Plasminogen Activator Inhibitor-1 Contains a Cryptic High Affinity Binding Site for the Low Density Lipoprotein Receptor-related Protein*

Steingrimur StefanssonDagger , Shabazz Muhammad, Xiang-Fei Cheng§, Frances D. Battey§, Dudley K. Strickland§, and Daniel A. Lawrence

From the Departments of Biochemistry and § Vascular Biology, J. H. Holland Laboratory, American Red Cross, Rockville, Maryland 20855

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Much of the controversy surrounding the binding of plasminogen activator inhibitor-1 (PAI-1) to the low density lipoprotein receptor-related protein (LRP) may be due to the labile structure of PAI-1 and the distinct conformations that it can adopt. To examine this possibility and to test the hypothesis that PAI-1 contains a specific high affinity binding site for LRP, a sensitive and quantitative assay for PAI-1 binding to LRP was developed. This assay utilizes a unique PAI-1 mutant that was constructed with a hexapeptide tag at the NH2 terminus, which is recognized by the protein kinase, heart muscle kinase and can be specifically labeled with 32P. Our results show that only 32P-PAI-1 in complex with a proteinase binds LRP with high affinity and is efficiently endocytosed by cells, indicating that a high affinity site for LRP is generated on PAI-1 only when in complex with a proteinase. In addition, PAI-1 in complex with different proteinases is shown to cross-compete for LRP binding, demonstrating that the binding site is independent of the proteinase and therefore must reside on the PAI-1 portion of the complex. Finally, mutagenesis of PAI-1 results in loss of LRP binding, confirming that the high affinity binding site is located on PAI-1 and suggesting that the LRP binding site lays within a region of PAI-1 previously shown to contain the heparin binding domain.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

The serine proteinase inhibitor (serpin)1 PAI-1 is the most efficient in vivo inhibitor known of both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) (1). Under normal physiological conditions PAI-1 expression is primarily limited to smooth muscle cells and megacaryocytes. However, many cells induce PAI-1 expression in response to cytokines associated with defensive processes such as inflammation (2). The active form of PAI-1 is thought to have its reactive center loop (RCL) fully exposed and thus available for interaction with proteinases (3, 4). However, the active conformation is unstable and decays to an inactive latent conformation which in turn can be partially reactivated by treatment with denaturants (5). Cleaved PAI-1 can be generated either by deacylation of the covalent enzyme-inhibitor complex (3, 6) or by reaction with a non-target protease such as elastase, which cleaves the RCL at a site other than the P1-P1' reactive center bond (7). In the latent and cleaved forms of PAI-1, the RCL is fully inserted into beta -sheet A of the inhibitor, making these forms of PAI-1 inactive against target proteinases (8, 9). Upon complex formation with a target proteinase the RCL is cleaved at the P1-P1' reactive center bond and the amino-terminal portion of the RCL is inserted into beta -sheet A (3, 10). Although the precise extent of RCL insertion in the stable complex is not known, recent data suggest that the insertion may be less than that of the latent and cleaved forms of PAI-1 (4, 11, 12). The integration of the RCL into beta -sheet A is an essential step in the serpin inhibitory mechanism and results in a conversion of beta -sheet A from a 5-stranded to a 6-stranded sheet, altering the conformation of PAI-1 at sites distant from the actual point of loop insertion (13). This change has been shown to result in the loss of high affinity binding of PAI-1 for vitronectin (14). The physiological implications of this conformational dependence of the PAI-1-vitronectin interaction are important since they affect the functions of both proteins. Vitronectin influences PAI-1 function by stabilizing its active conformation and converting it to a very efficient inhibitor of thrombin (15, 16). Conversely, PAI-1 influences vitronectin function by inducing a conformational change in vitronectin (17) and by blocking the binding of both integrins and the urokinase receptor (uPAR) to vitronectin. This latter interaction results in the inhibition of cell migration on vitronectin-containing matrices (18-20).

