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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3277-3285
Three Complement-Type Repeats of the Low-Density Lipoprotein
Receptor-Related Protein Define a Common Binding Site for RAP,
PAI-1, and Lactoferrin
By
Brian Vash,
Neil Phung,
Sima Zein, and
Dianne DeCamp
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ABSTRACT |
The low-density lipoprotein receptor-related protein
(LRP) is a 600-kD scavenger receptor that binds a number
of protein ligands with high affinity. Although some ligands do not
compete with each other, binding of all is uniformly blocked by the
39-kD receptor-associated protein (RAP). RAP is normally found in the
endoplasmic reticulum and seems to function as a chaperone for LRP. To
identify the binding sites for RAP, lactoferrin, and plasminogen
activator inhibitor-1 (PAI-1), a bacterial expression system has been
developed to produce soluble LRP fragments spanning residues 783-1399. These residues overlap most of the CNBr fragment
containing the second cluster of complement-type repeats (C). Solid
phase binding assays show that 125I-RAP binds to fragments
containing three successive complement-type repeats: C5-C7. PAI-1 and
lactoferrin bind to the same fragments. A fragment containing C5-C7
also blocks uptake and degradation of 125I-RAP by
fibroblasts in a concentration-dependent manner. Binding competition
experiments show that RAP, PAI-1, and lactoferrin each inhibit the
binding of the others, suggesting that at this site in LRP, RAP acts as
a competitive, rather than an allosteric, inhibitor of PAI-1 and
lactoferrin binding.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE LOW-DENSITY lipoprotein
receptor-related protein (LRP) is a large (600 kD) scavenger receptor
that is found on the surface of many types of cells, including
fibroblasts, hepatocytes, macrophages, and neurons.1 The
receptor contains four clusters of disulfide-bonded, complement-type,
and epidermal growth factor (EGF)-like repeats that identify it as a
member of the low-density lipoprotein (LDL) receptor
family.2 LRP is an endocytic receptor with high
selectivity; it is responsible for the uptake and degradation of more
than a dozen proteins, most of which are evolutionarily
unrelated.3 LRP ligands include
 2-macroglobulin
(2M).proteinase and tissue plasminogen
activator (t-PA).PA inhibitor-1 (PAI-1) complexes,
lactoferrin, lipoprotein lipase, and apoE-containing
lipoproteins.3 More recently, it was discovered that LRP
internalizes  -amyloid precursor protein4 and
thrombospondin.5
A 39-kD protein with high affinity for LRP, known as
receptor-associated protein (RAP), was first encountered as a
contaminant of 2M affinity-purified
receptor.6,7 RAP is resident in the endoplasmic reticulum
and functions as a chaperone for LRP folding and
secretion.8-10 By binding to several regions of newly synthesized LRP, RAP prevents intracellular ligand-induced aggregation and degradation of the receptor. Although RAP is not normally found
outside of the cell, RAP is a useful tool because it competes with all
known ligands,1 although the reverse is not true. Estimates
of the total number of RAP binding sites on LRP range from two to
five.7,11 The number of binding sites for other ligands has
not been determined. Examples of RAP competitors include lactoferrin,
which binds to the chicken oocyte receptor,12 and lipoprotein lipase, which binds to LRP.13 Other ligands,
such as t-PA and 2M, which bind to LRP on hepatoma
cells,14 and very low-density lipoprotein (VLDL)
and vitellogenin, which bind to the chicken oocyte
receptor, do not block the binding of RAP to the same
receptors.12 These observations have led to the proposal
that RAP blocks binding of all LRP ligands by causing allosteric
changes in the receptor.15-17
One or more RAP binding sites were identified in an approximately
600-amino acid, CNBr-generated fragment of LRP that
corresponds to a cluster of EGF and complement-type repeats near the
N-terminus of the molecule. Willnow et al16,18 showed that
this fragment can be expressed as a recombinant minireceptor in
mammalian cells and exhibits RAP-binding activity.
