Molecular Analysis of Ligand Binding to the Second Cluster of Complement-type Repeats of the Low Density Lipoprotein Receptor-related Protein
EVIDENCE FOR AN ALLOSTERIC COMPONENT IN RECEPTOR-ASSOCIATED PROTEIN-MEDIATED INHIBITION OF LIGAND BINDING*

(Received for publication, January 23, 1997, and in revised form, March 23, 1997)

Ivo R. Horn , Birgit M. M. van den Berg , Petronel Z. van der Meijden , Hans Pannekoek and Anton-Jan van Zonneveld Dagger

From the Department of Biochemistry, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor gene family, mediates the cellular uptake of a diversity of ligands. A folding chaperone, the 39-kDa receptor-associated protein (RAP) that resides in the early compartments of the secretory pathway inhibits the binding of all ligands to the receptor and may serve to prevent premature binding of ligands to the receptor during the trafficking to the cell surface. To elucidate the molecular interactions that underlie the interplay between the receptor, RAP, and the ligands, we have analyzed and delineated the binding sites of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA)·PAI-1 complexes, RAP, and the anti-LRP Fab fragment Fab A8. To that end, we have generated a series of soluble recombinant fragments spanning the second cluster of complement-type repeats (C3-C10) and the amino-terminal flanking epidermal growth factor repeat (E4) of LRP (E4-C10; amino acids 787-1165). All fragments were expressed by stably transfected baby hamster kidney cells and purified by affinity chromatography. A detailed study of ligand binding to the fragments using surface plasmon resonance revealed the presence of three distinct, Ca2+-dependent ligand binding sites in the cluster II domain (Cl-II) of LRP. t-PA·PAI-1 complexes as well as PAI-1 bind to a domain located in the amino-terminal portion of Cl-II, spanning repeats E4-C3-C7. Adjacent to this site and partially overlapping is a high affinity RAP-binding site located on repeats C5-C7. Fab A8, a pseudo-ligand of the receptor, binds to a third Ca2+-dependent binding site on repeats C8-C10 at the carboxyl-terminal end of Cl-II. Next, we studied the RAP-mediated inhibition of ligand binding to LRP and to Cl-II. As expected, we observed a strong inhibition of t-PA·PAI-1 complex and Fab A8 binding to LRP by RAP (IC50 congruent  0.3 nM), whereas in the reverse experiment, competition of t-PA·PAI-1 complexes and Fab A8 for RAP binding to LRP could only be shown at high concentrations of competitors (>= 1 µM). Interestingly, even though the equilibrium dissociation constants for the binding of RAP to LRP and to Cl-II are similar, the binding of the ligands to Cl-II is only prevented by RAP at concentrations that are at least 2 orders of magnitude higher than those required for inhibition of ligand binding to LRP. Our results favor models that propose RAP-induced allosteric inhibition of ligand binding to LRP that may require LRP moieties that are located outside Cl-II of the receptor.


INTRODUCTION

The low density lipoprotein receptor-related protein (LRP),1 a 600-kDa glycoprotein, is a multifunctional endocytic receptor (1-3). LRP binds and internalizes a broad spectrum of structurally unrelated ligands including two important components of the fibrinolytic system, tissue-type plasminogen activator (t-PA) and its specific inhibitor plasminogen activator inhibitor type-1 (PAI-1) (4, 5). In rodents, LRP was shown to be one of the in vivo receptor systems involved in the clearance of t-PA (6, 7).

LRP is synthesized as a 600-kDa precursor protein that is subsequently processed in the trans-Golgi compartment to a 515-kDa alpha -chain and a 85-kDa beta -chain. Both chains remain associated in an unusual noncovalent fashion (8). Ligand binding by the receptor is thought to be mediated by the so-called complement-type repeats. These 40-residue-long repeats are characterized by a highly conserved spacing pattern of six cysteine residues, and they are found in all other members of the low density lipoprotein receptor gene family (1). In the low density lipoprotein receptor, these complement-type repeats were shown to mediate ligand binding (9, 10). The alpha -chain of LRP contains four potential ligand-binding domains assembled as sequential series of 2, 8, 10, and 11 complement-type repeats (cluster I, II, III, and IV). The first evidence for a role of the cluster II domain (Cl-II) in ligand binding was presented by Moestrup et al., who demonstrated the binding of alpha 2-macroglobulin-complexes, urokinase-type plasminogen activator· PAI-1 complexes, and receptor-associated protein (RAP) to a 75-kDa CNBr-generated LRP fragment, spanning Cl-II, on a ligand blot (11). Using a recombinant DNA approach, Willnow et al. expressed LRP minireceptors and showed t-PA·PAI-1 complex and RAP binding to Cl-II and a low affinity binding of RAP to a truncated receptor harboring the cluster IV domain (12). In a previous study, we provided evidence for an additional ligand binding site in Cl-II by the selection of a high affinity Cl-II-binding Fab fragment (Fab A8) that bound the receptor in a Ca2+-dependent fashion and completely inhibited the binding of pro-urokinase to LRP (13).