A number of studies have examined the cellular clearance of uPA·PAI-1 complexes and established that cell surface uPA is rapidly endocytosed and degraded when in complex with PAI-1 (21-24). This clearance was demonstrated to be mediated through high affinity binding to low density lipoprotein receptor-related protein (LRP) (25) or to other members of the low density lipoprotein receptor family (26-28). The exact nature of the high affinity binding of uPA·PAI-1 complexes to LRP is unclear. To date, the most detailed study on the binding of uPA·PAI-1 to LRP was performed by Nykjaer et al. (29) who observed that active, latent, or cleaved PAI-1 as well as uPA alone could each individually inhibit the binding of uPA·PAI-1 complexes to LRP. The EC50 values for each of these competitors was approximately 100-fold higher than that obtained with the complex, and based on these results they proposed that the high affinity binding of uPA·PAI-1 complexes to LRP was due to contributions from the two low affinity sites present on the proteinase and on the inhibitor. Consistent with their data, single chain uPA and active PAI-1 have also been reported to bind to purified LRP with Kd values of 45 nM (30), and 35 nM (31), respectively. We recently demonstrated that thrombin in complex with PAI-1 was endocytosed and degraded by cells via the LRP much more efficiently than either thrombin alone or thrombin in complex with other proteinase inhibitors (32). These data led us to suspect that PAI-1 is unique among serpins and contains a specific high affinity binding site for LRP that is not present on thrombin or the other inhibitors examined. Thus in the present study we test the hypothesis that PAI-1 contains a specific high affinity binding site for LRP by analyzing the catabolism of different conformational forms of 32P-PAI-1 by mouse embryonic fibroblasts, and by direct binding to purified LRP. In addition, competitive inhibition assays and mutational analysis examine the relative contributions of both the inhibitor and proteinase in the binding of complexes to LRP. These results indicate that a high affinity binding site for LRP is generated on PAI-1 only upon complex formation with an enzyme, and that this site is not expressed on either the enzyme alone or on the active, latent or elastase cleaved forms of PAI-1. Finally, analysis of a PAI-1 variant with arginine 76 mutated to glutamic acid suggests that this cryptic site in PAI-1 is located in a region previously shown to contain the heparin binding domain.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Proteins and Reagents-- PIPES was purchased from Boehringer Mannheim. Bovine heart muscle kinase-catalytic subunit (HMK), BSA, dithiothreitol, aminophenylmethylsulfonyl fluoride (APMSF), phenylmethylsulfonyl fluoride (PMSF), gelatin, and casein were purchased from Sigma. Na125I and RedivueTM [gamma -32P]ATP (6000 Ci/mmol) were purchased from Amersham Corp. Phastgels, heparin-Sepharose, and phenyl-Sepharose were purchased from Pharmacia Biotech Inc. Human pro-uPA and low molecular weight uPA (L-uPA) were generous gifts from Dr. Jack Henkin (Abbott). High molecular weight uPA (H-uPA) (Ukidan, Serono AB of Switzerland) was a generous gift from Dr. Tor Ny (Department of Medical Biochemistry and Biophysics, Umeå University, Sweden) or generated from pro-uPA by incubation with immobilized plasmin followed by purification on benzamidine-Sepharose. Bovine beta -trypsin was a generous gift from Dr. Steven T. Olson (Center for Molecular Biology of Oral Diseases, University of Illinois, Chicago, IL). Human 39-kDa receptor-associated protein (RAP) was expressed in bacteria as a fusion protein with glutathione S-transferase as described in Williams et al. (33). LRP was purified by affinity chromatography using alpha 2-macroglobulin:methylamine-Sepharose as described by Ashcom et al. (34). Fully active wtPAI-1 and native vitronectin were purchased from Molecular Innovations (Royal Oak, MI). Pfu polymerase was purchased from Stratagene (La Jolla, CA). LRP-deficient fibroblasts (PEA-13) and normal mouse embryo fibroblasts (MEF) (35) were a generous gift from Dr. Joachim Herz (Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX).

