From the Departments of Vascular Biology and
¶ Biochemistry, Jerome H. Holland Laboratory for the Biomedical
Sciences, American Red Cross, Rockville, Maryland 20855
Received for publication, December 10, 2002, and in revised form, March 5, 2003
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ABSTRACT |
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The low density lipoprotein receptor-related
protein (LRP) is a large endocytic receptor that recognizes more than
30 different ligands and plays important roles in protease and
lipoprotein catabolism. Ligand binding to newly synthesized LRP is
modulated by the receptor-associated protein (RAP), an endoplasmic
reticulum-resident protein that functions as a molecular
chaperone and prevents ligands from associating with LRP via an
allosteric-type mechanism. RAP is a multidomain protein that contains
two independent LRP binding sites, one located at the amino-terminal
portion of the molecule and the other at the carboxyl-terminal portion
of the molecule. The objective of the present investigation was to gain
insight into how these two regions of RAP interact with LRP and
function to modulate its ligand binding properties. These objectives
were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the
carboxyl-terminal domain that are necessary for binding of this region
of RAP to LRP and to heparin. RAP molecules in which either of these
two lysine residues was mutated still bound LRP but with reduced
affinity. Furthermore, the mutant RAPs were significantly impaired in
their ability to inhibit The low density lipoprotein receptor-related protein
(LRP)1 is one of twelve or
more receptors that make up the LDL receptor superfamily (for reviews,
see Refs. 1 and 2) and is essential for embryonic development in mice
(3). LRP recognizes more than 30 different ligands and plays important
roles in protease and lipoprotein catabolism. The deduced amino acid
sequence of LRP reveals that it is composed of a cytoplasmic domain
containing two copies of an NPXY consensus sequence,
a transmembrane domain, and a large extracellular region containing a
total of 22 growth factor repeats and 31 complement-type repeats that
are arranged into four clusters (clusters I-IV). Most ligands seem to
bind to repeats located within clusters II and IV (4-6). One ligand, the activated form of While purifying LRP by ligand affinity chromatography, a 39-kDa
protein, termed the receptor-associated protein (RAP) was identified
(8-10). Analysis of the primary sequence of RAP revealed a possible
internal triple repeat structure (11, 12) leading to the suggestion
that this molecule may contain three domains, termed D1, D2, and D3,
that roughly correspond to thirds of the molecule. The independent
nature of the domains was confirmed by expressing individual fragments
and performing structural analysis by 3H NMR spectroscopy
(12) and differential scanning calorimetry (DSC) (13). Interestingly,
the DSC measurements suggested that D2 might be composed of two
subdomains (13). The structure of the amino-terminal domain (residues
18-112, termed D1) of RAP has been solved by NMR spectroscopy (14) and
contains three helices composed of residues 23-34, 39-65, and 73-88
that are arranged in an anti-parallel topology.
Within the cell, RAP is localized primarily to the endoplasmic
reticulum due to the presence of an HNEL sequence at its carboxyl terminus (9, 15-17). Here RAP appears to function as a molecular chaperone for LRP and other LDL receptor family members by binding to
the newly synthesized receptors and preventing them from associating with ligands also present within the ER (15-19). The mechanism by
which RAP antagonizes the binding of all known ligands to LRP is not
fully understood. LRP contains at least three independent binding sites
for RAP, one each in cluster II, cluster III, and cluster IV. By
preparing soluble fragments encompassing portions of cluster II, Vash
et al. (20) demonstrated that RAP binds complement repeats
5-7 within this repeat. Repeats 5-7 are also responsible for binding
two LRP ligands, uPA·PAI-1 complexes and lactoferrin. Thus,
one mechanism by which RAP antagonizes ligand binding is by direct
competition for their LRP binding site. However, other ligands, such as
The binding of RAP to LRP is complicated by the fact that two
independent binding regions within RAP bind LRP (11). One of these
binding sites is localized at the amino-terminal region of RAP
containing D1 and a portion of D2 (and includes amino acid residues
1-164) that binds to LRP with a KD of 9 nM (13). In addition, the carboxyl-terminal domain of RAP,
which encompasses amino acid residues 216-323, termed D3, binds to LRP
with a KD of 6 nM (13, 22). The
objective of the present investigation was to gain insight into how
these two regions of RAP interact with LRP and function to modulate its
ligand binding properties.