The 39-kDa folding chaperone RAP is a molecule with a high affinity for LRP that resides in the endoplasmic reticulum and the cis-Golgi compartments (14). RAP inhibits the binding of all known ligands to LRP, and it has been proposed that this activity may serve to prevent premature binding of ligands to the receptor during the maturation and trafficking of LRP (14, 15). Little is known about the mechanism by which RAP inhibits the binding of the diversity of structurally unrelated ligands. One model assumes a close spatial association of a RAP binding site to each of the independent LRP ligand-binding sites (16, 17). Consequently, RAP would inhibit ligand binding by steric hindrance of each of the LRP ligands. An alternative model for the regulation of ligand binding by RAP proposes a RAP-induced conformational change in the LRP molecule (18). A unique feature of RAP is its repeated triplicate structure, endowing the protein with three potential LRP-binding sites as determined by cross-linking studies (14). Binding studies performed with amino-terminal and carboxyl-terminal fragments of RAP have indicated that at least two high affinity LRP binding sites are present in RAP (19). The presence of RAP-binding sites in the different clusters of LRP (11, 12, 20), as well as the presence of multiple receptor-binding sites in RAP, may support a model in which RAP binds to different clusters synchronically, thereby inducing a conformational change in LRP that would be incompatible with ligand binding. Such a model would imply that competition by RAP for ligand binding is not on the level of the individual clusters but requires the intact LRP molecule. In this study, we have performed a detailed analysis of ligand-binding sites in the cluster II domain of the receptor. Using recombinant fragments spanning Cl-II, we demonstrate that Cl-II contains three distinct binding sites for t-PA·PAI-1 complexes, RAP, and the pseudo-ligand Fab A8. In addition, we provide evidence for an allosteric component in RAP-mediated inhibition of ligand binding to LRP.


EXPERIMENTAL PROCEDURES

Materials

Oligonucleotides were from Pharmacia Biotech Inc. Restriction and other DNA-modifying enzymes were from Life Technologies, Inc. All other chemicals used were reagent grade (Sigma or Merck).

Proteins

Human LRP was kindly provided by Dr. S. K. Moestrup (Institute of Medical Biochemistry, University of Aarhus, Aarhus, Denmark). Recombinant glutathione S-transferase-fused RAP was kindly provided by Dr. J. Kuiper (Sylvius Laboratory, University of Leiden, Leiden, The Netherlands). Two-chain t-PA was from Biopool (Umea, Sweden) and recombinant t-PA (Actilyse) was from Boehringer Ingelheim (Ingelheim/Rhein, Germany). Active PAI-1 purification procedures were essentially as described (21, 22). t-PA·PAI-1 complexes were prepared as follows. Recombinant t-PA was incubated with an excess of PAI-1 for 2 h at 37 °C. Complexed t-PA was then separated from the excess of unreacted PAI-1 by affinity chromatography using lysine-Sepharose. Complexes bound to the lysine-Sepharose were washed with 20 mM HEPES (pH 7.4), 150 mM NaCl, and 0.001% (v/v) Tween 80 (HBST) followed by washings with HBST containing 500 mM NaCl and subsequent elutions with HBST containing 1 M NaCl and 0.2 M epsilon -aminocaproic acid. Preparations were concentrated 20-fold by centrifugation in a Sorvall high speed centrifuge at 7,000 rpm for 1 h at 4 °C using Centricon 30 concentrators (Amicon, Beverly, MA) and subsequently dialyzed against filtered (0.45-µm filters, Schleicher & Schuell) and degassed, modified HBST buffer (containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM CaCl2, 0.001% (v/v) Tween 80). Preparations revealed a single protein band on a silver-stained SDS-polyacrylamide gel and were stored at -20 °C until use. Isolation of the monoclonal Fab fragment Fab A8 as well as the isolation of the anti-factor VIII monoclonal antibody CAg69 were essentially as described (13, 23).

SPR Reagents and Instrumentation

The BIAcoreTM2000 biosensor system and reagents, including an amine-coupling kit containing N-hydroxysuccinimide, N-ethyl-N'-(3-diethylamino-propyl)carbodiimide, ethanolamine hydrochloride, and CM5 sensor chips (research grade), were from Biacore AB (Uppsala, Sweden).