Construction of Mutant PAI-1 cDNA-- The coding sequence for the six-residue peptide tag, Ala-Arg-Arg-Ala-Ser-Val, which contains the recognition sequence for the specific protein kinase HMK (36) was introduced at the 5'-end of the mature PAI-1 cDNA by a polymerase chain reaction with the following oligonucleotides: forward, 5'-GAG CAT ATG GCG AGA AGA GCG AGC GTG CAC CAT CCC CCA TCC TAC G-3', and reverse 5'-GCG GTG GCA GCA GCC AAC TC-3'. The polymerase chain reaction product was then restricted with NdeI and NsiI, and ligated into the pET3aPAI-1 prokaryotic vector (37). DNA sequence analysis of the entire coding region indicated that no additional sequence changes were introduced during the polymerase chain reaction. The construct was then transformed into Escherichia coli strain BL21 (DE3), and high levels of recombinant PAI-1 containing the HMK tag at its amino terminus were produced and purified in the fully active form as described previously (38), except that HMK-PAI-1 was eluted from the heparin-Sepharose with 2.5 M NaCl in 0.05 M potassium phosphate, 1 mM EDTA, pH 6.5, and the active and latent forms of HMK-PAI-1 were separated on phenyl-Sepharose using a gradient of 0 to 10% glycerol in phosphate-buffered saline, pH 6.6. The PAI-1 mutant, arginine 76 to glutamic acid (R76E), was constructed with the following oligonucleotide 5'-GCC CCC GCC CTC GAG CAT CTG TAC AAG-3' using the transformer site-directed mutagenesis kit (CLONTECH) according to the manufacturer's instructions. To produce PAI-1 exclusively in the active conformation, the R76E mutant was constructed on a stable PAI-1 background, 14-1b, that has been previously shown to have significantly enhanced functional stability, but to be indistinguishable from wild-type PAI-1 with respect to inhibitory activity, heparin binding, and vitronectin binding (39). Its binding to LRP is also indistinguishable from wild-type PAI-1 (compare Figs. 3 and 5 to Figs. 8 and 9). The R76E mutant was purified in two steps by hydroxylapatite chromatography (40) followed by phenyl-Sepharose (38).

Radiolabeling of Proteins-- Purified active HMK-PAI-1 (20 µg) was added to a tube containing 20 µl of labeling buffer (200 mM PIPES, pH 6.5, 10 mM dithiothreitol, 200 mM NaCl, and 120 mM MgCl2). The reaction mixture was brought to 200 µl with water followed by addition of HMK (250 units in 40 µl of water), 20 µl of BSA (1% in water), and 25 µl of [gamma -32P]ATP. The sample was incubated at room temperature for 60 min, after which the radiolabeled protein was separated from unreacted nucleotides using a PD-10 column equilibrated with TBS containing 1% boiled BSA, 0.01% Tween 20, and 5 mM CaCl2. The specific activity of the HMK-PAI-1 labeled by this procedure was typically 1-5 × 106 cpm/µg. Latent 32P-PAI-1 was generated from active 32P-PAI-1 as described by Sancho et al. (41). The sample was concentrated by the addition of solid ammonium sulfate to a final concentration of 55% followed by centrifugation at 13,000 rpm for 10 min at 4 °C. Cleaved 32P-PAI-1 was generated by treating active 32P-PAI-1 with pancreatic elastase at a 10/1 molar ratio (PAI-1/elastase) for 1 h at room temperature in 20 mM PIPES, pH 6.0, containing 500 mM NaCl and 3% BSA. The reaction was stopped by the addition of PMSF in dimethyl sulfoxide to a final concentration of 1 mM. Complexes of 32P-PAI-1 with H-uPA and L-uPA are made by incubating the proteins at a 1/1.5 molar ratio (PAI-1/uPA) for 30 min at room temperature in TBS, pH 7.5, containing 1% boiled BSA, 0.01% Tween 20, and 5 mM CaCl2 followed by the addition of 5 µM APMSF. Complexes of trypsin 32P-PAI-1 were made by incubating the proteins in a 3/1 molar ratio (PAI-1/trypsin) for 30 min at room temperature in TBS, pH 7.5, containing 1% boiled BSA, 0.01% Tween 20, and 5 mM CaCl2 followed by addition of 5 µM APMSF. Complex formation with 125I-labeled trypsin, H-uPA, and L-uPA in complex with wtPAI-1 and PAI-1 mutants was performed using a molar ratio of 2/1 (PAI-1/PA) and 4/1 (PAI-1/trypsin) followed by addition of 5 µM APMSF.