We have now expressed the same soluble minireceptor in the periplasm of
Escherichia coli and have shown its specific binding activity
towards RAP. Further, by the use of bacterially expressed fragments of
this minireceptor, we have localized the binding site for RAP to a
small region in LRP consisting of complement-type repeats C5-C7. These
residues also constitute the binding site for PAI-1 and lactoferrin.
RAP competes with both ligands suggesting that, in this instance, RAP
inhibition is due to direct competition rather than allosteric effects.
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MATERIALS AND METHODS |
Materials.
Human PAI-1 was expressed and purified using the pPAI-1.HIS
vector.19 The GST 39-kD expression plasmid,6
pLRPex192 containing the LRP cDNA, and mouse fibroblast
cell lines20 were obtained from Dr Joachim
Herz (University of Texas Southwestern Medical Center,
Dallas). Plasmid pTMN was obtained from the American Type Culture
Collection (Bethesda, MD), catalog #87197. All 125I-labeled
proteins were radiolabeled with Iodobeads (Pierce Chemical Co,
Rockford, IL) to a specific activity of 2,000 to 5,000 cpm/ng. Radionuclides were purchased from Amersham Life Sciences, Inc (Arlington Heights, IL). Protein concentrations were determined using
the Bio-Rad Protein Assay (Hercules, CA).
Generation of LRP constructs.
cDNA encoding LRP fragments was inserted into a periplasmic expression
vector using a polymerase chain reaction (PCR) cloning strategy.
Fragment inserts were amplified from the LRP cDNA using the
oligonucleotides shown in Table 1. The
inserts were digested with BamHI and XhoI (Life
Technologies, Gaithersburg, MD) and ligated into vector pSecTagB
(Invitrogen) that had been digested with the same enzymes.
Recombinant proteins expressed from this vector contain a myc
tag followed by a 6-His tag at the C-terminus. For insertion into the
periplasmic expression vector pTMN,21 the mammalian
constructs were amplified a second time with Expand taq polymerase
(Boehringer Corp, Indianapolis, IN) to change the restriction enzyme sites. Forward primers were identical to those listed in Table 1, except that the BamHI sequence (GGATCC) was replaced with an NcoI site (CCATGGGA) in the same frame as the pTMN polylinker. The reverse primer was
5 -GGCGGATCCTCAATGATGATGATG-3, which adds a stop codon
followed by a BamHI site after the 6-His tag coding region of
the inserts. On cleavage of the OmpA signal peptide, all LRP fragments
had "Met-Gly-" as the amino terminus.
Expression and purification of LRP fragments.
Plasmids encoding the LRP fragments were transformed into strain
BL21/DE3(pLysS) competent cells (Novagen, Madison, WI)
for protein expression. Cultures were grown in Luria-Bertani medium broth containing 0.4% glucose, 15 µg/mL
chloramphenicol, and 25 µg/mL ampicillin (LCA) at 37°C.
Expressing clones were stored frozen as glycerol stocks and streaked
out on LCA plates when required. For each liter of culture, 100 mL of
LCA was inoculated with freshly streaked colonies and grown overnight.
The bacteria were pelleted, resuspended in a liter of fresh media, and
grown to an optical density of 0.5 to 0.6 at 600 nm. After induction with 0.5 mmol/L isopropylthio--D-galactoside (IPTG)
and 5-hour growth, the cells were obtained by
centrifugation at 4,000g for 20 minutes and periplasmic
proteins were isolated by cold osmotic shock.22 Stock
solutions were added to the osmotic shock fluid to give a final
concentration of 50 mmol/L Tris-HCl, pH 8; 150 mmol/L NaCl; 0.1 mmol/L
phenylmethylsulfonyl fluoride; 2 mmol/L CaCl2; and 10 mmol/L imidazole (buffer A). The osmotic shock fluid was stirred
overnight with Ni-NTA agarose (Qiagen Inc, Santa Clarita, CA) at 4°C and pelleted by centrifugation at
500g. The pellet was then washed four times with fresh buffer
A, followed by four washes with buffer A containing 500 mmol/L NaCl.