Construction of Expression Plasmids for Recombinant Cluster II Fragments

Plasmids encoding recombinant LRP Cl-II fragments (Cl-II, amino acids 786-1165; Cl-II-1, amino acids 786-914; Cl-II-2, amino acids 915-1034; and Cl-II-3, amino acids 1041-1165; numbering according to Herz et al. (24)) were obtained by polymerase chain reaction using a plasmid clone containing the human LRP cDNA (a gift from Dr. J. Herz, University of Texas Southwestern Medical Center, Dallas) as a template. DNAs encoding recombinant-LRP cluster II fragments were amplified using primers LRP 3 (5'-GGACTCGAGAACAAATGCCGGGTGAAC-3') and LRP 8 (5'-AACTAGTCTGGTCGCAGAGCTCGCC-3'); LRP3 and LRP4 (5'-GACTAGTGGCTGAACAAGTGGCATT-3'); LRP5 (5'-TTACTCGAGCGCACCTGCCCCCCCAAC-3') and LRP 6 (5'-AACTAGTGTTGGTGCAGTTGGCGTG-3'); and LRP 7 (5'-TTACTCGAGGGTGGCTGCCACACTGAT-3') and LRP8 for LRP Cl-II fragments Cl-II, Cl-II-1, Cl-II-2, and Cl-II-3, respectively. Polymerase chain reaction products were digested with XhoI and SpeI and subsequently cloned into the HindIII/XbaI-digested plasmid pRc/CMV (Invitrogen, La Jolla, California) together with a 192-base pair HindIII/XhoI fragment, encoding the entire signal and pro-sequences of t-PA (amino acids -35 to 1) (25) followed by a 16-amino acid tag (KKEDFDIYDEDENQSP) that contains the antigenic determinant of the mAb CAg69 (26). Subsequently, DNA preparations encoding Cl-II fragments were obtained from the pRc/CMV constructs using HindIII and BclII, blunt-ended using T4 DNA polymerase, and cloned into the SmaI site of vector pZEM229R (27) to yield expression plasmids encoding LRP Cl-II fragments Cl-II, Cl-II-1, Cl-II-2, and Cl-II-3. Expression plasmids encoding Cl-II-1/2 and Cl-II-2/3 were obtained by digestion of expression plasmids coding for Cl-II and Cl-II-2 using either HindIII and AflIII or AflIII and BclII combinations and ligating the proper DNA fragments. All constructs were verified by DNA sequence analysis using an ALF express automated DNA sequencer (Pharmacia).

Expression and Purification of Recombinant LRP Cl-II Fragments

Baby hamster kidney cells (thymidine kinase-deficient BHK-570 cells, ATCC CRL 10314) were grown to subconfluency (80%) in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), containing 5% (v/v) fetal calf serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml fungizone. Cells were transfected with 20 µg of DNA using the calcium precipitation method, essentially as described (28). DNA-calcium-phosphate coprecipitates were added, and after 5 h the cells were incubated for 1 min with 15% (v/v) glycerol. Cells were allowed to recover in Dulbecco's modified Eagle's medium for 12 h and subsequently cultured for 12 days on selective medium (Dulbecco's modified Eagle's medium, supplemented with 1 µM methotrexate (amethopterin; Sigma)). Individual colonies were isolated and tested for the expression of recombinant LRP cluster II fragments by ELISAs using mAb CAg69, or a polyclonal antibody directed to LRP (a gift from Dr. S. K. Moestrup). Cl-II producing clones were grown to subconfluency (80%) in 500 cm2 flasks (NUNC, Kamstrup, Denmark). Next, the cells were cultured for several days in Optimem medium (Life Technologies, Inc.), and conditioned media were harvested every 24 h. Cl-II fragments were purified from the conditioned media by affinity chromatography using Sepharose-coupled mAb CAg69. After binding, columns were washed with HEPES-buffered saline (HBS, 20 mM HEPES (pH 7.4), 150 mM NaCl) and eluted with HBS containing 1 M NaCl. If required, another affinity chromatography purification step was included using either RAP- or Fab A8-Sepharose columns. These columns were washed with HBS and eluted with HBS containing 10 mM EDTA. Cl-II preparations were concentrated in a HEPES-buffered 1 M NaCl solution by centrifugation in a Sorvall high speed centrifuge at 7,000 rpm for 1 h at 4 °C, using Centricon 10 or 30 concentrators (Amicon, Beverly, MA) and dialyzed against filtered and degassed, modified HBST buffer (containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM CaCl2, 0.005% (v/v) Tween 80). All Cl-II fragment preparations were analyzed by ELISA, SDS-polyacrylamide gel electrophoresis, and immunoblotting with mAb CAg69 and anti-LRP antibodies.

