From the Department of Molecular and Structural
Biology, University of Aarhus, DK-8000 Aarhus C, Denmark, the
§ Departments of Cell Biology and Medicine, Baylor College
of Medicine, Houston, Texas 77030, and the
¶ Friedrich-Miescher Institute, P.O. Box 2543, CH-4002
Basel, Switzerland
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ABSTRACT |
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The very low density lipoprotein receptor (VLDLR)
binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine
proteinase-serpin complexes, including complexes of the proteinase
urokinase-type plasminogen activator (uPA) with the serpins plasminogen
activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have
analyzed the binding of RAP, uPA·PAI-1, and uPA·PN-1 to two
naturally occurring VLDLR variants, VLDLR-I, containing all eight
complement-type repeats, and VLDLR-III, lacking the third
complement-type repeat, encoded by exon 4. VLDLR-III displayed
~4-fold lower binding of RAP than VLDLR-I and ~10-fold lower
binding of the most C-terminal one of the three domains of RAP. In
contrast, the binding of uPA·PAI-1 and uPA·PN-1 to the two VLDLR
variants was indistinguishable. Surprisingly, uPA·PN-1, but not
uPA·PAI-1, competed RAP binding to both VLDLR variants. These
observations show that the third complement-type repeat plays a crucial
role in maintaining the contact sites needed for optimal recognition of
RAP, but does not affect the proteinase-serpin complex contact sites,
and that two ligands can show full cross-competition without sharing
the same contacts with the receptor. These results elucidate the
mechanisms of molecular recognition of ligands by receptors of the low
density lipoprotein receptor family.
The low density lipoprotein receptor
(LDLR)1 family comprises a
group of multiligand endocytosis receptors with a characteristic domain
structure. Besides LDLR, the known mammalian family members are
VLDLR binds a variety of serine proteinase-serpin complexes (13-16),
the proenzyme form of urokinase-type plasminogen activator (uPA) (14),
apolipoprotein E-containing lipoproteins (17, 18), apo(a) (19),
lipoprotein lipase (14, 20), and thrombospondin-1 (21). VLDLR contains
one CTR cluster (positioned at the N terminus), two EGF-like repeats
that are separated from a third one by a YWTD spacer region, an
O-linked sugar domain, a transmembrane domain, and a
cytoplasmic domain (17, 22-24). Mature VLDLR mRNA is
differentially spliced. Fig. 1 shows a
presentation of the four corresponding receptor variants. One variation
affects exon 16, encoding the O-linked sugar domain.
VLDLR-II, lacking the O-linked sugar domain, binds the
Mr 40,000 receptor-associated protein (RAP) and
serine proteinase-serpin complexes with the same affinity as VLDLR-I
(25). A second variation affects exon 4, encoding the third (CTR-3) out
of the total eight CTRs. VLDLR mRNAs with and without exon 4 have
been demonstrated in human brain (26) and in a human neuroblastoma cell
line (27), suggesting that VLDLR without CTR-3 is expressed in neural
tissue.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin receptor/low density lipoprotein
receptor-related protein (
2MR/LRP), gp330/megalin, and
very low density lipoprotein receptor (VLDLR) (for reviews, see Refs.
1-3) and apolipoprotein E receptor-2 or LR8B
(LDLR-relative with 8
binding repeats) (4, 5). All members of the family contain
a C-terminal cytoplasmic domain with at least one copy of the sequence
NPXY, directing internalization via clathrin-coated pits,
followed by a single transmembrane domain. The N-terminal extracellular
part of the receptors contains clustered complement-type repeats (CTRs)
and epidermal growth factor (EGF) precursor homology domains, the latter consisting of multiple copies of cysteine-rich EGF-like repeats
and spacer regions with the consensus sequence YWTD (for a review, see
Ref. 2). The members of the family show distinct although partially
overlapping binding specificities and bind with high affinity,
Ca2+-dependently, a variety of ligands like
proteinases, proteinase-inhibitor complexes, apolipoproteins,
extracellular matrix proteins, and viruses (for reviews, see Refs. 1,
2, 6, and 7). Ligand binding seems to be mediated by the CTR clusters
(8-12).