LRP fragments were eluted from the Ni-NTA gel with buffer A containing
250 mmol/L imidazole and bound to a RAP affinity column (see below).
RAP-Sepharose was washed with 50 mmol/L Tris-HCl, pH 7.5, containing
150 mmol/L NaCl, 2 mmol/L CaCl2, and 0.02%
NaN3. LRP fragments were eluted with calcium-free Tris-HCl
buffer containing 10 mmol/L EDTA. The correct amino acid sequence of
the fragments was confirmed by double-stranded DNA sequencing using the
Sequenase 7-Deaza-dGTP DNA Sequencing Kit (US Biochemical Corp,
Cleveland, OH). N-terminal amino acid sequencing and
electrospray mass spectrometry were performed on selected fragments by
Dr Clive Slaughter (HHMI Biopolymer Facility, University of Texas
Southwestern, Dallas, TX).
Preparation of RAP affinity column.
To obtain recombinant rat RAP in an unfused state, the GST-RAP
expression plasmid was used as a PCR template with the following pair
of primers: 5-GCAGGATCCTACTCGCGGGAGAAG-3 (forward) and 5-ACGCTCGAGCTAGAGTTCGTTGTGCCGAGCCCT-3 (reverse). The
resulting insert was digested with BamHI and XhoI and
ligated into the BamHI/XhoI-digested pET-28a vector
(Novagen). RAP expressed from this plasmid contains a 6-His tag
followed by a T7 epitope tag at the N-terminus. The pET28-RAP vector
was transformed into strain BL21/DE3(pLysS) for protein expression.
Cultures were induced and obtained as described for LRP fragments.
Purification on Ni-NTA gel followed by a heparin column was essentially
as described for active PAI-1.19 RAP was coupled to
CNBr-activated Sepharose 4B (Pharmacia Biotech, Inc, Piscataway,
NJ) according to the manufacturer's instructions.
Direct binding assays.
Solid-phase binding assays using 125I-RAP were performed in
96-well microtiter plates (Immulon-4, Removawell; Dynatech, Chantilly, VA). Wells were coated with 5 pmol purified receptor in 100 µL Tris-HCl-buffered saline and allowed to bind overnight at 4°C. The
plates were then blocked for 4 hours at room temperature
with 300 µL binding buffer (50 mmol/L Tris-HCl, pH 7.5; 150 mmol/L NaCl; 0.02% NaN3; 0.05% Tween-20; 2 mmol/L
CaCl2; 5% bovine serum albumin [BSA]) and incubated
overnight at 4°C with increasing amounts of 125I-RAP
diluted in binding buffer. The plates were washed three times with 200 µL binding buffer and the wells counted in a  -counter. To assay
nonspecific binding, an equal number of control wells were prepared
with calcium-free binding buffer containing 40 mmol/L EDTA. Nonspecific
binding was normally less than 5% of the total and was subtracted from
the total cpm. Binding data were fit to a Langmuir isotherm by
nonlinear regression using SigmaStat version 2.0 (Jandel
Scientific, San Rafael, CA). In every case binding data were fit to a
single-site model; multisite models failed to produce statistically
significant (P < .05) improvements in  2.
Dissociation constants (Kd) were determined by nonlinear regression of
the single-site Langmuir isotherm rather than using linear Scatchard
analysis.23 Because iodinated lactoferrin and PAI-1 are
insoluble at high concentrations, we were unable to determine their
dissociation constants by direct binding.
Binding competition assays.
Binding competitions of 2 nmol/L 125I-RAP, 10 nmol/L
125I-lactoferrin, and 15 nmol/L 125I-PAI-1 with
unlabeled RAP, lactoferrin, and PAI-1 were performed using
RAP-affinity-purified C5-C7 and E4-C10. To rule out nonspecific interactions, unlabeled RAP, lactoferrin, BSA, and PAI-1 were coated in
wells and assayed to ensure that they did not bind to the iodinated
ligands. The competition experiments were performed using 96-well
microtiter plates in the same manner as were direct binding assays. The
wells were counted in a -counter and the IC50 values
were determined by nonlinear regression using a single-site model in
the SigmaStat program.