Solid Phase Binding Assays-- Solid phase binding assays were performed by either adding increasing concentrations of radiolabeled ligand to immobilized receptor (direct binding assays) or incubating trace concentrations of radiolabeled ligand with immobilized receptor in the presence of increasing concentrations of unlabeled ligand or inhibitor (competition binding assays) (33). Purified LRP or BSA (3 µg/ml) were coated onto microtiter wells and incubated for 18 h at 4 °C. Plates were then blocked with TBS, pH 7.5, containing 1% boiled BSA, 0.01% Tween 20, and 5 mM CaCl2 for 16-18 h at 4 °C or 2 h at 37 °C. Direct binding assays were performed by adding increasing concentrations of 32P-PAI-1 conformers or proteinase complexes to immobilized LRP or BSA coated plates in TBS, pH 7.5, containing 1% boiled BSA, 0.01% Tween 20, and 5 mM CaCl2 for 16-18 h at 4 °C. Wells were washed three times with TBS, pH 7.5, containing 0.01% Tween 20 and 5 mM CaCl2. Competition binding assays were performed by adding increasing concentrations of unlabeled competitor to wells containing 2.5-5 nM 32P-PAI-1 conformers or proteinase complexes in TBS, pH 7.5, containing 1% boiled BSA, 0.01% Tween 20, and 5 mM CaCl2 for 16-18 h at 4 °C. Wells were washed three times with TBS, pH 7.5, containing 0.01% Tween 20 and 5 mM CaCl2. Binding of 32P-PAI-1 to native vitronectin was performed using competition assays. Vitronectin was coated onto microtiter wells (1 µg/ml) for 18 h at 4 °C followed by blocking unoccupied sites with TBS, pH 7.5, containing 3% BSA. The binding of 32P-PAI-1 conformers or proteinase complexes were performed in the presence of increasing concentrations of corresponding unlabeled PAI-1 conformers or proteinase complexes. For all assays, bound ligand was removed from wells using 0.1 ml of 0.1 M NaOH prior to liquid scintillation analysis. Binding of 32P-PAI-1 conformations and proteinase complexes to BSA-coated plates was subtracted from the values obtained for native vitronectin or LRP. Kd values for the solid phase binding of proteinase·32P-PAI-1 complexes to immobilized LRP were calculated using the following forms of the standard binding equation. Equation 1 was used for a single binding site model
y=<UP>Cap</UP>(L/(K<SUB>d</SUB>+L)) (Eq. 1)
where y is the amount of bound ligand Cap is the capacity for binding, and L is the concentration of ligand added. Equation 2 was used for a two binding site model
y=<UP>Cap</UP>(L/(K<SUB>d</SUB>+L))+<UP>Cap</UP>2(L/(K<SUB>d</SUB>2+L)) (Eq. 2)
where y is the amount of bound ligand, Cap and Kd are the capacity and dissociation constant of binding site 1, Cap2 and Kd2 are the capacity and dissociation constant of binding site 2, and L is the concentration of ligand added. Inhibition constants (Ki) for receptor-ligand interactions were calculated using a four-parameter logistic fit (42)
y=a/(1+(x/K<SUB>i</SUB>)<SUP><IT>s</IT></SUP>)+b (Eq. 3)
where a is the Y range, Ki is the IC50, s is the slope factor, and b is the background binding. Competition binding assays using 125I-trypsin in complex with PAI-1 and mutant PAI-1 were prepared as described above for the 32P-PAI-1 complexes, and the assay was performed as described previously (32).

