Ligand Binding Properties of the Very Low Density Lipoprotein Receptor
ABSENCE OF THE THIRD COMPLEMENT-TYPE REPEAT ENCODED BY EXON 4 IS ASSOCIATED WITH REDUCED BINDING OF Mr 40,000 RECEPTOR-ASSOCIATED PROTEIN*

Peter M. RettenbergerDagger , Kazuhiro Oka§, Lars EllgaardDagger , Helle H. PetersenDagger , Anni ChristensenDagger , Pia M. MartensenDagger , Denis Monard, Michael EtzerodtDagger , Lawrence Chan§, and Peter A. AndreasenDagger parallel

From the Dagger  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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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.

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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 alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 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).

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.


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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 alpha 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, alpha 2-macroglobulin-proteinase complexes and uPA-serpin complexes do not compete for binding to alpha 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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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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Fig. 2.   Reverse transcription-PCR analysis of VLDLR mRNA in CHO cells transfected with VLDLR-I and VLDLR-III cDNAs, respectively. The primers used for PCR were positioned around exon 4 (A) or around exon 16 (B). Lane 1, the PCR products from cells transfected with VLDLR-I cDNA; lane 2, the PCR products from cells transfected with VLDLR-III cDNA; lane 3, no cDNA added. The positions of the products corresponding to VLDLR mRNA with (390 bp) and without (266 bp) exon 4 and with exon 16 (377 bp) are indicated by arrows.

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 lambda  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 gamma -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 right-left-arrows 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 right-left-arrows RL and R + I right-left-arrows 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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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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Fig. 3.   SDS-PAGE, immunoblot analysis, and ligand blot analysis of the binding of RAP and uPA-inhibitor complexes to VLDLR-I and VLDLR-III. VLDLR-I and VLDLR-III were affinity-purified from crude CHAPS extracts of the corresponding transfected CHO cells on RAP-coupled Sepharose 4B columns and subjected to SDS-PAGE. A shows the silver-stained bands of ~500 ng of receptor protein (8-fold more receptor per lane than in B-E). In B-E, parallel samples of VLDLR-I and VLDLR-III were transferred to PVDF filters after electrophoresis. B shows immunoblot analysis with rabbit polyclonal anti-VLDLR antibodies. In C-E, the filters were incubated with 20 pM 125I-RAP, 125I-uPA·PN-1, and 125I-uPA·PAI-1, respectively. Bound radioactive ligand was visualized by autoradiography. The exposure times were 1 h (B), 7 h (C), and 22 h (D). The migration of Mr markers is indicated to the left.

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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Table I
Binding of 125I-RAP and 125I-uPA/PN-1 to VLDLR variants
The purified receptors were immobilized by the use of antibodies against their C terminus, coated onto the wells. The immobilized receptors were incubated for 16 h at 4 °C with 20 pM 125I-RAP or 20 pM 125I-uPA/PN-1. The binding of each of the radioactively labeled ligands to each individual receptor variant is expressed as a percentage of the binding to VLDLR-I. Means ± S.D. and numbers of experiments (n) are indicated.

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 right-left-arrows 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 right-left-arrows 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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Fig. 4.   Binding of RAP to intact CHO cells transfected with VLDLR-I and VLDLR-III cDNAs, respectively. CHO cells transfected with VLDLR-I and VLDLR-III cDNAs, respectively, at a cell density of ~5 × 104 cells/well, were incubated for 16 h on ice with 1-5 pM radiolabeled RAP and varying concentrations of the same ligand in nonradioactive form. Shown are the ratios between the amounts of cell-bound and free ligand (B/F) as a fraction of the B/F value at 1 pM RAP, plotted semilogarithmically versus the free ligand concentration. Each point represents a single determination. The lines drawn were obtained by fitting the data to the equation (B/F)/(B/F)1pM = ([R]/[L])/([R]t/Kd) = Kd/(Kd + [L]). The Kd values found in this typical experiment were 16 and 48 pM for VLDLR-I and VLDLR-III, respectively. For further explanation, see the "Results."