Cell degradation assays.
Mouse fibroblasts (2 × 105 per well) were seeded in
12-well plates and grown for 24 hours in Dulbecco's modified Eagle's
medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were washed with DMEM (without glutamine or serum) containing 0.2% (wt/vol) BSA and incubated with 1 mL of the same media containing 1.5 µg
125I-RAP.20 Competing ligands dissolved in 25 mmol/L HEPES, pH 7.4; 150 mmol/L NaCl; and 2 mmol/L CaCl2
were added to the wells to give a final volume of 1.2 mL; controls
received 200 µL HEPES buffer only. Cells were allowed to take up
125I-RAP for 4 hours at 37°C. Degradation was assessed
by measuring 125I-labeled trichloroacetic acid-soluble
(noniodide) material released into the culture medium.24
Amounts of degradation products were normalized to the values for
wild-type mouse embryonic fibroblast (MEF 1) cells
without competitors added. These values ranged from 520 to 866 ng/mg
total cell protein/4 hours in different experiments. Background ligand
degradation was measured in control wells lacking cells and was
subtracted.
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RESULTS |
Periplasmic expression of LRP fragments.
To identify the minimal binding sites for various LRP ligands, we
cloned and expressed receptor fragments spanning most of the region
that corresponds to the CNBr fragment (amino acids 776-1399), which
contains the second cluster of complement-type repeats in
LRP.16 The oligonucleotides used as PCR primers are listed
in Table 1. All fragments comprised multiples of entire, contiguous
repeats followed by a myc epitope tag and a 6-His tag at their
C-termini. The fragments were secreted into the periplasm, isolated by
cold osmotic shock, and subjected to further chromatographic purification. An approximately equal amount of inactive recombinant protein secreted into the media or the cytoplasm was discarded. LRP
fragments separated on sodium dodecyl sulftate (SDS)-polyacrylamide gel
electrophoresis and stained with Coomassie Brilliant blue (Bio-Rad) are
shown in Fig 1. The mobility of the
proteins under reducing conditions, particularly the smaller fragments,
is lower than expected (Fig 1A). For example, the mass of E4-C3
measured by protein trap electrospray is 13,155 atomic mass units (amu) versus 13,160 amu computed from the amino acid sequence,
although it seems to be greater than 18 kD according to the molecular
weight markers. The anomalous mobility must be due to the presence of the 6-His tag. Figure 1B shows two of the RAP-binding fragments on a
nonreducing SDS gel; both appear to have a major band consisting of the
monomer. It is predicted that the fragments will migrate faster under
nonreducing conditions because disulfide bonds prevent complete
denaturation of the protein. The mobility of the smaller protein C5-C7
barely increases, presumably because the effect of the 6-His tag
predominates over the presence of intact disulfides. E4-C10 migrates
much faster on the nonreducing gel, indicating a more compact structure
in the nonreduced state.
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| Fig 1.
Recombinant LRP fragments from region II. (A)
Electrophoresis of fragments on a 13.5% reducing SDS polyacrylamide
gel. Lane 1 contains prestained low molecular weight markers (Life
Technologies). The remaining lanes (2 through 6) contain
the LRP fragments in the following order: E4 (8.2 kD), E4-C3 (13.2 kD),
C5-C7 (16.8 kD), E5-AA1399 (29.5 kD), and E4-C10 (44.8 kD). The smaller
proteins migrate abnormally slowly and appear to be larger than the
actual molecular weights shown in parentheses due to the presence of
the 6-His tag. (B) RAP affinity-purified fragments on a nonreducing
13.5% SDS polyacrylamide gel. Lane 1 contains low molecular weight
markers, lane 2 contains C5-C7, and lane 3 contains E4-C10. Both gels
were stained with Coomassie Brilliant blue.
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Analysis of RAP binding.