Cell Assays-- LRP-deficient fibroblasts derived from an LRP null mouse embryo (PEA-13) and normal MEF were grown in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum (Hyclone, Logan, UT), penicillin, and streptomycin. For the endocytosis and degradation assays, cells were plated for 18 h at 37 °C, 5% CO2 on 24- or 12-well tissue culture plates precoated with 0.1% gelatin to exclude any effects of serum vitronectin (43, 44). Prior to addition of radiolabeled ligand, the cells were washed twice in serum-free Dulbecco's modified Eagle's medium and incubated for 30 min in Dulbecco's modified Eagle's medium containing 0.5% BSA and 0.5% casein (assay medium). 32P-PAI-1 conformers or proteinase complexes were added to cell layers in assay medium and incubated for 4-6 h at 37 °C, 5% CO2. Where indicated, RAP (1 µM) was added to the assay 30 min prior to addition of radiolabeled ligands. Quantitation of endocytosis and degradation was done essentially as described (45). Briefly, following the incubation period, the assay medium was removed from cells and precipitated with 10% trichloroacetic acid. Cell-mediated degradation was determined by subtracting trichloroacetic acid-soluble radioactivity in cell media from trichloroacetic acid-soluble radioactivity in media in the absence of cells. Cell layers were washed twice with serum-free medium and incubated in medium containing trypsin (0.5 mg/ml), proteinase K (0.5 mg/ml), and 0.5 mM EDTA for 2-5 min at 4 °C. The cells were then centrifuged at 6000 rpm for 2 min, and the radioactivity in the cell pellet was taken to represent the amount of endocytosed ligand. In experiments following the metabolic fate of 32P-PAI-1 in tissue culture, MEF cells were seeded onto 6-well tissue culture plates in the presence of 10% serum and allowed to adhere for 16 h. Cells were washed and incubated with active 32P-PAI-1 (5 nM) in assay medium for 30 min. After washing away unbound 32P-PAI-1, the cells were incubated either with assay medium alone, medium containing RAP (1 µM), medium containing H-uPA (200 nM), or medium containing uPA and RAP (200 nM and 1 µM, respectively). After 1 h the medium was removed, and cell layers were washed and incubated with 1 ml of trypsin, EDTA, and proteinase K, and both cell pellet (internalized ligand) and trypsin/proteinase K soluble supernatant (matrix bound ligand) were recovered and counted. LRP-mediated cellular endocytosis and degradation of 125I-trypsin, in complex with PAI-1 and mutant PAI-1, were prepared as described above for the 32P-PAI-1 complexes, and the experiments were performed as described previously (32).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

HMK-PAI-1 Is Indistinguishable from wtPAI-1-- Previously we showed that 125I-thrombin in complex with PAI-1, but not in complex with other serpins, binds to LRP with high affinity and that the individual components of the complex (thrombin and PAI-1) were unable to compete for this high affinity interaction (32). These results suggested that PAI-1, when in a covalent complex with an enzyme, contained the high affinity binding site for LRP. To test this hypothesis, the interaction of PAI-1 with LRP was examined. Since PAI-1 is extremely sensitive to oxidative radiolabeling (46, 47), it was necessary to develop an alternative approach to monitor PAI-1 binding to LRP. Therefore, a mutant form of PAI-1 was constructed with the HMK recognition sequence, ARRASV, at the NH2 terminus. This tag provides a sensitive and nondenaturing way to radiolabel PAI-1. The specificity of this procedure is seen in Fig. 1A. When crude lysates from E. coli expressing either HMK-PAI-1 (Fig. 1A, lane 1) or wtPAI-1 (Fig. 1A, lane 2) or lysates from control E. coli cells (Fig. 1A, lane 3) were incubated with HMK and [gamma -32P]ATP and analyzed, a major band with the same mobility as PAI-1 was detected by autoradiography only in lysates from cells expressing HMK-PAI-1. Following purification and separation of the active and latent HMK-PAI-1, the active HMK-PAI-1 was efficiently labeled with 32P using this method (Fig. 1B, lane 4). The labeling procedure did not affect the ability of HMK-PAI-1 to form SDS-stable complexes with H-uPA or tPA (Fig. 1B, lanes 5 and 6, respectively). Densitometric analysis revealed that greater than 95% of the 32P-PAI-1 formed complexes with the enzymes in contrast to latent 32P-PAI-1, which formed less than 10% SDS-stable complexes with either tPA or uPA (data not shown). Finally, like active wtPAI-1, 32P-PAI-1 is a substrate for pancreatic elastase that cleaves PAI-1 in the reactive center loop at Val343-Ser344 (7) and destroys inhibitory activity (data not shown). These data demonstrate that the addition of ARRASV to the NH2 terminus of PAI-1 neither affects its inhibitory properties nor generation of latent or cleaved forms of PAI-1.