View larger version (27K):
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Fig. 1.
Schematic presentation of the structure of
VLDLR. A, schematic showing the domain structure
of VLDLR-I, VLDLR-II, VLDLR-III, and VLDLR-IV. Boxes
1-8 represent the ligand-binding complement-type repeats.
Also indicated are the EGF precursor homology domain region with the
EGF-like repeats A and B separated from repeat C by the YWTD spacer,
the O-linked sugar domain (short bar), the
transmembrane domain (long bar), and the cytoplasmic domain
with the NPXY sequence (×). Exons encoding the respective
regions are indicated above the presentation of the VLDLR-I domain
structure. B, nucleotide and derived amino acid sequences
around exon 4 of VLDLR-I, VLDLR-II, VLDLR-III, and VLDLR-IV.
The Mr 40,000 receptor-associated protein (RAP)
binds with high affinity to 2MR/LRP and gp330/megalin
(for reviews, see Refs. 1 and 28), VLDLR (18, 29), and LR8B (5), but
with only low affinity to LDLR (30, 31). The biological function of RAP
may be that of a molecular chaperone in the endoplasmic reticulum
(32-34). Mature human RAP consists of 323 amino acids (35). Due to
internal sequence homology, a three-domain structure has been proposed
(36, 37). The three-dimensional structure of the N-terminal domain was
determined by NMR spectroscopy (38).
Many of the ligands binding to one and the same family member do not
show any obvious structural similarity, and there is no general
cross-competition between the different ligands. For example,
2-macroglobulin-proteinase complexes and uPA-serpin complexes do not compete for binding to
2MR/LRP (42).
RAP is able to inhibit the binding of all known ligands, whereas almost all other ligands do not inhibit RAP binding (for reviews, see Refs. 1
and 28). Equilibrium binding and competition patterns between different
ligands have shown that different domains of RAP and of serine
proteinase-serpin complexes make independent contacts with the
receptors (15, 36, 39, 40, 41, 43-45). Thus, these receptors exhibit
unique principles of molecular recognition.
To elucidate the mechanisms of molecular recognition of ligands by this
receptor family, we have now compared the binding of RAP and of
complexes between the serine proteinase uPA and the serpins plasminogen
activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1) to the VLDLR
variants VLDLR-I and VLDLR-III, differing with respect to the presence
of CTR-3 (Fig. 1).
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EXPERIMENTAL PROCEDURES |
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VLDLR cDNA Constructs-- cDNA of the VLDLR types II and III was generated by PCR-based mutagenesis of phV58, the human VLDLR-I (full-length) cDNA cloned in pBluescript KS (22). VLDLR-II cDNA, lacking exon 16, was generated with primers 5'-CTG GAT CAG AGC TAG CCA CTC-3' (upstream primer), 5'-AGG AAA ATG GCC GAG ACT GTC AAA GGA TCA ATG TGA CCA CAG CAG TAT-3' (mutagenesis primer), and 5'-TCC TAT TGC CAT TGT CCC AAC CAT TGA A-3' (downstream primer). The NheI/HindIII fragment of the PCR product and the EcoRI/NheI fragment of phV58 were ligated into the EcoRI/HindIII sites of pBluescript KS. VLDLR-III cDNA, lacking exon 4, was generated with primers 5'-TTC TTC CTC CTT TCG GAA GGG CTG-3' (upstream primer), 5'-CAG ATG AAA GCC CAG AAC AGT GCC GCA ATA TAA CAT GTA GTC CCG ACG-3' (mutagenesis primer), and 5'-GAC ACT CTT TCA GGG GCT C-3' (downstream primer). The NcoI fragment of phV58 was exchanged with the NcoI fragment of the PCR product. VLDLR-IV cDNA was generated by ligation of the VLDLR-III EcoRI/NheI fragment and the VLDLR-II NheI/HindIII fragment into the EcoRI/HindIII sites of pBluescript KS. The cDNAs of the VLDLR variants were excised from the cloning vectors by BamHI/ClaI digestion and cloned into the BglII/ClaI sites of expression vector pCMV4 (46). The intended changes were confirmed by nucleotide sequencing.