Proteins and Antibodies--
LRP was isolated from human
placenta as described by Ashcom et al. (8). Human RAP was
expressed in bacteria as fusion proteins with glutathione
S-transferase and was cleaved and purified as described
previously (19). Monoclonal antibody 8G1 has been described previously
(23). Rabbit anti-RAP IgG (R438) was prepared against recombinant human
RAP, and the IgG fraction was purified. RAP mutants were prepared using
the ExSiteTM PCR-based site-directed mutagenesis kit from
Stratagene (24). Full-length RAP in the pGEX-2T vector was used as the
template for the PCR. The D4 RAP mutant (206-323) inserts were
prepared by PCR using the following primers:
5'-GACCGCGGATCCAGGGTCAGCCACCAG-3' and
5'-CAGAATTCTCAGAGTTCGTTGTGCCGAGC-3'. A denaturing temperature of
94 °C was used with an annealing temperature of 74 °C. The inserts were cleaved with the restriction enzymes BamH1 and
EcoR1 and ligated into the pGEX-2T vector. All
plasmids were sequenced in entirety. Protein concentrations in all
experiments were determined spectrophotometrically using the following
absorption coefficients (A280,1%) and molecular
weight values (13): RAP, 0.926 and 37,915; D3
(RAP206-323), 0.688 and 13,995; RAP1-164, 1.38 and 19,408; and D1-D2 (RAP1-216), 1.11 and
25,298.
Random Mutagenesis of the D3 of RAP--
To identify amino acid
residues within the carboxyl-terminal domain of RAP (D3) that are
critical for its binding to LRP, a library of random RAP mutants was
constructed. The library contained clones with randomly occurring
mutations within the sequence of RAP encoding for residues 206-323.
For construction of the RAP random mutant library, the D3 coding
sequence (9) was amplified by error-prone PCR as described by Lawrence
et al. (25). Sequence analysis of 24 clones revealed a
mutation frequency of 1:92 bp (~3.8 per clone). To identify clones
that were deficient in LRP binding, a receptor ligand blotting protocol
was utilized. In a typical assay, 160 unique clones were screened for
LRP binding by expressing the mutated D3 as a fusion with glutathione
S-transferase. Bacterial extracts containing the expressed
protein were analyzed for antigen by immunoblot analysis and for LRP
binding by receptor blot analysis. The majority (~77%) of the clones
were completely negative for LRP binding, likely due to folding
mutations introduced into RAP. Approximately 16% of the clones were
strongly positive for LRP binding, whereas, interestingly, ~10% of
the clones were weakly positive for LRP binding as revealed by receptor
blot analysis. The weakly positive clones were selected for further
analysis, because we reasoned that these molecules were defective in
LRP binding but were still appropriately folded. Sequence analysis of
all of these clones revealed that most clones had multiple mutations as
expected from the mutation frequency.
Receptor Blotting--
5 µg of RAP was subjected to SDS-PAGE
on 4-20% Tris-glycine gradient gels. Following electrophoresis,
proteins were transferred overnight to nitrocellulose and were
visualized with Ponceau stain. After blocking with 20 mM
Tris, 150 mM NaCl, pH 7.5, containing 3% milk for
1 h, the blot was incubated with 14 nM LRP in TBS containing 3% milk, 0.05% Tween 20 and 5 mM
CaCl2 for 2 h at room temperature and then washed and
incubated with 1 µg/ml 8G1 for 45 min. The blot was washed and
developed using Bio-Rad rabbit anti-mouse horseradish peroxidase conjugate.
Ligand Blot--
10 ng of LRP was subjected to SDS-PAGE using
4-20% Tris-glycine gels. Proteins were then transferred to
nitrocellulose for 3 h on 75 V at 0 °C, and the nitrocellulose
was then blocked with TBS containing 3% milk for 1 h. The blot
was incubated with 1 or 10 nM RAP, RAP K256A, or RAP K270E
in TBS containing 3% milk, 0.05% Tween, and 5 mM
CaCl2 overnight at 4 °C. The blot was washed and
incubated with 1 µg/ml R438 anti-RAP IgG for 1 h. The blot was
then washed and developed using Bio-Rad goat anti-rabbit horseradish peroxidase conjugate.
Solid-phase Binding Assay--
Microtiter wells were coated with
human LRP (4 µg/ml in TBS, pH 7.5, 100 µl/well) overnight and then
blocked with 300 µl of 3% BSA in TBS. Wells were washed three times
with TBS containing 3 mg/ml BSA, 0.05% Tween, and 5 mM
CaCl2. 100 µl of 125I-labeled RAP,
uPA·PAI-1 complex, or Heparin Binding--
The fluorescence anisotropy of 0.1 µM fluorescein-labeled heparin in TBS was measured while
a concentrated solution of D3 (WT), D3 K256A, or D3 K270E was added
continuously with a motorized syringe controlled by the same computer
controlling the fluorometer. Measurements were made at 25 °C with an
SLM-8000C spectrofluorometer in the T format using excitation and
emission wavelengths of 493 and 524 nm, respectively. Anisotropy
(A), as a function of titrant concentration, was fit to a
single class of equivalent binding sites using the following
equation,
Heparin-Sepharose Affinity Chromatography--
Approximately 73 µg of D3 and its mutants were injected onto a 1.7-ml
heparin-Sepharose column equilibrated with TBS, pH 7.4, at a flow rate
of 1 ml/min controlled by an Amersham Biosciences fast-protein liquid
chromatography system. A linear gradient of NaCl from 0.15 to 1 M was applied to elute bound proteins. Elution was
monitored using a Shimadzu 535 fluorescence monitor to detect intrinsic
fluorescence at 340 nm using an excitation wavelength of 280 nm.