When we initiated our study, the 39-kD protein RAP was known to have a
high affinity for cellular LRP and to bind to recombinant fragments
corresponding to region II of the receptor, as shown by ligand blots
and coimmunoprecipitation experiments.9,16 Therefore, we
examined the ability of periplasmically expressed recombinant receptor
fragments to bind 125I-RAP as an assay of native structure.
A schematic of the repeat structure of the receptor fragments showing
their Kd values are shown in Fig 2. EGF
repeat E4 was not essential for RAP binding, in agreement with previous
ligand blot experiments. The C-terminal EGF-precursor homologous
domain, which contains E5 and E6, also did not seem to bind RAP. By
comparison of the Kd values, the minimal RAP binding site can be
localized to the three complement-type repeats, C5-C7 (Fig 3). Every
protein containing these three repeats had a Kd of approximately 2 nmol/L, including protein fragments beginning with E4 and terminating
at C7 or C8 (data not shown). The affinity of C5-C7 for RAP is similar
to affinity constants reported for the binding of 125I-RAP
to immobilized LRP: 14 nmol/L7 and 18 nmol/L.25
Complement-type repeats contain conserved and essential disulfide
bonds.6 As a control, we tested the RAP binding activity in
the presence and absence of reducing
agents. RAP has no cysteines
and is unaffected by reducing agents. Five pmol of fragments C5-C7 and
E4-C10 were coated on plates and assayed for binding to 2 nmol/L
125I-RAP in the presence or absence of 10 mmol/L
dithiothreitol (DTT). For C5-C7, counts per minute
corrected for background were 7,959 ± 198 (no DTT) and 0 ± 101 (+ DTT); for E4-C10, 7,013 ± 231 (no DTT) and 0 ± 31 (+ DTT).
DTT totally abolished the binding of 125I-RAP to the
receptor fragments, suggesting that binding activity is dependent on
the presence of intact disulfides in the receptor fragments.
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| Fig 2.
RAP binding to LRP region II fragments. A schematic
drawing of the recombinant LRP fragments shows their constituent EGF
precursor type repeats ( ) and complement-type repeats ( ). The LRP
repeats have been numbered according to Herz et al.2 The
Kds determined by direct binding assays are shown at left; ND, no
detectable binding.
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| Fig 3.
Direct binding of 125I-RAP to LRP fragments
E4-C10 (A) and C5-C7 (B). 125I-RAP was added to receptor
fragments (250 ng E3-C10 or 100 ng C5-C7) coated in 96-well microtiter
plates as described in Materials and Methods. Triplicate experiments
were used to calculate a mean and SD for each data point. In each
experiment the total amount of 125I-RAP added ranged from
0.5 nmol/L to 200 nmol/L and the concentration of free
125I-RAP was calculated by counting the well buffer after
overnight binding. The amount of 125I-RAP bound was
determined by counting the dry wells after washing. Nonspecific binding
was determined by subtracting the value of wells in which
125I-RAP was bound without calcium and in the presence of
40 mmol/L EDTA. The Kd values were determined to be 2.13 ± 0.67 for
the fragment E3-C10 (A) and 1.60 ± 0.09 for C5-C7 (B). Inset to each
panel is the Scatchard plot of the Langmuir isotherm calculated from
the data.
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Effect on LRP fragments on uptake of 125I-RAP by
fibroblasts.
Most of the protein ligands internalized through LRP are degraded in a
lysozomal pathway. To confirm the results of the plate assays, we
examined the ability of soluble receptor fragments to bind
125I-RAP and block its uptake and degradation by mouse
fibroblast cells. The activity of five receptor fragments is shown in
Table 2. MEF 1 express large amounts of LRP
and the rate at which they take up and degrade 125I-RAP is
considered 100% of full activity. The data shown in Table 2 mirror the
findings of the binding assays in that only fragment E4-C10, which
contains repeats C5-C7, is able to prevent 125I-RAP uptake
and degradation. Unlabeled RAP is included as a control and is also
effective at decreasing the amount of degradation. PEA 13 cells are homozygous LRP-deficient but take up and
degrade 125I-RAP at one-seventh the rate observed in
wild-type cells.20 This residual uptake activity is
presumably LRP independent and thus cannot be blocked by ligands that
bind exclusively to LRP, indicating that 400 nmol/L E4-C10 is able to
block about 70% of the LRP-dependent degradation. The activity of
E4-C10 to interfere with degradation is proportional to its
concentration, as shown in Fig 4.