Analysis of N-linked Glycosylation of Recombinant LRP Cl-II Fragments

Cl-II fragments were analyzed by a nonreducing silver-stained SDS-polyacrylamide gel (12% (w/v) polyacrylamide). To assess the contribution of N-linked glycosylation to the observed migration of the fragments, the Cl-II fragment preparations were treated with glycanase (N-glycosidase F, Boehringer Mannheim). Briefly, the protein samples were incubated for 30 min at 45 °C in 0.4% (w/v) SDS-containing 100 mM potassium phosphate buffer (pH 8). Subsequently, the preparations were diluted 1:1 with 2% Triton X-100 and incubated with 0.1 unit of glycanase for 2 h at 37 °C. Treated samples were reduced and analyzed on a silver-stained SDS-polyacrylamide gel.

Concentration Determinations Using SPR

The tag-binding mAb (CAg69) was immobilized to a CM5 sensor chip, using the amine-coupling kit following the instructions of the supplier at a high density of approximately 50 fmol/mm2 to determine binding of the samples under conditions of mass-transport limitation. A control channel on the sensor chip was activated and blocked using amine-coupling reagents without immobilization of protein. The binding of protein preparations to this noncoated channel was subtracted from specific binding. To calibrate the system, standard Cl-II fragment preparations with known concentrations (BCA kit, Pierce), were passed over the sensor chip at 25 °C at a flow rate of 5 µl/min, using HBST as running buffer. Regeneration of the sensor chip surface was performed with 100 mM H3PO4. For each concentration, the slope of the binding curve was determined by linear fitting using the BIAevaluation software (Biacore AB). Using the BIAconcentration evaluation software, the system was calibrated to determine unknown Cl-II fragment concentrations.

Kinetic Determinations Using SPR

Purified human LRP was immobilized on a CM5 sensor chip by amine coupling at a low density of 6.7 fmol/mm2. Using modified HBST as running buffer, RAP and Fab A8 were passed over three separate channels with immobilized LRP and one control (nonimmobilized) channel at 25 °C at a flow rate of 20 µl/min. Each determination was performed at least in triplicate at different concentrations (n = 4) in the appropriate concentration range (around Kd values). RAP/LRP interaction constants were determined by performing fittings according to a double site model (a single site model did not appropriately describe the interaction) according to the following equation:
R<SUB>t</SUB>=R<SUB><UP>eq1</UP></SUB>(1−e<SUP><UP>−</UP>k<SUB><UP>s1</UP></SUB>t</SUP>)+R<SUB><UP>eq2</UP></SUB>(1−e<SUP><UP>−</UP>k<SUB><UP>s2</UP></SUB>t</SUP>) (Eq. 1)
where Rt is the measured response at time t (t is the elapsed time t - t0), Req1 and Req2 are the responses at equilibrium for the first and second interaction, ks1 and ks2 are the observed rate constant for the first and the second interaction. The kon values are subsequently derived from secondary graphs of ks values plotted against the concentration (C). The kon values are represented by the slopes of the linear curves described by the following equations:
k<SUB><UP>s1</UP></SUB>=k<SUB><UP>on1</UP></SUB> · C+k<SUB><UP>off1</UP></SUB> (Eq. 2)
k<SUB><UP>s2</UP></SUB>=k<SUB><UP>on2</UP></SUB> · C+k<SUB><UP>off2</UP></SUB> (Eq. 3)
The dissociation phase of the curve was fitted according to the following equation:
R<SUB>t</SUB>=R<SUB>1</SUB> · e<SUP><UP>−</UP>k<SUB><UP>off1</UP></SUB>t</SUP>+(R<SUB>0</SUB>−R<SUB>1</SUB>) · e<SUP><UP>−</UP>k<SUB><UP>off2</UP></SUB>t</SUP> (Eq. 4)
where Rt is the measured response at time t, R0 is the maximal response at t0 of the dissociation phase, R1 and R0 - R1 represents maximal binding responses to receptor sites 1 and 2, respectively, and koff1 and koff2 are dissociation rate constants for the binding sites 1 and 2, respectively. A single site model was used to describe all other interactions; the mathematical description of this model is derived from that of the two-site model (in the case of a single site model ks2 = 0).

Recombinant Cl-II fragment-ligand interactions were measured as follows: RAP, purified t-PA·PAI-1 complexes, PAI-1, and Fab A8 were immobilized to the sensor chip at low densities (approximately 15-30 fmol/mm2). Measurements were performed at 25 °C at a flow rate of 20 µl/min. Six different analyses were performed at least at four different concentrations. Kinetic constants were obtained by fitting to single site models (describing the interaction appropriately, as judged from residual plots and statistical parameters) and calculated as indicated above. The data were validated by subjecting them to the tests of selfconsistency (29).