Calorimetric Measurements--
Differential scanning calorimetry
(DSC) measurements were made with a DASM-4M calorimeter (26) at a scan
rate of 1 °C/min essentially as described previously (13). Protein
concentrations varied from 3 to 4 mg/ml in 20 mM Gly, pH
8.7, with 0.25 M guanidinium chloride. Under these
conditions, the endotherms were completely reversible (13). The DSC
curves were corrected for the instrumental baseline obtained by heating
the solvent. Deconvolution analysis was performed as described
previously (26, 27). Melting temperature (Tm)
and enthalpies were determined from the DSC curves using the same software.
CD Measurements--
Circular dichroism (CD) spectra were
recorded on a Jasco J-715 spectropolarimeter with a Peltier PFD-350S
unit for temperature control. Proteins were dissolved at a
concentration of 0.28 mg/ml in phosphate buffer (10 mM, pH
8.65). A 1-mm path length cell was used, and CD spectra data points
were recorded every 0.5 nm for the wavelength range 300-180 nm at
25 °C with 20 nm/min scans and a 2-s response time. Four scans were
accumulated per spectrum.
Cell Uptake Assays--
Cellular internalization assays were
generally conducted as described previously (7). Human WI-38
fibroblasts were seeded into 12-well culture dishes (5 × 104 cells per well) and grown in Dulbecco's modified
Eagle's medium supplemented with 10% bovine calf serum and
penicillin/streptomycin for 2 days. Cells were washed and incubated in
assay media (Dulbecco's modified Eagle's medium containing 1.5% BSA,
1% nutradoma, and 20 mM HEPES, pH 7.5). Assay media
containing 3 nM 125I-labeled Random Mutagenesis of the D3--
To identify amino acid residues
within D3 that are critical for its binding to LRP, a library of random
D3 mutants was constructed. This library was screened for deficiency in
LRP binding using a receptor ligand blotting protocol. Those clones
with impaired binding in this assay were selected and sequenced. As
expected from the mutation frequency of the library, most clones had
multiple mutations. Of interest, we found that the mutation frequency
was significantly higher at three lysine residues located within D3: Lys-256, Lys-270, and Lys-306. One mutant that displayed impaired LRP
binding had a single mutation in which Lys-270 was converted to
glutamic acid. D3 is known to contain an important LRP binding site
(13, 28), and because prior studies have implicated charged residues in
LRP binding (29), the current results implicate a role for these
residues in binding to LRP. To test this hypothesis, we introduced the
individual K256A, K270E, and K306A mutations into RAP and subjected the
purified mutant molecules to further analysis.
Binding Analysis of RAP Point Mutants to LRP--
Fig.
1 shows a receptor blot analysis
measuring the binding of LRP to wild-type RAP and the three mutant RAP
molecules following SDS-PAGE and transfer to nitrocellulose. It is
apparent that both the K256A mutant and the K270E mutant have impaired
LRP binding in this assay. Although the protein load on the gel for the
K256A mutant was slightly lower in this experiment than the other
proteins, in several repeats of this experiment, the K256A mutant
binding was always deficient in binding LRP. In contrast, to the K256A and K270E mutants, the K306A mutant appeared normal in its binding to
LRP. Thus, for the remainder of this study, experiments focused on the
K256A and K270E mutants.
The direct binding of RAP and the K256A and K270E RAP mutants to
purified LRP was also measured by ligand blotting approaches in which
various concentrations of RAP were incubated with LRP following
SDS-PAGE and transfer to nitrocellulose. The results (Fig.
2A) demonstrate that even at
low RAP concentrations (1 nM) wild-type RAP binds to LRP,
whereas neither of the two mutant RAP molecules bind very effectively
to LRP at this concentration. The mutant RAP molecules do bind to LRP,
but even at higher concentrations (10 nM) the extent of
binding is somewhat less than that of wild-type RAP (Fig.
2B).