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| Fig 4.
Dependence of inhibition of 125I-RAP
degradation on soluble E4-C10. Replicate monolayers of MEF 1 fibroblasts were incubated with serum-free DMEM containing
125I-RAP and different concentrations of LRP fragment
E4-C10. After incubation at 37°C for 4 hours, the total amount of
125I-RAP degradation products secreted into the media was
measured. The points are the mean of triplicates from one to three
experiments and are normalized to the amount of degradation products
released in the absence of E4-C10.
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Identification of ligand binding sites for RAP, PAI-1, and
lactoferrin.
RAP is able to interfere with the binding of all known LRP ligands,
including PAI-1 and lactoferrin, although it has been more problematic
to show reciprocal competition between individual ligands.15 This has been interpreted as an indication of
multiple independent or partially overlapping binding sites on LRP. To compare the binding sites of RAP, PAI-1, and lactoferrin, iodinated proteins (25 nmol/L) were incubated with various LRP fragments and
assayed for binding activity (Fig 5). The
data show that all three ligands interact predominately with C5-C7; a
longer fragment (E4-C10) containing all eight complement-type repeats
does not show appreciably better binding for any of the proteins. A
cluster of three repeats seems to be the minimal binding site
detectable by this assay, because C6-C7 and C5 alone have negligible
binding. This is consistent with the observation that LRP ligand
binding domains contain at least three consecutive complement-type
repeats. The unusual cluster of two repeats found at the N-terminus of LRP (region I) is unable to bind RAP.9
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| Fig 5.
Qualitative binding of lactoferrin ( ), PAI-1 ( ),
and RAP ( ) to LRP fragments. The ability of various LRP fragments to
bind iodinated protein ligands at a single concentration (25 nmol/L)
was investigated by direct binding assay. 125I-RAP,
125I-lactoferrin, and 125I-PAI-1 were bound to
5 pmol of various LRP fragments in the presence of 2 mmol/L
CaCl2. BSA was used as a nonreceptor control. Nonspecific
binding was measured in parallel wells without calcium and in the
presence of 40 mmol/L EDTA. Fragment E4-C10 was used as the 100%
binding control and had the following cpm: 125I-RAP = 31,707 ± 1433; 125I-lactoferrin = 14,072 ± 255; and
125I-PAI-1 = 4,098 ± 60. Triplicate points in duplicate
experiments were used to calculate a mean and SD for each receptor.
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Reciprocal competition of RAP, PAI-1 and lactoferrin on C5-C7 and
E4-C10.
To confirm that RAP, PAI-1, and lactoferrin bind to a single region
encompassed by C5-C7, we performed competition experiments between
iodinated RAP, PAI-1, lactoferrin, and unlabeled ligands. The effect of
increasing concentrations of the two unlabeled ligands on
125I-RAP and 125I-lactoferrin binding is shown
in Fig 6, and the corresponding IC50 values are summarized in
Table 3 along with values from experiments
with 125I-PAI-1. Each ligand is able to reduce binding of
each of the others by nearly 100%. The IC50s for unlabeled
RAP against the other ligands are similar to its Kd, consistent with
its high affinity for the receptor. Cold PAI-1 competes equally well
against 125I-RAP bound to either receptor, suggesting that
C5-C7 contains the entire PAI-1 binding site. Differences in affinity
for the two receptors are mainly observed with lactoferrin. Unlabeled lactoferrin is more efficient at competing with 125I-RAP
bound to E4-C10, suggesting that its binding site may extend outside of
C5-C7. Consistent with this, higher concentrations of PAI-1 are
required to compete with 125I-lactoferrin bound to the
larger receptor fragment. Further competition experiments are required
to determine the exact boundaries of the lactoferrin binding site.
