25% of the added tracer. The bars represent the mean values of
triplicates.
Figure 5:
Inhibition by the LpL fragment and LpL of
the binding of established MR/LRP ligands. A,
MR/LRP immobilized in microtiter wells was incubated
for 16 h at 4 °C with
I-pro-uPA
(
--
),
I-uPA
PAI-1
(
--
), and
I-
M*
(
--
), all at approximately 10 pM, and
varying concentrations of LpL-(378-448). The amount of
immobilized receptor was about 150 fmol/well when using
I-pro-uPA and
I-
M*, and
about 15 fmol/well when using
I-uPA
PAI-1. The
amount of tracer bound in the absence of unlabeled competitor was 12%
for
I-
M*, 15% for
I-uPA
PAI-1, and 5% for
I-pro-uPA. The
binding of each of the tracers in the absence of the LpL fragment was
set at 100%. The calculated EC
values for the LpL fragment
were as follows:
I-uPA, 54 nM;
I-uPA
PAI-1, 35 nM;
I-
M*, 214 nM. B, the
experiment was performed as in panelA using
I-tPA (
,
) or the
I-tPA
PAI-1 complex (
--
).
The amount of immobilized receptor was about 150 fmol/well when using
tPA and about 15 fmol/well when using the tPA
PAI-1 complex. The
amount of tracer bound in the absence of competitor was 5% for
I-tPA and 19% for
I-tPA
PAI-1. The
competitor was either bovine LpL (opensymbols) or
LpL-(378-448) (closedsymbols). The results are
the means of triplicate values.
The present results show that the binding of RAP can be
completely inhibited by peptides containing the receptor-binding domain
of LpL. This strongly suggests interaction of the LpL fragment with an
important site on the receptor also available for binding of RAP. In
addition, the peptide blocked the MR/LRP-mediated
uptake of RAP in cells. Furthermore, it became important to determine
the site specificity in view of the separate sites on the receptor for
binding of amino and carboxyl-terminal RAP fragments(36) .
Since amino-terminal RAP fragments were poor competitors for binding of
both carboxyl-terminal RAP fragments and the LpL fragment, whereas
carboxyl-terminal RAP fragments and the LpL fragment cross-competed
completely, the data indicate binding of LpL and the carboxyl-terminal
RAP fragment to the same site or to strongly overlapping sites.
The
COOH-terminal regions of LpL and RAP exhibit about 29% sequence
identity when allowing for several gaps(34) , but sequence
alignment provides no immediate clue as to where interaction with the
receptor may occur. Positively charged residues have been shown to be
important for binding of several ligands to
MR/LRP(2, 51) , and both the LpL
fragment and the carboxyl-terminal RAP fragment contain clusters of
basic residues. We suggest that both fragments achieve affinity by
interacting with the receptor via two patches containing such clusters.
For the LpL fragment, these may include residues 379-383 (KLKWK
in human LpL) and residues 403-407 (KIRVK in human LpL), the
former being supported by the observation that amino acids
378-391 are necessary for binding (35) and the latter by
mutational studies(34, 35) . For binding of the RAP
fragment, it is likely that amino acids 200-203 (RLRR in rat and
human RAP) (37) participate in the binding together with
several more COOH-terminally located basic residues (e.g. Lys
, Lys
, Lys
,
Lys
, Lys
, Lys
, Lys
in the rat sequence). The COOH-terminal residues 312-319
are important determinants for the inhibitory pattern since
RAP-(187-311) is noninhibitory toward
M* and tPA
binding (37) even though it binds to
MR/LRP
and inhibits pro-uPA binding with about the same affinity as
RAP-(187-319) (Table 1). Further COOH-terminal truncation
including residue 305 results in loss of binding affinity ((37) , Table 1). This may be due to a disruption of the
structure of the carboxyl-terminal domain, although the possibility of
direct participation of residues 305-311 in the binding cannot be
excluded.
RAP binds strongly to the domain on
MR/LRP containing a cluster of eight complement-type
repeats (cluster 2) and more weakly to the cluster containing 11
complement-type repeats (cluster 4)(20) . Unfortunately, it is
not known whether the strong binding to the cluster 2 domain is due to
high affinity or to a large number of sites. The finding of Iadonato et al.(18) that the calculated number of RAP sites on
MR/LRP of hepatoma cells is about 6 times higher than
the number of tPA sites argues for the presence of multiple RAP sites
in the cluster 2 domain. Within the RAP sites, nonreciprocal inhibition
occurs between RAP fragments since RAP-(115-319) inhibits binding
of RAP-(1-114), whereas the reverse inhibition is incomplete and
only occurs with a very low inhibitory potency. This pattern may be
explained by a close juxtaposition of the sites for binding of the
carboxyl- and amino-terminal RAP domains, with steric hindrance for
binding of the latter provided by residues in the former. Such a model
may also explain the ability of the carboxyl-terminal RAP fragment to
inhibit the binding of pro-uPA as a putative ligand for the site on
MR/LRP that binds the amino-terminal RAP domain.