VLDLR Expression and Purification-- CHO-ldlA7 cells, a line of mutant Chinese hamster ovary cells lacking LDLR (47), were stably transfected with cDNA for the various VLDLR variants (13). The transfected cells were maintained in Ham's F-10 medium with 5% fetal bovine serum and 0.4 mg/ml G418. The expression of the correct VLDLR mRNA (and only the correct ones) by the transfected cells was confirmed by reverse transcription-PCR with primers spanning exons 4 and 16, respectively. The primers spanning exon 16 (VLR-1 and VLR-2) were those described previously (26). PCR products of 377 and 293 base pairs (bp) were expected from mRNAs with and without exon 16, respectively. The primers spanning exon 4 (vle3 and vle5) were those described previously (27). PCR products of 390 and 266 bp were expected from mRNAs with and without exon 4, respectively. The transfected cells expressed mRNA giving the expected reverse transcription-PCR products, as shown in Fig. 2 in the case of VLDLR-I and VLDLR-III cDNA-transfected cells. The transfected cell lines expressed a VLDLR immunoreactivity on Western blots (see below) that was 10-20-fold higher than that of the parental cell line (data not shown).
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Cell extracts were prepared by washing confluent cell monolayers with
Tris-buffered saline (TBS; 50 mM Tris-HCl, pH 7.4, and 150 mM NaCl), scraping the cells from the culture dishes with a
rubber policeman, pelleting the cells by centrifugation at 1000 × g, lysing the cells in ice-cold binding buffer (20 mM HEPES, 2.5 mM
Na2HPO4, pH 7.4, 124 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, and 1.2 mM MgSO4) with 1% CHAPS and 1 mM
phenylmethylsulfonyl fluoride using 0.5 ml of buffer/15-cm diameter
dish, and centrifuging at 15,000 × g at 4 °C. The
supernatant, representing the cell extract, was used directly or stored
at 20 °C with glycerol at a final concentration of 50%.
The VLDLR variants were purified from extracts of transfected CHO cells
by RAP affinity chromatography on Sepharose 4B columns coupled with
~2 mg of RAP/ml of Sepharose, equilibrated in binding buffer with 1%
CHAPS. About 0.5-1 mg of total protein from cell extracts was applied
per mg of immobilized RAP. After the application, the columns were
washed with 0.1 M Tris-HCl, pH 7.8, 0.14 M
NaCl, 2 mM CaCl2, and 1% CHAPS. Bound protein
was eluted with 0.1 M CH3COOH, pH 3.5, 0.5 M NaCl, 1% CHAPS, and 10 mM EDTA. The eluates were neutralized by addition of 1 M Tris-HCl, pH 9.0, and
25 mM CaCl2; concentrated in a dialysis bag by
applying polyethylene glycol 35,000 to the outside; and dialyzed
against TBS supplemented with 1% CHAPS and 2 mM
CaCl2. The receptor preparations were stored at 20 °C
in 50% glycerol.
Miscellaneous Proteins--
Recombinant human RAP was prepared
as described (42). The RAP domains were expressed as fusion proteins.
The constructs (carrying the first 34 N-terminal residues of the phage CII repressor, a hexahistidine affinity tag, and a factor Xa
recognition site at the N terminus of the RAP sequences) were
constructed, expressed, and purified as described (37). The construct
D1RAP contained amino acids 18-112, D2RAP
amino acids 113-218, D3RAP amino acids 219-323,
D1D2RAP amino acids 18-218, and D2D3RAP amino
acids 113-323. RAP and D3RAP were labeled with
125I as described (48).
Rat PN-1 and human PAI-1 were prepared as described (49, 50). Human uPA was purchased from Serono (Aubonne, Switzerland). 125I-uPA·PAI-1 complex was prepared as described (45). 125I-uPA·PN-1 complex was prepared by reacting 125I-labeled uPA with a 5-fold excess of PN-1 and then removing the excess of PN-1 by immunoaffinity chromatography with a monoclonal anti-uPA antibody immobilized on Sepharose 4B. The preparation was treated with diisopropyl fluorophosphate to block any contamination with uncomplexed uPA (15).