We next measured the ability of RAP molecules containing these
individual mutations to inhibit the binding of 125I-RAP to
purified LRP using a solid-phase binding assay in which LRP was coated
to the surface of microtiter wells. The results of this experiment
(Fig. 3) reveal that wild-type RAP
competes for 125I-labeled RAP binding with a
KI,app of 3.3 nM. Both the K256A and K270E mutants showed a significant defect in their ability to
inhibit the binding of 125I-labeled RAP to LRP, with
KI,app values of 86 and 94 nM, respectively. These results indicate that RAP molecules
containing point mutations at residues 256 and 270 are defective in
competing for the binding of 125I-RAP to LRP.
Analysis of D3 Point Mutants--
To determine if mutations at
Lys-256 and Lys-270 alter the binding of D3 to LRP, we expressed and
purified recombinant D3 containing the K256A and K270E point mutations.
The binding of the wild-type and mutant D3 to purified LRP was assessed
by solid-phase assays. The results (Fig.
4A) demonstrated that in
contrast to wild-type D3, mutant D3 molecules containing the K270E
mutation failed to bind to LRP, whereas only weak binding of the K256A mutant to LRP is apparent. Together, these results reveal that mutation
at Lys-256 and Lys-270 impact binding of the carboxyl-terminal domain
of RAP to LRP, implying that these residues contribute significantly to
this interaction. D3 is also known to bind to heparin (29), and thus we
performed titrations to measure the effect of these mutations on
heparin binding. Wild-type D3 bound heparin with a
KD of 15.4 µM, whereas the K256A
mutant showed a diminished affinity for heparin and the K270E mutants failed to bind (Fig. 4B). To further examine this, these
mutant molecules were subjected to affinity chromatography on
heparin-Sepharose (Fig. 4C). Wild-type D3 bound to the
column and was eluted at 0.4 M NaCl. In contrast, the K256A
mutant had markedly reduced affinity for heparin-Sepharose and was
retarded by the column, whereas the K270E mutant did not bind to the
column at all. Thus, not only do mutations at Lys-256 and Lys-270 alter
the ability of this fragment to bind LRP, but they also alter the
interaction of the carboxyl-terminal portion of RAP with heparin.
Characterization of the Folding of D3 Point Mutants--
The loss
of binding activity in D3 associated with the Lys-256
Previous studies have shown that D3 exhibits a CD spectrum typical for
The K256A and K270E Mutations in RAP Effect Its Ability to Inhibit
In contrast to the data obtained for inhibition of uPA·PAI-1 binding,
both the K256A and K270E mutants were unable to completely inhibit the
binding of
We also examined the effect of these mutants on the ability of WI-38
fibroblasts to internalize 125I-labeled Inhibition of Mutant RAP Binding to LRP by the Amino- and
Carboxyl-terminal Domains of RAP--
To gain insight into the
relationship between the amino- and carboxyl-terminal LRP binding sites
on RAP, competition experiments were performed to determine if these
two RAP fragments are capable of competing with one another for binding
to LRP. The results indicate that RAP and both RAP fragments were
effective competitors for the binding of 125I-labeled
RAP1-164 to LRP (Fig.
8A). In contrast, only RAP and
D3 were able to compete for the binding of 125I-labeled D3
to LRP (Fig. 8B). The inability of RAP1-164 to
block the binding of D3 to LRP reveals that these two regions of RAP
bind to distinct sites on LRP. Thus, the binding of D3 to LRP must
induce a conformational change in LRP that blocks binding of the
amino-terminal RAP domain to its site on LRP but at the same time
increases binding of
Mutation of either Lys-256 or Lys-270 impaired binding of the
carboxyl-terminal region of RAP to LRP. Thus RAP molecules containing these mutations are likely to interact with LRP primarily via sites on
the amino-terminal region of the molecule. To test this, we therefore
examined the ability of D1-D2 and D3 to compete for the binding of
wild-type and mutant RAP to LRP (Fig.
9A). D1-D2 does not
effectively compete for the binding of wild-type RAP to LRP. In
contrast, D1-D2 inhibits the binding of the mutant RAP molecules to
LRP. We also examined the ability of D3 to compete with RAP and RAP
mutants for LRP binding. The results (Fig. 9B) reveal that
D3 was ~10-fold more effective as a competitor for the binding of
mutant RAPs to LRP than the wild-type RAP, consistent with the ability
of this RAP domain to block binding of the amino-terminal domain of RAP
to LRP.