Analysis of RAP Inhibition of Ligand Binding by Competitive ELISA

ELISAs were performed at half-maximal saturating concentrations of ligands versus immobilized LRP or Cl-II. To determine half-maximal saturating concentrations, 50 ng of LRP or 160 ng of Cl-II was immobilized to 96-well plates for 30 min at 37 °C. Wells were blocked with 3% (w/v) bovine serum albumin for 30 min at 37 °C, washed, and incubated with a range of ligand concentrations in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.005% (v/v) Tween 20 (TBST). Bound proteins were detected using either biotinylated anti-PAI-1 mAb CLB 2C8, biotinylated rat anti-mouse kappa light chain mAb (CLB products, Amsterdam, The Netherlands), or goat anti-glutathione S-transferase polyclonal antibodies (Pharmacia Biotech Inc.) for t-PA·PAI-1 complexes, Fab A8, and glutathione S-transferase-RAP, respectively. Staining was performed as described (13). Competitive ELISAs were subsequently performed under identical conditions, and experiments were performed in triplicate.


RESULTS

Construction and Expression of Soluble Cl-II Fragments

In a previous study, we confirmed and extended the evidence for the importance of Cl-II for ligand binding by the isolation and analysis of a "phage display" derived Fab fragment (Fab A8) (13). This Fab fragment behaves like a pseudo-ligand because it binds LRP in a Ca2+-dependent fashion and its binding is completely inhibited by RAP. Using this Fab fragment, we provided evidence for a binding site for pro-urokinase in Cl-II, distinct from the binding sites for alpha 2-macroglobulin and t-PA·PAI-1 complexes. To investigate ligand binding by Cl-II in more detail, we have expressed a series of soluble recombinant cluster II fragments (Fig. 1) in stably transfected baby hamster kidney cell lines. Each of the fragments is preceded by a t-PA-derived signal pro-sequence for targeting the fragments into the secretory pathway and a tag sequence to facilitate detection and purification. The epidermal growth factor repeat (E4) that precedes Cl-II is included in all fragments corresponding to the amino-terminal end of Cl-II, because this repeat has been postulated to be important for the binding of methylamine activated alpha 2-macroglobulin to LRP (11, 12). Purified recombinant fragments were analyzed by SDS-polyacrylamide gel electrophoresis, and gels were stained with silver (Fig. 2A). The mobility of the fragments was somewhat slower than would be expected on the basis of the calculated molecular masses (approximately 15.9, 14.9, 15.4, 28.9, 29.3, and 43.5 kDa for Cl-II-1, Cl-II-2, Cl-II-3, Cl-II-1/2, Cl-II-2/3, and Cl-II, respectively). This observation might be readily explained by N-linked glycosylation of several of the complement-type repeats of Cl-II (20). To show that N-linked glycosylation is indeed responsible for the altered mobility of the fragments, we treated the fragments with glycanase (N-glycosidase F) and compared the migration pattern to that of the untreated fragments. As shown in Fig. 2B, after glycanase treatment Cl-II-2/3 migrates with the mobility of a protein of the expected molecular mass (29.3 kDa). Similar results were obtained for the other recombinant fragments (data not shown), and we conclude that the mobility of the fragments during SDS-polyacrylamide gel electrophoresis is reduced as a result of carbohydrate moieties attached to the fragments by N-linked glycosylation. These results can also explain the observed minor bands (Fig. 2A), which may represent unglycosylated forms of the respective fragments.


Fig. 1. Ligand mapping strategy. Recombinant Cl-II fragments and subfragments were designed based on the numbering according to Herz et al. (24). The Roman numbers indicate the four clusters of complement-type repeats. The recombinant fragments span the indicated epidermal growth factor repeat and complement-type repeats. All fragments are preceded by the signal/pro-peptide of t-PA that are cleaved off upon secretion and a tag sequence to facilitate detection and purification.
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Fig. 2. Purified recombinant cluster II fragments. A, silverstaining of a nonreducing 12% (w/v) SDS-polyacrylamide gel. The lanes represent the Cl-II fragments as indicated. B, silverstaining of a reducing 10% (w/v) SDS-polyacrylamide gel showing untreated Cl-II-2/3 (lane 1) and glycanase-treated Cl-II-2/3 (lane 2). Molecular mass markers are indicated in kDa.
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Analysis of Ligand Binding to LRP Using SPR

In this study we have focused on the binding of the cluster II ligands RAP, t-PA·PAI-1 complexes, PAI-1, t-PA, and Fab A8. First, we determined the Kd values for interactions of these ligands with the intact receptor. To that end, LRP was immobilized on a sensor chip and the association and dissociation rate constants were determined by SPR measurements. As shown in Table I, RAP binding to LRP could only be described using a double exponential decay model, indicating the presence of at least two RAP-binding sites on LRP, as reported by Williams and co-workers (30). The two sites have Kd values of 39.4 and 275.0 nM, respectively, which are in good agreement with similar SPR measurements described by Moestrup et al. (high and low affinity sites of 41.7 and 240 nM, respectively) (31). However, Kd values for the RAP-LRP interaction determined by conventional binding studies turn out to be lower than the ones calculated here. This apparent discrepancy may be due to the different experimental approach. The interactions of t-PA·PAI-1 complexes, PAI-1, t-PA, and Fab A8 could accurately be described by a single site model, yielding Kd values of 6.1, 35, 158, and 3.3 nM, respectively.