Antibodies-- A rabbit polyclonal antibody against the amino acid sequence ASVGHTYPAISVVSTDDLA, representing the C terminus of human VLDLR, was raised and purified as described (13).
Electrophoresis, Immunoblot Analysis, and Ligand Blot Analysis-- SDS-PAGE was performed by standard methods on 4-16% gradient gels using a Sigma prestained SDS Mr marker kit. Silver staining was performed with a Bio-Rad silver stain kit. For immunoblot and ligand blot analyses, samples were subjected to SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride (PVDF) filters. Immunoblot analysis was performed by standard methods using the Amersham ECLTM detection system. Ligand blot analysis was performed by incubating the filters overnight with radioactively labeled ligands in binding buffer (see above) supplemented with 0.5% bovine serum albumin and visualizing bound ligand by autoradiography (29).
Binding Assays in Microtiter Wells--
VLDLR-ligand binding
assays in microtiter wells were performed using anti-VLDLR antibodies
for receptor immobilization. Rabbit polyclonal anti-VLDLR antibodies
against the C terminus of the receptor were coated onto microtiter
wells (Maxisorb, NUNC, Roskilde, Denmark) by incubating the wells with
100 µl of 2 µg/ml antibody in 50 mM
NaHCO3/Na2CO3, pH 9.6, for 2 h
at room temperature. After blocking nonspecific binding sites with 5%
skim milk powder in TBS with 0.5% Tween 20 (TBS-T), the wells were
incubated with 100 µl of cell extract or solution of partially
purified receptor, diluted 10-fold with TBS, for 3 h at room
temperature. The wells were then incubated with radioactive and
nonradioactive ligands in binding buffer with 0.5% bovine serum
albumin for 16 h at 4 °C. The incubation media were collected;
the wells were washed three times with ice-cold TBS-T; and bound
radioactivity was eluted with 1 M NaOH for 30 min at
37 °C. The radioactivity in the incubation media and the eluates was
determined by -counting.
Whole Cell Binding Assays-- The cells were grown in 1.5-cm diameter wells to a density of ~5 × 105 cells/well. The desired ligands were added to the cells in 2 ml of binding buffer with 0.5% bovine serum albumin. The cells were incubated for 16 h on ice. The media were then collected; the cells were washed with ice-cold binding buffer and solubilized with 1 M NaOH; and the radioactivity in the media and bound to the cells was determined. The cell densities were chosen in a range in which the amount of bound ligand did not exceed 15% of the amount added.
Mathematical Analysis of Receptor-Ligand Binding
Data--
Dissociation constants (Kd) for
receptor-ligand binding were found by analyzing the concentration
dependence of steady-state binding. The ratios between the
concentrations of bound and free ligand (B/F) were determined with
1-10 pM radiolabeled ligand plus several concentrations of
nonradioactive ligand. The B/F ratios were then plotted
semilogarithmically versus the free ligand concentrations.
The data were fitted by the method of least squares to the equation
B/F = [RL]/[L] = ([R]t/(Kd + [L])) + C, derived under the assumption of a simple
binding equilibrium R + L RL. C is a constant
representing non-displaceable, non-receptor binding. R represents the
receptor, L the ligand, and RL the receptor-ligand complex. This
procedure yields the Kd and [R]t values
giving the best fit to the data.
In competition studies, a picomolar concentration of a radioactively
labeled ligand was incubated with the receptors in the presence of
varying concentrations of another, nonradioactive ligand. Assuming
simple binding equilibria for both ligands, R + L RL and R + I
RI, the B/F ratio for the radioactively labeled ligand is, in this
case, related to the concentration of the nonradioactive ligand ([I])
by the equation B/F = [RL]/[L] = ([R]t/(Kd + [L] + (Kd/Ki)[I])) + C.
Ki is the dissociation constant for the binding of I
to R. Ki can be derived by fitting the binding data
by the method of least squares to the above equation when the values
for [L] and Kd are known.