RAP is an endoplasmic-resident protein that associates tightly to
newly synthesized LRP, gp330/megalin, and very low density lipoprotein
receptor and prevents them from binding endogenously produced ligands
(15-17). Recent studies reveal that RAP also acts in concert with
MESD, another ER resident protein, to facilitate the export of LRP5 and
LRP6 from the ER (30). The importance of RAP was revealed when the gene
was deleted in mice (31), and it was found that the functional levels
of LRP in the liver and brain were significantly reduced. Although the
reason for this is not entirely clear, it appears that RAP is required
to prevent receptor aggregation suggesting that it may assist in protein folding (16). Furthermore, association of LRP with certain ligands in the ER, which occurs in the absence of RAP, leads to degradation of the receptor, thereby reducing the amount of LRP on the
cell surface (31). Mechanisms by which RAP prevents ligands from
binding to LDL receptor family members are not fully understood at this
time. This is an exceptionally complex problem, because LRP binds to 30 or more structurally distinct ligands. The objective of this study was
to gain insight into mechanisms by which RAP so effectively antagonizes
the binding of all known ligands to members of the LDL receptor family
using LRP as the model system.
In the current investigation, we employed a random mutagenesis approach
to identify critical residues in the carboxyl-terminal domain of RAP
(D3) that are important for its interaction with LRP. This domain
contains one of the two sites on RAP that bind to LRP (12, 13, 32, 33)
and was chosen because the isolated domain can completely inhibit RAP
binding to LRP (12) and can prevent some, but not all, ligands from
binding to LRP (33). Furthermore, transfected U87 cells are unable to
secrete soluble LRP mini-receptors unless RAP or D3 is co-expressed
(28). A second LRP binding site on RAP is located at the amino-terminal portion of RAP within D1-D2. In contrast to D3, a fragment containing this site is unable to compete for RAP binding to LRP, and it does not
promote secretion of soluble fragments of LRP but can partially inhibit
ligands like We first identified critical residues within D3 that are important for
LRP binding. The results from the current study reveal that alteration
of lysine 256 or lysine 270 abolishes binding of the isolated
carboxyl-terminal RAP domain to LRP. It is somewhat surprising that
single point mutations have such a dramatic effect on the ability of D3
to bind to LRP and to heparin. The RAP D3 domain is unusual in that it
unfolds at a low temperature (Tm = 43 °C)
indicating that it may be highly flexible at physiological temperatures. Secondary structure analysis of D3 predicts a high content of When we introduced these mutations into full-length RAP, mutant RAP
molecules were generated that are defective in their ability to compete
for RAP binding and for 2M* binding to LRP via
allosteric mechanisms. In contrast, the mutant RAP molecules were still
effective at inhibiting uPA·PAI-1 binding to LRP. These results
confirm that both LRP binding sites within RAP cooperate to inhibit
ligand binding via an allosteric mechanism.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
2-macroglobulin
(
2M*), requires repeats from both cluster I and cluster
II for binding (7).
2M*, bind to a different set of complement-type repeats
found in clusters I and II, and thus the
2M* binding site does not strictly overlap with the RAP binding site (7). These
results indicate that RAP also inhibits ligand binding to LRP by
allosteric-type mechanisms. Further evidence for an allosteric inhibition mechanism is also derived from studies showing that, although RAP binds tightly to a soluble fragment spanning the second
cluster of complement-type repeats (C3-C10) and the amino-terminal flanking epidermal growth factor repeat of LRP (amino acids 787-1165), it is not a very effective inhibitor of ligand binding to this LRP
fragment (21).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2M* in wash buffer were then
added to the wells in the absence or presence of competitor as
indicated in the figure legends. Direct binding of D3 and D3 mutants
(K256A and K270E) was measured by adding 100 µl of
125I-labeled fragment in wash buffer at concentrations
indicated in the figure legend. The binding was carried out overnight
at 4 °C. Following incubation, the microtiter wells were washed and counted.
with [titrant] being the free concentration of D3,
(Eq. 1)
Amax being the maximum anisotropy change
occurring at saturating concentrations of titrant, and
KD being the apparent dissociation constant of the
heparin·protein complex. Because the concentration of
fluorescein-heparin was low compared with the range of concentrations
of D3, the concentration of free protein was taken as the total.
2M*
and 200 nM RAP, RAP mutants, or RAP fragments was added to
the corresponding wells and incubated for 3 h at 37 °C. Following incubation the cells were washed with phosphate-buffered saline and detached from plastic using trypsin (0.5 mg/ml), proteinase K (0.5 mg/ml), and EDTA (5 mM) containing buffer.
Internalized 125I-
2M* was defined as
radioactivity associated with the cell pellet. The cell numbers for
each experimental condition were measured in parallel wells that did
not contain radioactivity.
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ABSTRACT
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Fig. 1.
LRP receptor blot of RAP and various RAP
point mutants. 5 µg of RAP, and the K256A, K270E, and K306A
mutants were subjected to SDS-PAGE on 4-20% gradient gels under
non-reducing conditions. Following electrophoresis, the proteins were
transferred to nitrocellulose. Left panel, Ponceau stain of
nitrocellulose following transfer of proteins; right panel,
the nitrocellulose sheet was incubated with 14 nM human
LRP, and bound LRP was detected with monoclonal antibody 8G1 (1 µg/ml).