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| Fig 6.
Reciprocal competition of LRP ligands on binding to C5-C7
or E4-C10. Binding of 10 nmol/L 125I-lactoferrin (A and B)
was competed with RAP ( ) and active PAI-1 (); binding of 2 nmol/L
125I-RAP (C and D) was competed with lactoferrin () and
active PAI-1 (). Triplicate experiments were used to calculate a
mean and SD for each data point. In each experiment, the concentration
of the competing unlabeled ligand ranged from 0.01 pmol/L to 10 µmol/L. Nonspecific binding was measured in parallel wells without
calcium and in the presence of 40 mmol/L EDTA. The IC50
value for each competing ligand was determined by using a single-site
competition model and is shown in Table 3.
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DISCUSSION |
To obtain sufficient quantities of LRP receptor to perform
structure-function studies, we have developed a prokaryotic secretion system that is capable of producing fragments of active LRP. Folding and disulfide formation occur in bacteria despite the absence of the
mammalian molecular chaperone, RAP. This is not totally unexpected,
because RAP-deficient mice have about 25% of the normal amount of
LRP.26 Recombinant proteins representing portions of the
LRP region II were purified from bacterial periplasm and tested for
their ability to bind iodinated RAP, lactoferrin, and PAI-1 in a
solid-phase assay. Binding activity of the fragments is dependent on
the presence of their disulfide bonds and Ca2+, and can be
reversibly blocked by EDTA. Comparison of different-sized fragments
indicates that all three protein ligands bind to a group of
complement-type repeats in the middle of region II, namely C5-C7. This
is the first demonstration of the position of a lactoferrin binding
site on LRP and that a single binding site on LRP is capable of binding
three different proteins, including RAP. The fact that a
three-complement repeat binds ligands as well as the eight-complement repeat cluster shows that the binding activity of the site is independent of its position or context within the cluster and is
probably due to unique properties of the C5-C7 repeats.
LRP region II had previously been identified as a binding site for
1M light chain (and presumably 2M),
uPA.PAI-1, and RAP on the basis of ligand
blots.27 While this work was in progress, two reports
further defining the RAP and PAI-1 binding sites of recombinant LRP
region II were published. Obermoeller et al11 used ligand
blots to examine binding of RAP and RAP subdomains to all four regions
of LRP containing complement-type repeats, and Horn et al17
analyzed RAP, PAI-1, t-PA.PAI-1, and Fab A8 binding by
means of surface plasmon resonance. With respect to RAP, our results
agree well with those of Horn et al because we both identify C5-C7 as
the binding site, although Kds determined by the solid-phase binding
assay are lower than those measured by surface plasmon resonance.
However, Horn et al failed to observe PAI-1 binding to C5-C7 alone, and
concluded that the PAI-1 binding site only partially overlaps the RAP
binding site and extends towards the fourth EGF-like repeat at the
N-terminus of region II. We did not measure the Kd values for
125I-PAI-1 or 125I-lactoferrin by direct
binding due to technical limitations, although we did examine their
competition of equimolar amounts of 125I-RAP bound to
different receptors that had the same Kd for RAP. In our experiments,
cold PAI-1 gave a similar IC50 when competing against
125I-RAP bound to either the eight complement-type repeat
fragment (E4-C10) or C5-C7. This suggests that both fragments contain
the entire PAI-1 binding site, and that RAP and PAI-1 may bind to a
similar surface of the receptor. Possible reasons for discrepancies between Horn et al's results and our solid-phase binding assays are
that the ligands were immobilized for the surface plasmon resonance measurements, whereas we immobilized the
receptor fragments,17 the presence of an epitope tag on the
N-termini of their fragments versus on the C-termini of ours, and
absence of glycosylation in the bacterial proteins.