Chemical Cross-linking--
Affinity-purified VLDLR variants and
125I-RAP were dialyzed against 100 mM sodium
citrate, pH 7.0, 150 mM NaCl, 2 mM
CaCl2, and 0.5% CHAPS. An ~0.5 nM
concentration of each of the VLDLR variants was incubated with 4 nM 125I-RAP in a final volume of 50 µl for
16 h at 4 °C in the absence or presence of 50 mM
EDTA. Then, 50 µl of 0.2% glutaraldehyde in 2 mM
CaCl2 was added, and the incubation was continued for an
additional 1.5 h. After addition of 10 µl of 2 M
glycine, incubation for another 1.5 h, and reduction of the
volumes by vacuum centrifugation, the samples were subjected to
SDS-PAGE and autoradiography.
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RESULTS |
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Affinity Purification and Ligand Blot Analysis of VLDLR Variants-- We purified each of the VLDLR variants VLDLR-I, VLDLR-II, VLDLR-III, and VLDLR-IV from CHAPS extracts of CHO cells transfected with the corresponding cDNAs by affinity chromatography on a column of Sepharose 4B-immobilized RAP. The receptor yield was estimated to ~0.5-1.5% of the total CHAPS-extractable protein and was indistinguishable for the four receptor types, showing that all the variants were able to bind RAP. The purity of VLDLR-I and VLDLR-III is presented by a silver-stained gel (Fig. 3A), showing distinct bands with the same migration as the bands observed in immunoblot analysis with an antibody against the VLDLR C terminus, which is identical in the four variants (Fig. 3B).
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The relative affinities of RAP and serine proteinase-serpin complexes for each of the four VLDLR variants were screened by ligand blot analysis of the affinity-purified preparations. The relative amount of VLDLR protein of the different types was evaluated by immunoblot analysis. More than 95% of the binding of radiolabeled ligands in the position of VLDLR immunoreactivity could be displaced by an excess of nonradioactive RAP (data not shown). With equal amounts of immunoreactivity of VLDLR-I and VLDLR-III (Fig. 3B), the signal obtained with 20 pM 125I-RAP was ~5-fold stronger with VLDLR-I than with VLDLR-III (Fig. 3C). However, the signals obtained after incubation of each of the two variants with 20 pM 125I-uPA·PN-1 or 20 pM 125I-uPA·PAI-1 were indistinguishable (Fig. 3, D and E). VLDLR-II and VLDLR-IV showed binding properties identical to those of VLDLR-I and VLDLR-III, respectively (data not shown).
Microtiter Well Binding Assays-- To confirm the above-mentioned results with receptors, which had not been exposed to the denaturing conditions used in the ligand blot analysis, we performed binding analysis with receptors immobilized in microtiter wells. A convenient immobilization procedure was coating of the wells with anti-VLDLR antibody, followed by binding of the receptors to the antibodies. Incubation of the antibody-coated wells with an excess of receptor over antibody was assumed to assure immobilization of equal amounts of receptor, irrespective of the exact concentration in the receptor-containing sample, allowing direct comparison of results obtained with the different receptor types. This assumption was shown to be true by demonstrating equal binding of 125I-RAP in two antibody-coated wells, successively incubated with the same receptor preparation (data not shown). As source of receptor, both purified preparations and crude cell extracts could be used, with indistinguishable results (data not shown). By this method, the receptor variants without CTR-3 were found to have an ~3-fold reduced 125I-RAP binding, whereas the binding of 125I-uPA·PN-1 was indistinguishable between the variants (Table I).
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The same assay was used for studying the concentration dependence of
steady-state binding of RAP to VLDLR-I and VLDLR-III in the
concentration range 1-105 pM. The use of lower
concentrations was limited by the specific activity of the radiolabeled
RAP. The concentration dependence of binding did not deviate
significantly from that expected from the simple binding reaction R + L
RL (data not shown). Analyzing the data accordingly, the estimated
Kd values were 5.6-13.7 pM for VLDLR-I
and 5.4-12.5 pM for VLDLR-III, with an average of 9 pM for both VLDLR-I and VLDLR-III. In contrast, the
estimated total concentration of binding sites ([R]t) was
3.4-fold lower with VLDLR-III than with VLDLR-I.