Table I. kon, koff values, and Kd values for the interactions of RAP, t-PA · PAI-1 complexes, PAI-1, t-PA, and Fab A8 with LRP

Data from the SPR analyses represent the means ± S.E. of at least four experiments. Data from the SPR analyses represent the means ± S.E. of at least four experiments.

Ligand kon1 kon2 koff1 koff2 Kd1 Kd2

M-1s-1 M-1s-1 s-1 s-1 nM nM
RAP 1.8  × 104 1.6  × 105 7.1  × 10-4 4.4  × 10-2 39.4  ± 11.0 275.0  ± 28.0
t-PA · PAI-1 3.8  × 105 2.3  × 10-3 6.1  ± 3.3
PAI-1 1.2  × 105 4.2  × 10-3 35.0  ± 12.4
t-PA 1.2  × 105 1.9  × 10-2 158.0  ± 20.0
Fab A8 6.3  × 105 2.1  × 10-3 3.3  ± 0.3

Mapping of Ligand Binding Sites for RAP, t-PA·PAI-1 Complexes, PAI-1, and Fab A8 on Cl-II

To delineate the binding sites for RAP, t-PA·PAI-1 complexes, PAI-1, and Fab A8 on Cl-II, we immobilized these LRP ligands at a low density on sensor chips and determined the rate constants for the interactions with the Cl-II fragments. As presented in Table II, RAP binds to a subset of Cl-II fragments according to a single site model with Kd values of 20.9 nM for the whole Cl-II and 57.9 nM for the smallest fragment (Cl-II-2). The fragments Cl-II-1/2 and Cl-II-2/3 bind to RAP with similar Kd values. These results indicate the presence of a single RAP-binding site in Cl-II, localized on a fragment spanning complement-type repeats C5, C6, and C7.

Table II. kon and koff values and calculated Kd values for the interactions of RAP, t-PA · PAI-1 complexes, PAI-1, and Fab A8 with recombinant cluster II fragments

Data from the SPR analyses represent the means ± S.E. of at least four experiments. * > 105, no binding detected. Data from the SPR analyses represent the means ± S.E. of at least four experiments. * > 105, no binding detected.

LRP ligand LRP cluster II fragment kon koff Kd

M-1s-1 s-1 nM
RAP Cl-II 4.7  × 104 9.8  × 10-4 20.9  ± 2.9
Cl-II-1/2 3.4  × 104 2.1  × 10-3 61.8  ± 21.4
Cl-II-2/3 3.4  × 104 2.9  × 10-3 85.3  ± 26.8
Cl-II-1 >105
Cl-II-2 1.9  × 105 1.1  × 10-2 57.9  ± 3.9
Cl-II-3 >105
t-PA·PAI-1 Cl-II 2.7  × 104 1.2  × 10-3 44.4  ± 5.1
Cl-II-1/2 5.0  × 104 2.7  × 10-3 54.0  ± 12.6
Cl-II-2/3 >105
Cl-II-1 >105
Cl-II-2 >105
Cl-II-3 >105
PAI-1 Cl-II 2.7  × 104 5.3  × 10-3 196.3  ± 91.2
Cl-II-1/2 4.2  × 104 9.2  × 10-3 219.1  ± 56.9
Cl-II-2/3 >105
Cl-II-1 >105
Cl-II-2 >105
Cl-II-3 >105
Fab A8 Cl-II 4.1  × 104 1.2  × 10-3 29.3  ± 12.0
Cl-II-1/2 >105
Cl-II-2/3 4.1  × 104 1.5  × 10-3 36.6  ± 14.2
Cl-II-1 >105
Cl-II-2 >105
Cl-II-3 9.6  × 104 1.1  × 10-2 114.6  ± 28.4

a >105, no binding detected.

t-PA·PAI-1 complexes bind to recombinant fragments Cl-II and Cl-II-1/2. The respective Kd values are 44.4 (Cl-II) and 54.0 nM (Cl-II-1/2), indicating an interaction site on Cl-II localized on a fragment spanning epidermal growth factor repeat E4 and complement-type repeats C3-C7. Cl-II and Cl-II-1/2 interact with PAI-1 as well, although with higher Kd values (196.3 and 219.1 nM for Cl-II and Cl-II-1/2, respectively). We could not detect any significant binding of Cl-II fragments to immobilized t-PA. We conclude that t-PA·PAI-1 complexes as well as PAI-1 bind to a site that is distinct from but partially overlapping the RAP binding site in Cl-II.