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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Fig. 5.   Ligand blot analysis of cross-competition between RAP and D3RAP for binding to VLDLR variants. VLDLR-I and VLDLR-III were subjected to SDS-PAGE and transferred to PVDF filters. The migration of Mr markers is indicated to the left. A, equal immunoreactivity was demonstrated by use of polyclonal antibody against the VLDLR C terminus (left). A parallel blot was incubated with 20 pM 125I-labeled D3RAP (right). B, the amounts of VLDLR-I and VLDLR-III were adjusted to result in equal signals after incubation with 20 pM 125I-RAP (left). A parallel blot was incubated with 20 pM 125I-RAP and 200 nM nonradioactive D3RAP (right). The exposure time was 24 h.

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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Fig. 6.   Ligand blot analysis of competition of RAP binding to VLDLR variants by individual RAP domains. Parallel samples of VLDLR-I and VLDLR-III were subjected to SDS-PAGE and transferred to PVDF filters. The amounts of VLDLR-I and VLDLR-III were adjusted to result in equal signals after incubation with 20 pM 125I-RAP. Parallel blots were incubated with 20 pM 125I-RAP and 200 nM nonradioactive D1RAP, D2RAP, D1D2RAP, and D2D3RAP, as indicated. Bound radioactive ligand was visualized by autoradiography.

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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Fig. 7.   Concentration dependence of competition of 125I-RAP binding to VLDLR-I and VLDLR-III by D3RAP as estimated by microtiter well assay. Affinity-purified VLDLR-I and VLDLR-III were immobilized in microtiter wells coated with rabbit polyclonal antibodies against the VLDLR C terminus and incubated with 20 pM 125I-RAP and the indicated concentrations of nonradioactive D3RAP. In A, the ratio between bound and free 125I-RAP (B/F) was plotted semilogarithmically versus the concentration of D3RAP. In B, the B/F ratios in the absence of D3RAP were set equal to 100%, and the B/F values at the various D3RAP concentrations are expressed as a percentage thereof. Each data point shows the mean ± S.D. for three determinations. The lines drawn were obtained by fitting the data by the method of least squares to the equation B/F = [RL]/[L] = ([R]t/(Kd + [L] + (Kd/Ki)[I])) + C, where Kd = 9 pM (see "Results") and [L] = 20 pM. The estimated Ki values for VLDLR-I and VLDLR-III were 0.3 and 3 nM, respectively. The estimated total receptor concentration ([R]t) was 3.6-fold lower with VLDLR-III than with VLDLR-I. This experiment is one out of a total of two.

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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Fig. 8.   Ligand blot analysis of cross-competition between RAP and uPA-inhibitor complexes. Parallel samples with equal amounts of immunoreactive VLDLR-I and VLDLR-III were subjected to SDS-PAGE; transferred to PVDF filters; and incubated with 20 pM 125I-RAP (left), 20 pM 125I-RAP and 200 nM uPA·PAI-1 (center), and 20 pM 125I-RAP and 200 nM uPA·PN-1 (right). Bound radioactive ligand was visualized by autoradiography.

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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Fig. 9.   Chemical cross-linking of VLDLR·RAP complexes. VLDLR-I, VLDLR-III, and VLDLR-II were incubated with 125I-RAP in the absence or presence of EDTA. Following cross-linking with glutaraldehyde, the samples were subjected to SDS-PAGE. The radioactivity in the gels was visualized by autoradiography with an exposure time of 1 h.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha 2MR/LRP and gp330/megalin (40-44) and of other serine proteinase-serpin complexes to alpha 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-alpha 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 alpha 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 alpha 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 right-left-arrows 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 alpha 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.

    FOOTNOTES

* 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.

parallel 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.

    ABBREVIATIONS

The abbreviations used are: LDLR, low density lipoprotein receptor; alpha 2MR/LRP, alpha 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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