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Fig. 2.
Ligand blotting with RAP and RAP point
mutants. LRP (10 ng) was subjected to SDS-PAGE on 4-20% gradient
gels under non-reducing conditions and then transferred to
nitrocellulose. Following blocking with BSA, 1 nM
(A) or 10 nM (B) RAP, RAP K256A, or
RAP K270E was incubated with immobilized LRP, and, following washing,
the amount of binding was detected with anti-RAP polyclonal IgG
R438 (1 µg/ml).
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Fig. 3.
Inhibition of 125I-RAP binding to
LRP by wild-type RAP and RAP point mutants. 125I-RAP
(2 nM) was incubated with immobilized LRP (100 µl, 4 µg/ml) in the presence of increasing concentrations of unlabeled RAP
(circles), RAP K256A (open squares), or RAP K270E
(closed squares). Each plotted value represents the average
of duplicates with the range indicated by bars.
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Fig. 4.
Binding of D3, D3-K256A, and D3-K270E point
mutants to LRP (A) fluorescent-labeled heparin
(B), or heparin-Sepharose (C).
A, 125I-RAP D3 (circles),
125I-RAP D3 K256A (squares), or
125I-RAP D3 K270E (triangles) were incubated
with immobilized LRP at increasing concentrations. Following incubation
overnight at 4 °C, the wells were washed, and the amount of cpm in
each well was measured. Each plotted value represents the average of
duplicates. In the case of wild-type D3, the solid lines
represent the best-fit curve to a binding isotherm determined by
non-linear regression analysis. B, anisotropy of
fluorescently-labeled heparin (0.1 µM) was measured in
the presence of increasing concentrations of RAP D3 (WT),
RAP D3 K256A (K256A), or RAP D3 K270E (K270E).
The solid curve for wild-type D3 represents the best fit to
a binding isotherm by non-linear regression analysis. C,
approximately 73 µg of D3 or its mutants were injected onto a 1.7-ml
heparin-Sepharose column equilibrated with TBS, pH 7.4, at a flow rate
of 1 ml/min controlled by a Amersham Biosciences fast-protein liquid
chromatography system. Bound proteins were eluted with a linear
gradient of NaCl from 0.15 to 1 M, and elution was
monitored by measuring intrinsic fluorescence at 340 nm.
Ala or
Lys-270
Glu mutations could result from a general unfolding of this
domain induced by the amino acid changes, because this region is known
to be relatively unstable (13). Thus, we compared the unfolding
properties of these two mutant proteins with wild-type D3 by
differential scanning calorimetry (Fig.
5A). As reported earlier (13),
D3 unfolds as a single two-state transition with an enthalpy of 45.3 kcal/mol and a Tm of 41.5 °C. The endotherm of the K256A mutant was virtually identical to wild-type D3 and revealed that the K256A mutation unfolded with a enthalpy of 43.2 kcal/mol and a Tm of 43.1 °C, confirming that
this mutation does not alter the stability or unfolding properties of
the domain. Interestingly, the K270E mutant was actually stabilized
when compared with wild-type D3 and unfolded with an enthalpy of 59.5 kcal/mol and a Tm of 49.1 °C. Lys-270 is
predicted to occur in a large
-helix, and it is possible that
changing lysine to glutamic acid replaces a charge repulsion that
occurs between two helical regions with a charge attraction leading to
stabilization. Regardless of the mechanism, these two mutations in the
RAP carboxyl-terminal domain do not lead to destabilization or
unfolding of this region.
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Fig. 5.
Differential scanning calorimetry
(A) and CD spectra (B) of wild-type
RAP D3 and point mutants. A, DSC curves obtained upon
heating of RAP D3 (3.9 mg/ml), RAP D3 K256A (3.2 mg/ml), and RAP D3
K270E (4.2 mg/ml) in 20 mM glycine buffer, pH 8.7, containing 0.25 M guanidinium chloride. Data shown has a
buffer baseline subtracted. B, CD spectrum of RAP D3, RAP D3
K256A, and RAP D3 K270E (0.28 mg/ml) in phosphate-buffered saline, pH
7.4, at a temperature of 20 °C. Curve 1, RAP D3;
curve 2, RAP D3 K256A; and curve 3, RAP D3
K270E.
-helical proteins that is abolished when the temperature is raised
(13). We therefore compared the CD-spectra of wild-type D3 domain with
the mutant molecules and the results, shown in Fig. 5B, reveal that
both mutant D3 molecules contain extensive
-helical content,
confirming that the secondary structure of these mutant molecules is
not significantly altered. Together with the data of Fig. 5A, the
results indicate that these mutations in the carboxyl-terminal domain
of RAP do not lead to general unfolding of the structure nor to a
decrease in the helical content of the molecule.