Whole Cell Binding Assays--
To substantiate further the
difference in RAP binding properties between VLDLR variants with and
without CTR-3, we studied the concentration dependence of steady-state
binding of RAP, in the concentration range 1-105
pM, to intact CHO cells transfected with VLDLR-I or
VLDLR-III (Fig. 4). The concentration
dependence of the binding did not deviate significantly from that
expected from the simple binding reaction R + L RL. The following
Kd values were found: VLDLR-I, 16.2 pM
(range 8.7-34.7 pM, n = 6); and VLDLR-III,
79.4 pM (range 49.2-151.0 pM,
n = 3), corresponding to an ~5-fold and statistically
significant (p < 0.001) difference between the
Kd values. It was not possible to analyze whether
the variants had different numbers of RAP-binding sites per receptor
molecule by the whole cell binding assays, as the relative number of
receptor molecules expressed on the cell surfaces of individual
transfected cell lines could not be determined with sufficient accuracy
by immunochemical methods.
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Binding of Individual Domains of RAP to the VLDLR Variants-- In ligand blot analysis with the radiolabeled C-terminal domain of RAP (D3RAP), the difference in binding between VLDLR-I and VLDLR-III was even more pronounced than with the entire RAP molecule (Fig. 5A). To obtain a clear signal with D3RAP and VLDLR-III, longer exposure times or more VLDLR-III had to be applied (Fig. 5B). We did not perform ligand blot analysis with the N-terminal (D1RAP) and middle (D2RAP) domains, as they could not be radioactively labeled with sufficiently high specific activity.
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To evaluate further the importance of individual RAP domains, we screened 200 nM concentrations of unlabeled domains for their ability to compete for the binding of 20 pM 125I-RAP by ligand blot analysis. For these experiments, the amounts of VLDLR-I and VLDLR-III loaded onto the gels were adjusted to result in equal signals with 125I-RAP as ligand. D3RAP competed the binding of 125I-RAP to both VLDLR-I and VLDLR-III, but the competition was less efficient with VLDLR-III than with VLDLR-I, not only with 200 nM D3RAP (Fig. 5B), but also with lower D3RAP concentrations (data not shown). We did not find competition of RAP binding with D1RAP, D2RAP, or a fragment consisting of these domains together (D1D2RAP). In contrast, a fragment consisting of the middle and C-terminal domains (D2D3RAP) inhibited 125I-RAP binding to VLDLR-I and VLDLR-III (Fig. 6), with equal efficiency for the two receptor variants (data not shown).
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We used the microtiter well assay to analyze quantitatively the competition of 125I-RAP binding by D3RAP. We found that half-inhibition of the binding of 20 pM 125I-RAP required ~10-fold higher D3RAP concentrations with VLDLR-III than with VLDLR-I (Fig. 7). The Ki values estimated according to a simple competitive binding model were 0.3 and 3 nM for VLDLR-I and VLDLR-III, respectively.
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Cross-competition between RAP and uPA-Inhibitor Complexes-- Unexpectedly, 200 nM uPA·PN-1 competed the binding of 20 pM 125I-RAP efficiently, whereas 200 nM uPA·PAI-1 was unable to compete 125I-RAP binding detectably (Fig. 8).
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Chemical Cross-linking of VLDLR·RAP Complexes-- To analyze whether VLDLR contains multiple RAP-binding sites, we chemically cross-linked complexes of 125I-RAP and affinity-purified VLDLR variants. Fig. 9 shows an autoradiogram of SDS-PAGE of cross-linked complexes of 125I-RAP with VLDLR-I, VLDLR-II, and VLDLR-III, respectively. The faster migration of VLDLR-II was used as a control for identification of the complexes. As an additional control, we used the fact that EDTA could inhibit complex formation, in agreement with the Ca2+ dependence of the binding. The Mr values of the complexes, estimated on the basis of their migration on the gel, corresponded to one RAP molecule binding to each VLDLR molecule.
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DISCUSSION |
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We have compared the ligand binding properties of VLDLR variants resulting from translation of an mRNA with all known exons and of an mRNA without exon 4, encoding CTR-3. We found that VLDLR variants without CTR-3 bound RAP weaker than those with CTR-3, whereas all variants displayed indistinguishable binding of the serine proteinase-serpin complexes uPA·PAI-1 and uPA·PN-1. Our results are compatible with the notion that the CTR cluster is implicated in ligand binding, as expected from results with other receptors of the family. Our work represents the first study of the influence of individual CTRs on the ligand binding properties of VLDLR. Our findings suggest that also other ligands may have different affinities for VLDLR variants with and without CTR-3. VLDLR mRNA without exon 4 has so far only been shown to occur naturally in brain (26) and in a neuroblastoma cell line (27).