The anti-LRP Fab fragment Fab A8, which inhibits the binding of pro-urokinase-type plasminogen activator to LRP, binds to another subset of Cl-II fragments including Cl-II, Cl-II-2/3, and Cl-II-3. The Kd values are 29.3, 36.6, and 114.6 nM for Cl-II, Cl-II-2/3, and Cl-II-3, respectively, indicating a unique binding site for Fab A8 localized on complement-type repeats C8, C9, and C10.

The results of these studies are schematically represented in Fig. 3. We conclude that the recombinant fragments of Cl-II harbor functional ligand binding sites and Cl-II contains a linear array of at least three distinct binding sites for t-PA·PAI-1 complexes (PAI-1), RAP, and Fab A8.


Fig. 3. Schematic representation of delineation of ligand binding sites on Cl-II. t-PA·PAI-1 complexes and PAI-1 bind to a fragment spanning E4, C3-C7; RAP binds to a fragment spanning C5-C7; Fab A8 interacts with a fragment spanning C8-C10.
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Determination of IC50 Values by Competitive ELISA

We previously demonstrated that RAP can completely prevent the binding of Fab A8 to LRP, whereas Fab A8 is unable to compete for the RAP/LRP interaction (13). Furthermore, it has been shown that RAP also inhibits t-PA·PAI-1 complex binding to LRP (4), whereas the reverse has not been observed. One possible mechanism to explain RAP inhibition of ligand binding to LRP is the direct sterical hindrance due to the close spatial association of the RAP- and ligand-binding sites. To test this hypothesis, we performed a series of competition experiments to assess the cross-competition of RAP-, t-PA·PAI-1 complex-, and Fab A8-binding to LRP and to Cl-II. First, we determined the concentration required for half-maximal binding to the immobilized LRP and to Cl-II. As can be seen in Fig. 4A, half-maximal saturation of RAP binding to either LRP or Cl-II is obtained at nearly identical concentrations, being 0.3 or 0.5 nM, respectively. From panels B and C in Fig. 4, it can be deduced that the same conclusion is valid for t-PA·PAI-1 complexes and Fab A8, with half-maximal saturation at concentrations of 0.5 and 0.8 nM for t-PA·PAI-1 complex and 0.4 and 0.8 nM for Fab A8 binding to LRP and Cl-II, respectively. These results again demonstrate that the functional integrity of the ligand-binding sites of Cl-II is fully maintained. Next, we studied the cross-competition of RAP-, t-PA·PAI-1 complex-, and Fab A8-binding to LRP and to Cl-II. As shown in Fig. 4D, t-PA·PAI-1 complex binding to LRP is totally inhibited by RAP at an IC50 value of 0.3 nM. Surprisingly, an approximately 250-fold higher concentration of RAP is required to inhibit t-PA·PAI-1 complex binding to Cl-II (IC50 = 76 nM). The latter residual inhibition is likely to be the result of sterical hindrance of RAP for t-PA·PAI-1 complex binding as a consequence of the partial overlap of the binding sites. This is illustrated by the reverse experiment shown in Fig. 4F, where similarly high concentrations are required to compete for RAP binding to Cl-II and to LRP. Even more pronounced is the difference in the RAP inhibition of Fab A8 binding to LRP and Cl-II (Fig. 4E). Whereas Fab A8 binding to LRP is efficiently prevented by RAP at an IC50 of 0.2 nM, RAP inhibition of Fab A8 binding to Cl-II was only partial and required high RAP concentrations (approximately 40% inhibition at 1 µM RAP). Results from the reverse competition experiment, depicted in Fig. 4G, show a complete lack of inhibition of RAP binding to either LRP or Cl-II by Fab A8. From these experiments we conclude that although RAP is fully able to bind to Cl-II, it is not able to display its ligand inhibitory role.


Fig. 4. Competition of ligands for binding to either LRP or recombinant Cl-II. A-C, determination of the concentrations of half-maximal saturation of the binding to immobilized LRP (black-triangle) or Cl-II (bullet ). D-G, competitive ELISA experiments. Ligands were incubated in the presence of indicated competitors on either immobilized LRP in the presence (black-triangle) or the absence (black-diamond ) of Ca2+ or immobilized Cl-II in the presence (bullet ) or the absence (black-square) of Ca2+. D and E represent incubations with t-PA·PAI-1 complexes and Fab A8, respectively, in the presence of an increasing concentration of RAP. F and G represent incubations with RAP in the presence of increasing concentrations of t-PA·PAI-1 complexes or Fab A8, respectively. Competition experiments were performed at least in triplicate.
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DISCUSSION