Interestingly, the bulky GST moiety can induce or strengthen the
inhibitory properties of the amino-terminal RAP domain ((37) , Fig. 4), probably due to steric hindrance of ligand binding to
neighboring sites.
It is established that M*, tPA,
pro-uPA, and complexes of PAI-1 and uPA or tPA bind in the region
containing the cluster 2 domain(19, 20, 52) .
The inhibition of
M* and tPA by carboxyl-terminal RAP
fragments is most likely due to steric hindrance. Residues
312-319 appear particularly important for the pattern of
inhibition since RAP-(187-311) is noninhibitory toward binding of
M* and tPA and since the removal of even a single
residue (leucine 319) alters the inhibitory pattern without changing
the binding affinity(45) . Residues 312-319 should
therefore be important for the putative function of RAP in preventing
binding of coexpressed ligands in the endoplasmic reticulum of e.g. hepatocytes(29, 30, 31) . The LpL
fragment appears to inhibit ligand binding in a manner similar to that
of the carboxyl-terminal RAP fragment with deleted leucine 319, i.e. inhibition of
M* binding but not tPA
binding(45) . It is remarkable that LpL itself (Fig. 5B) and the LpL fragment combined with a large
fusion partner inhibit tPA binding as well.
Although not proven, the
present data suggest that pro-uPA may bind to the sites available for
the amino-terminal RAP domain. In addition, complexes such as
uPAPAI-1 with binding patches both in the uPA and the PAI-1
moieties (40) may bind in a mixed fashion involving multiple
sites on
MR/LRP that can also interact with amino- or
carboxyl-terminal RAP fragments. Thus,
MR/LRP deserves
designation as a ``molecular flypaper'' even though this term
was first coined for another important endocytic receptor, the
macrophage scavenger receptor, that binds a multitude of negatively
charged ligands(1) .
RAP also binds to the homologous giant
receptor gp330/megalin(21, 22) , also known to bind
lipoprotein lipase(6, 53) . It is therefore
anticipated that the LpL fragment should block the binding of RAP to
gp330, and this result ()has in fact been obtained in
experiments analogous to that shown in Fig. 1A. In
addition, RAP has recently been shown to bind to the smaller members of
the LDL receptor family, the VLDL receptor that binds RAP with high
affinity(23, 24) , and the LDL receptor that exhibits
somewhat lower binding affinity(25) . It is therefore probable
that these receptors bind LpL as well.
Since RAP normally occupies
these receptors expressed at the cell surface to at most only a small
extent(27, 32) , we suggest that LpL is one of the
natural extracellular ligands for the multiple sites on
MR/LRP that also bind carboxyl-terminal RAP fragments.
Physiologically, this may imply that binding to the receptor is
strengthened when multiple copies of a ligand, which binds with
moderate affinity as a single entity, are assembled on a particle. This
may apply to LpL-lipoprotein complexes assembled on cell surface
proteoglycans(35, 54) . Since efficient apolipoprotein
E-mediated binding of lipoproteins to
MR/LRP appears
to require multiple copies of the apolipoprotein on the
particle(6, 55) , it will be important to determine
whether apolipoprotein E may also bind to multiple RAP sites in
MR/LRP. In addition, complexes of molecules (e.g. protease-inhibitor complexes) can achieve high affinity for
binding to
MR/LRP even though the individual
components bind with low affinities. As an example, uPA binds with low
affinity (via at least two binding patches) as does PAI-1, whereas the
affinity of the uPA
PAI-1 complex is much higher whether bound to
the cell surface urokinase receptor or free in solution(40) .
Binding of uPA
PAI-1 complexes may in part occur at sites that
also bind RAP, a hypothesis further supported by the finding that
uPA
PAI-1 complexes can inhibit binding of RAP by at least 80%. (
)In addition, we find that whereas tPA is not inhibited by
the LpL fragment, the binding of tPA
PAI-1 complex is inhibited (Fig. 4B), suggesting that important binding of the
PAI-1 moiety in the tPA
PAI-1 complex may occur to RAP sites.
In conclusion, we have provided evidence that LpL and the
carboxyl-terminal RAP fragment bind to the same sites on
MR/LRP, whereas pro-uPA and the amino-terminal RAP
fragment may bind to the same or strongly overlapping sites. We suggest
that LpL is one of the natural extracellular ligands for the RAP sites
and that interaction with the multiple sites on the receptor
strengthens the binding of complex ligands such as lipoproteins.