2M* Binding to LRP--
A major function of RAP is to
inhibit binding of all known ligands to LRP. We therefore measured the
ability of the mutant RAP molecules to inhibit two ligands whose
binding sites on LRP have been mapped: uPA·PAI-1 complexes and
2M*. Like wild-type RAP, both the K256A and K270E
mutants were potent inhibitors of 125I-labeled uPA·PAI-1
binding to LRP (Fig. 6A).
Although differences in the dose-response curves were noted,
importantly, at saturating amounts of competitor, uPA·PAI-1 binding
was completely inhibited by all RAP molecules.
View larger version (21K):
[in a new window]
Fig. 6.
Competition of
125I-uPA·PAI-1 and
125I- 2M* binding to
LRP by RAP and mutant RAPs. A,
125I-uPA-PAI-1 (2 nM) was added to immobilized
LRP (100 µl, 4 µg/ml) in the presence of increasing concentrations
of RAP (closed circles), RAP K256A (open
squares), or RAP K270E (open diamonds). Following
incubation, the wells were washed and counted. Each plotted value
represents the average of duplicates with the range indicated by
bars. B, 125I-
2M* (3 nM) was added to immobilized LRP (100 µl, 4 µg/ml) in
the presence of increasing concentrations of RAP (closed
circles), RAP K256A (open squares), or RAP K270E
(open diamonds). Following incubation, the wells were washed
twice and counted. Each plotted value represents the average of
duplicates with the range indicated by bars.
2M* to LRP and only reduced the binding to
about 50% at saturating concentrations (Fig. 6B),
indicating that mutations at residues 256 and 270 generate a RAP
molecule altered in its ability to antagonize
2M*
binding to LRP.
2M*
(Fig. 7). At a concentration of 200 nM, RAP completely blocked the internalization of 3 nM 125I-labeled
2M*. In
contrast, both the K256A and K270E mutants reduced the amount of
125I-labeled
2M* internalized but were
unable to completely block it, even at saturating concentrations of
inhibitor. The magnitude of the effect was similar to that seen with
saturating amounts of a fragment of RAP, which contains the
amino-terminal LRP binding site (RAP1-164) or with D1-D2
(data not shown). Curiously, D3 consistently increased the amount of
125I-labeled
2M* that was internalized,
suggesting that its binding to LRP alters the conformation of the
receptor, resulting in increased affinity for
2M*.
View larger version (14K):
[in a new window]
Fig. 7.
Inhibition of
125I- 2M*
internalization by RAP and mutant RAPs.
125I-
2M (3 nM) was incubated
with WI38 fibroblasts in the presence of 200 nM RAP, RAP
K270E, RAP K256A, D3, or RAP1-164. After 3 h the
amount of 125I-
2M* internalized was measured
as described under "Experimental Procedures." The graph
shows the average of duplicates with the range indicated by
bars.
2M* to LRP.
View larger version (18K):
[in a new window]
Fig. 8.
The carboxyl-terminal domain of RAP competes
with the amino-terminal RAP fragment for binding to LRP.
A, 2 nM 125I-labeled
RAP1-164 was incubated overnight at 4 °C with
immobilized LRP in the presence of increasing concentrations of RAP
(circles), RAP1-164 (squares), and
D3 (triangles). Following incubation and washing, the wells
were counted. B, 2 nM 125I-labeled
D3 was incubated overnight at 4 °C with immobilized LRP in the
presence of increasing concentrations of RAP (circles),
RAP1-164 (squares), and D3 (triangles).
Following incubation and washing, the wells were counted.
View larger version (18K):
[in a new window]
Fig. 9.
Inhibition of 125I-RAP and mutant
RAP binding to LRP by D1-D2 (A) or D3
(B). 2 nM 125I-RAP
(inverted triangles), 125I-RAP K256A
(circles), or 125I-RAP K270E
(squares) were added to immobilized LRP in the presence of
increasing concentrations of RAP D1-D2 (A) or RAP D3
(B). Following overnight incubation at 4 °C, the wells
were washed and the cpm in each well was measured. Each plotted value
represents the average of duplicates with the range indicated by
bars.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2M* from binding to LRP. The results of the
current investigation give insight into how these two domains function
in intact RAP to modulate LRP binding.