The following points strongly argue against the possibility that the lower RAP binding of the VLDLR variants without CTR-3 is an artifact due to abnormal folding of some of the molecules, for instance as a result of scrambling of disulfide bridges. 1) Differences in RAP binding could be demonstrated with receptors directly on the surface of intact cells. 2) The binding of the two serine proteinase-serpin complexes was indistinguishable, despite the different binding of RAP. 3) The residual RAP binding displayed by VLDLR-III had properties different from those of the RAP binding by VLDLR-I since it was more weakly competed by D3RAP. 4) VLDLR-III could be purified by affinity chromatography with immobilized RAP in a high yield, excluding the possibility that the low binding was due to a large fraction of the VLDLR-III molecules being in a misfolded state unable to bind to RAP. We are therefore confident that our data allow us to conclude that the weaker RAP binding of VLDLR-III is a property of a native, properly folded protein produced by translation of the mRNA without exon 4.
The observation that VLDLR-I and VLDLR-III show different RAP binding,
but indistinguishable serine proteinase-serpin complex binding,
strongly suggests that different contributions by different CTRs to
binding of different ligands are an important determinant of ligand
binding specificity of VLDLR and other LDLR family members. An
additional important, previously reported observation is that ligand
binding involves multiple contacts between ligand and receptor. This
observation was made by analyses of the binding of individual RAP
domains to 2MR/LRP and gp330/megalin (40-44) and of
other serine proteinase-serpin complexes to
2MR/LRP and
VLDLR (15, 45). The most simple binding model accounting for both the
observations of the contributions from different CTRs and the
multicontact hypothesis is one in which each of the multiple contact
sites is localized in a particular CTR. In the present case, this model would imply that a direct contact between VLDLR CTR-3 and RAP contributes strongly to RAP binding. An alternative binding model is
one in which binding of different ligands depends in different ways on
receptor conformation, the different binding properties of VLDLR-I and
VLDLR-III being due to a conformational effect of CTR-3, resulting in
presentation of different RAP-binding surfaces in the two variants.
This model would also imply that the spatial arrangement of the CTRs
determining the binding surface for the proteinase-serpin complexes is
unaffected by the lack of CTR-3. Along the same line, RAP binding could
result in a change in the spatial arrangement of the CTRs, with RAP
stabilizing a receptor conformation preventing the exposure of binding
sites for other ligands, in agreement with previous proposals for the
RAP-
2MR/LRP interaction (36, 39, 51, 52). The two models
are not mutually exclusive. With both, a combined contribution from
several CTRs to a multicontact binding of each ligand would explain how
the same receptor can exhibit high affinity binding of several,
structurally unrelated ligands and how the variations in amino acid
sequence in CTRs in different receptors can give rise to different, but overlapping binding specificities. This proposal is in agreement with a
previous hypothesis by Russell et al. (9), working with LDLR.
A second important observation described here is the ability of
uPA·PN-1 to compete RAP binding to VLDLR. RAP competes the binding of
all known ligands to 2MR/LRP, gp330/megalin, and VLDLR (for a review, see Ref. 2). In contrast, the only other ligand so far
reported to be able to inhibit RAP binding was lipoprotein lipase, in
the case of
2MR/LRP (40). The capability of uPA·PN-I for competition of VLDLR·RAP binding may be related to its unusually high affinity for VLDLR, the Kd being ~140
pM, ~6-fold lower than the Kd for
binding of uPA·PAI-1 (15). Since binding of RAP and uPA·PN-1 is
differently affected by the lack of CTR-3, we must contend that two
ligands can compete for binding without sharing the same contact sites.