In the case of lactoferrin, the approximately twofold higher
IC50 against 125I-RAP bound to the small
receptor fragment and its pattern of competition with cold PAI-1 could
mean that its binding site extends beyond C5-C7. These results also
suggest that, unlike RAP and PAI-1, lactoferrin interacts with a
different subset of the receptor residues. Despite this, unlabeled RAP
and PAI-1 are able to block binding of 125I-lactoferrin by
90% to 100%, indicating substantial overlap of the binding sites
resulting in steric hindrance. Our results showing reciprocal-competition between RAP and lactoferrin seem to contradict the conclusions of cell-uptake studies. It was previously noted in
uptake experiments on LDL-deficient fibroblasts that lactoferrin degradation was inhibited up to 60% by GST-RAP, but lactoferrin was
unable to block uptake of
125I-t-PA.PAI-1.15,28
Nonreceptor-mediated binding of LRP ligands to heparin and the presence
of additional lactoferrin receptors may complicate the results of cell
culture experiments.12,29 In addition, the
t-PA.PAI-1 complex seems to have a higher affinity for LRP
and recombinant LRP fragments compared with PAI-1 alone, which may
decrease its ability to be displaced by lactoferrin.17,30
In the study by Obermoeller et al,11 the eight
complement-type repeats of region II were divided into two halves and
assayed for RAP binding activity. Surprisingly, fragments representing C3-C6 and C7-C10 bound equally well to immobilized RAP. This suggested the possibility of two distinct RAP binding sites. However, our results
and those of Horn et al17 argue against this. The C8-C10 fragment17 and C9-E6 fragment do not bind RAP, indicating
that C7 must be crucial for the RAP binding observed on ligand blots. Because C7 is a part of C5-C7, region II may only contain one RAP
binding site, as is consistent with our results on the direct binding
assays. The number of binding sites presented by equimolar amounts of
E4-C10 and C5-C7 for 125I-RAP, the Bmax, was
actually less for E4-C10 than for C5-C7, lending support to the idea of
a single binding site. Of course, the Bmax might be
affected by the mode of binding of each fragment with the surface of
the plate. Another question is whether two molecules of 39-kD RAP would
be able to occupy adjacent binding sites on a series of four
complement-type repeats, representing an 18- to 19-kD fragment.
Currently little information is available about the complement-repeat
receptor:ligand interactions at the atomic level. The spatial
relationship between the repeats constituting LRP is unknown, although
structures of single repeats have been solved.31-33
Mutagenesis and peptide competition studies have been used in attempts
to identify the LRP binding sites on some of its ligands. The three binding sites on RAP have been most extensively
studied.11,34,35 We and others showed that replacement of
K1370 (human numbering) in the receptor binding fragment of
2M with an alanine effectively blocks receptor
binding.36,37 Lysine residues have also been implicated in
Pseudomonas exotoxin A,38 lipoprotein
lipase,39 and PAI-140 binding sites for LRP.
The recent x-ray structure of the LDL ligand-binding
repeat has ruled out the direct interaction of the lysines with the
conserved aspartate residues, because the latter are involved in
Ca2+ coordination.33 Details of the
interactions will be best answered by solving the three-dimensional
structure of an LRP ligand bound to a receptor fragment. The
recombinant LRP fragments that we have produced in bacteria should
prove useful in determining the stoichiometry of ligand binding and
help advance our understanding of the protein:protein interactions
between LRP and its ligands to the molecular level of detail.
| |
FOOTNOTES |
From the Department of Pharmacology, University of Texas
Southwestern Medical Center, Dallas, TX.
Submitted March 25, 1998; accepted June 25, 1998.
Supported by American Heart Association Texas Affiliate Grant-in-Aid
966-063.
Address reprint request to Dianne DeCamp, PhD, Department of
Pharmacology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75235-9041.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are indebted to our colleagues, Drs Elizabeth Goldsmith and Joachim
Herz, at the University of Texas Southwestern Medical Center, Dallas,
for providing plasmids and cDNA. We would also like to thank Dr Stephen
Sprang of the Howard Hughes Medical Institute at the University of
Texas Southwestern Medical Center, Dallas, for his comments on this
manuscript.
| |
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Abstract
Introduction
Abstract