-helix with a large helical region from residues 237 to
277 suggesting that Lys-256 and Lys-270 are likely to occur within a
helical region. The mutations do not lead to instability of the domain
as revealed by differential scanning calorimetric analysis. In fact,
one of the mutations (Lys-270
Glu) increased the stability of D3,
increasing its unfolding Tm by 6 °C. It is possible that the mutations induce a more rigid structure in this RAP
domain, which may dramatically impact its binding to LRP. Isothermal
titration calorimetry binding analysis revealed that association of the
carboxyl-terminal domain of RAP with two complement-type repeats from
cluster II of LRP is primarily driven by entropic contributions (34).
Furthermore, surface-plasmon resonance binding data are consistent with
a model in which this domain of RAP undergoes a conformational change
upon binding to LRP (34). A more rigid structure within RAP D3 may not
accommodate these necessary conformational changes upon association
with LRP.
2M* binding. Interestingly, the
mutant RAP molecules were still effective in inhibiting the binding of
uPA·PAI-1 complexes to LRP. Together, these data give insight into
the mechanisms by which RAP modulates ligand binding by LRP and suggest
that both D1-D2 and D3 domains are required for inducing
conformational changes in LRP that reduce ligand binding (Fig.
10). The conformation change induced in
LRP as a result of RAP binding must be initiated by D3. This is based
on evidence in the current study indicating that the binding of
isolated D3 to LRP actually increases the binding of
2M*
to LRP. It seems likely that the conformational change in LRP upon D3
binding alters the relationship between ligand binding clusters in LRP.
Increased binding of
2M* in the presence of D3 is
consistent with a rearrangement of cluster I and cluster II to
facilitate
2M* binding (Fig. 10C).
View larger version (32K):
[in a new window]
Fig. 10.
Model for RAP inhibition of LRP. RAP is
known to bind to at least three sites on LRP one each in cluster II,
III, and IV. For simplicity, only binding of RAP to cluster II is
depicted. Regions on LRP within cluster I and II that are responsible
for the binding of 2M* and uPA·PAI-1 complexes are
shown (B). Note that uPA·PAI-1 also binds repeats in
cluster IV (6), but for simplicity, this is not shown. RAP interacts
with LRP via its D3 domain and induces a conformational change in the
molecule, which is depicted by a change in the shape of
repeats in cluster II. This change is speculated to alter the
relationship of clusters I and II (A). However, when D3
alone is present, the conformational change is not complete, and only
ligands that recognize repeats in cluster II are blocked from binding.
However, the binding of ligands, such as
2M*, that
recognize cluster I and a portion of cluster II is actually enhanced
(C).
The results in the current study reveal that mutation of lysine residues in D3 impact not only LRP binding properties but also the heparin binding properties of D3. Previous studies have noted that mutations in RAP that lead to a loss of heparin binding also lead to a loss of LRP binding (29). These investigators found that mutation of basic amino acids within two clusters of basic residues (Arg-203 to Arg-206 along with Arg-282 to Lys-289) reduced binding of RAP to both LRP and heparin, suggesting that overlapping motifs within RAP are required for both LRP binding and heparin binding. Together with our study, it is apparent that basic residues within the carboxyl-terminal domain of RAP seem critical for binding of this portion of the molecule to LRP. A complete understanding of how RAP interacts with this receptor, however, will require solving the three-dimensional structure of the receptor· ligand complex.
In summary, we have identified critical residues on the
carboxyl-terminal domain of RAP important for the binding of this region to LRP. By introducing these mutations into full-length RAP, we
have generated a molecule that is defective in preventing ligands, such
as 2M*, from binding to LRP but is still very effective in antagonizing the binding of uPA·PAI-1. The data reveal that interaction of multiple regions of RAP with LRP are important for the
potent effect this molecule has on ligand binding to this receptor, and
impairment of just one of these sites significantly impacts its ability
to modulate ligand binding to LRP.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL50784 and HL54710.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Biacore, Inc., 200 Centennial Ave., Suite 100, Piscataway, NJ 08854.
To whom correspondence should be addressed: American Red
Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.:
301-738-0726; Fax: 301-738-0465; E-mail:
strickla@usa.redcross.org.
Published, JBC Papers in Press, March 12, 2003, DOI 10.1074/jbc.M212592200
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ABBREVIATIONS |
---|
The abbreviations used are:
LRP, low density
lipoprotein receptor-related protein;
2M,
2-macroglobulin;
2M*,
2-macroglobulin activated with trypsin;
RAP, receptor-associated protein;
RAP1-164, amino-terminal
fragment of RAP containing amino acids 1-164;
D1-D2, domains 1 and 2 of RAP consisting of amino acid residues 1-216;
D3, carboxyl-terminal
domain of RAP consisting of amino acid residues 206-323;
uPA, urokinase;
PAI-1, plasminogen activator inhibitor type 1;
DSC, differential scanning calorimetry;
CD, circular dichroism.
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