To further analyze ligand binding by Cl-II of LRP, we have generated a subset of Cl-II fragments. We show that expression of soluble Cl-II by baby hamster kidney cell lines yields preparations with fully functional Ca2+-dependent ligand binding sites. Using these fragments we revealed the presence of an array of three distinct binding sites for t-PA·PAI and PAI-1, for RAP, and for the pseudo-ligand Fab A8. In some cases, the Kd values for the ligand interactions with the fragments are somewhat higher than those for the intact LRP molecule. This may be explained by the fact that for the Kd determinations of the cluster II fragments, the ligands were immobilized in contrast to the analysis of ligand binding to LRP, where the receptor was immobilized to the sensor chip. Attempts to immobilize the Cl-II fragments to the sensor chips failed, possibly due to the acidic nature of the proteins that is incompatible with the immobilization protocol. The lack of t-PA binding to the fragments and the observation that t-PA·PAI-1 complexes and PAI-1 both bind to the same domain support our previous observation that the PAI-1 moiety of the complex is the major determinant responsible for binding to Cl-II of LRP.2 Because no binding of free t-PA to Cl-II could be detected, at present we cannot exclude that a t-PA binding site outside Cl-II is responsible for the binding to LRP (5, 33, 34).

The arrangement of the RAP binding site in Cl-II, in between a partially overlapping t-PA·PAI-1 complex binding site and an independent binding site for Fab A8, renders the recombinant Cl-II an interesting tool to study the mechanism of inhibition of ligand binding by RAP. Surprisingly, even though the binding of RAP to Cl-II and LRP are quite similar, RAP is a poor inhibitor of ligand binding to the isolated Cl-II compared with its inhibition of ligand binding to the intact molecule. This observation makes it unlikely that RAP regulates ligand binding by a mechanism that involves mutually exclusive binding to overlapping binding sites. Our data are compatible with a model of ligand inhibition by RAP that assumes RAP-induced allosteric changes in the receptor, as suggested by Willnow et al. (18). First, different studies, including this one, confirm the presence of at least two RAP binding sites on LRP (12, 16, 20, 30, 31). One site is localized in Cl-II, the second in the cluster IV domain. A third, low affinity site is present on cluster III, as reported recently by Bu and Rennke (20). Second, the RAP molecule may be multivalent for LRP binding based on the internal triplication of LRP binding domains (14). At least two of these domains, present at the amino- and carboxyl-terminal ends, have been shown to bind to LRP with high affinity (19). The presence of multiple receptor binding sites may endow a single RAP molecule with the property to interact with different RAP binding sites on LRP, thereby inducing a conformational change in the receptor that prevents ligand binding. Cl-II may lack the structural requirements to maintain the proposed allosteric model needed for efficient RAP inhibition of ligand binding. The observed residual cross-competition of RAP and t-PA·PAI-1 complexes for binding to Cl-II may be explained by steric hindrance, consistent with the findings that sites are partially overlapping.

In a study by Saito et al., the importance of Cl-II in the homologous glycoprotein 330/megalin was demonstrated (32). This region contains a major epitope involved in the pathogenesis of nephritis, suggesting a highly exposed localization that may be well suited for protein-protein interactions. Our studies focusing on ligand binding to Cl-II of LRP support this view. Therefore, the data reported here may serve as a paradigm for ligand binding to other members of the low density lipoprotein receptor gene family as well.

Recently it was demonstrated that RAP functions as a novel type of specialized chaperone, inhibiting premature ligand binding, which may result in subsequent degradation of the receptor in the endoplasmic reticulum/proteasome pathway (14, 15, 20). The model described here may apply to the intracellular function of RAP as well.


FOOTNOTES

*   This work was supported by Grant 902-26-128 from the Netherlands Organization for Scientific Research.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.
Dagger    Recipient of a Fellowship from the Royal Netherlands Academy of Arts and Sciences. To whom correspondence should be addressed: Academic Medical Center, Dept. of Biochemistry, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Tel.: 31-20-5665129; Fax: 31-20-691519; E-mail: a.j.vanzonneveld{at}amc.uva.nl.
1   The abbreviations used are: LRP, low density lipoprotein receptor-related protein; C, complement-type repeat; Cl-II, second cluster of complement-type repeats; E, epidermal growth factor-type repeat; mAb, monoclonal antibody; PAI-1, plasminogen activator inhibitor type-1; RAP, receptor-associated protein; SPR, surface plasmon resonance; t-PA, tissue-type plasminogen activator; ELISA, enzyme-linked immunosorbent assay; HBS, HEPES-buffered saline.
2   I. R. Horn, B. M. M. van den Berg, S. K. Moestrup, H. Pannekoek, and A. J. van Zonneveld, submitted for publication.

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