Important aspects of VLDLR-I and VLDLR-III ligand binding could be
explained by the simple binding model R + L RL. Thus, both the
concentration dependence of steady-state RAP binding and the
concentration dependence of inhibition of RAP binding by
D3RAP were in agreement with those predicted by the simple
binding model. Moreover, the whole cell binding assays showed an
~5-fold difference in apparent Kd for RAP binding
to VLDLR-I and VLDLR-III, and the microtiter well binding assay showed
an ~10-fold difference in the apparent Ki for
competition of RAP binding to VLDLR-I and VLDLR-III by
D3RAP, indicating a 10-fold difference in
Kd for D3RAP binding. The latter
observations were in agreement with our a priori expectancy
from the simple binding model of the different RAP binding resulting
from differences in Kd. But we were unable to detect
a difference in the apparent Kd for RAP binding to
VLDLR-I and VLDLR-III in the microtiter well binding assay, suggesting
that the difference in Kd was <3-fold under these
conditions and thus smaller than the difference observed in the whole
cell binding assay. Differences concerning Kd
determinations by whole cell and in vitro binding assays
have also been observed with other receptors (see, for instance, Ref.
53) and are most likely caused by differences in the microenvironment
rather than differences in the receptors themselves. In the present
case, this would mean that the same structural difference between the
two VLDLR variants would have different consequences for the difference
in apparent Kd in different microenvironments. The
difference in Kd for RAP binding to the variants was
also considerably smaller than the difference in Kd
for D3RAP binding. This observation can be explained by an
assumption of the N-terminal two and the C-terminal one of the three
domains of RAP, respectively, making independent binding contacts with VLDLR and only the contact of the C-terminal domain being affected by
the lack of CTR-3. This is in agreement with the observation that
D2D3RAP is a better competitor of RAP binding to VLDLR-III
than D3RAP. At the moment, however, we cannot explain the
unexpected observation that the total number of RAP-binding sites
([R]t) found with equal amounts of VLDLR-I and VLDLR-III
differed ~3-fold in the microtiter well binding assay. A possible
explanation is that two out of a total of three RAP-binding sites per
receptor molecule were inactivated by the lack of CTR-3, but although
multiple RAP-binding sites per
2MR/LRP molecule have
been reported (54, 55), our chemical cross-linking experiment gave no
indications of more than one RAP-binding site per VLDLR-I or VLDLR-III
molecule (Fig. 9). Alternatively, the observed [R]t
difference could be due to both VLDLR-I and VLDLR-III existing in two
conformations with a considerable difference in Kd
for RAP and D3RAP binding, with the high affinity
conformation being much less abundant in VLDLR-III than in VLDLR-I, but
both conformations having a RAP affinity high enough to cause retention
on RAP-Sepharose columns. This model is in line with the
above-mentioned proposal that CTR-3 affects the binding properties by
affecting receptor conformation.
Conclusively, our data help to gain insight into the mechanisms by
which this receptor class can bind many different ligands without
obvious structural similarity, but each of which may make several
independent contacts with the receptors and which do not show general
cross-competition.
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FOOTNOTES |
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* This work was supported by grants from the Danish Cancer Society, the Danish Medical Research Council, the Novo-Nordisk Foundation, and the Danish Biotechnology Program (to P. A. A.) and by National Institutes of Health Grant HL-16512 (to L. C.).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.
To whom correspondence should be addressed: Dept. of Molecular
and Structural Biology, University of Aarhus, 10C Gustav Wieds Vej,
DK-8000 Aarhus C, Denmark. Tel.: 4589425080; Fax: 4586123178; E-mail:
pa{at}mbio.aau.dk.
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ABBREVIATIONS |
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The abbreviations used are:
LDLR, low density
lipoprotein receptor;
2MR/LRP,
2-macroglobulin receptor/low density lipoprotein
receptor-related protein;
VLDLR, very low density lipoprotein receptor;
CTR, complement-type repeat;
EGF, epidermal growth factor;
uPA, urokinase-type plasminogen activator;
RAP, Mr
40,000 receptor-associated protein;
PAI-1, plasminogen activator
inhibitor-1;
PN-1, protease nexin-1;
PCR, polymerase chain reaction;
CHO, Chinese hamster ovary;
bp, base pairs;
TBS, Tris-buffered saline;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis;
PVDF, polyvinylidene
difluoride;
B/F, bound/free.
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REFERENCES |
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