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Fig. 1.   Autoradiography of 32P-PAI-1 and proteinase·32 P-PAI-1 complexes. Panel A, E. coli expressing either HMK-PAI-1 (lane 1), wtPAI-1 (lane 2), or control E. coli cells (lane 3) were lysed, incubated with HMK and [gamma -32P]ATP as described under "Materials and Methods," and precipitated with ammonium sulfate. Samples of precipitated cell lysates (5 µl) were then separated by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Panel B, purified active 32P-PAI-1 (5 ng, lane 4) was incubated with a 2-fold molar excess of either H-uPA (lane 5) or tPA (lane 6) in TBS for 30 min followed by SDS-polyacrylamide gel electrophoresis and autoradiography.

The HMK tag also does not affect the high affinity binding of active PAI-1 to vitronectin. As shown in Fig. 2A, active 32P-PAI binds to vitronectin with high affinity and is competed equally well by either unlabeled active HMK-PAI-1 or active wtPAI-1. In addition, latent and elastase cleaved 32P-PAI-1, or 32P-PAI-1 in complex with uPA bound to vitronectin with substantially reduced affinity (data not shown), consistent with previous observations that only the active form of PAI-1 binds vitronectin with high affinity (14) and confirming that the HMK tag does not affect vitronectin binding. Competitive binding assays also confirmed that the binding of H-uPA·32P-PAI-1 complexes to LRP is indistinguishable to that of H-uPA·wtPAI-1 complexes, since both unlabeled H-uPA·HMK-PAI-1 and H-uPA· wtPAI-1 complexes compete equally well for the binding of H-uPA·32P-PAI-1 complexes to LRP (Fig. 2B). The minor differences observed between the two curves in Fig. 2B are not significant. Together these data demonstrate that HMK-PAI-1 is indistinguishable from wtPAI-1 with respect to inhibitory activity and binding to either vitronectin or LRP. The high specific activity of HMK-PAI-1 achieved with this radiolabeling procedure together with its full retention of inhibitory activity makes this mutant an ideal reagent to examine the binding of different PAI-1 conformational forms to LRP.


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Fig. 2.   Competition binding assays of 32P-PAI-1 and proteinase·32P-PAI-1 complexes binding to vitronectin and LRP. Panel A, active 32P-PAI-1 (2 nM) was incubated with immobilized vitronectin in the presence of either increasing concentrations of wtPAI-1 (bullet ) or unlabeled HMK-PAI-1. (open circle ). Panel B, H-uPA·32P-PAI-1 (5 nM) was incubated with immobilized LRP in the presence of either increasing concentrations of H-uPA·wtPAI-1 complexes (bullet ) or unlabeled H-uPA·HMK-PAI-1 complexes (open circle ). The data presented are representative of at least three experiments. Each plotted value represents the average of duplicate determinations with the range indicated by bars.

Only PAI-1 in Complex with Proteinase Binds to LRP with High Affinity and Is Cleared by Cells-- Several studies have suggested that H-uPA is cleared from the cell surface only when in complex with the inhibitor PAI-1 (21-23, 25). Subsequently, LRP was found to bind the H-uPA·PAI-1 complex with high affinity and is likely the major clearance receptor for this complex in vivo (25). However, other studies have suggested that uPA alone can be cleared by cells (30). It has also been suggested that the binding of H-uPA·PAI-1 and H-uPA·proteinase nexin-1 to uPAR is an essential step for LRP-mediated endocytosis (48, 49). Whereas other reports have shown that PAI-1 and proteinase nexin-1 in complex with non-uPAR binding proteinases such as thrombin and L-uPA, still bind to LRP and are efficiently endocytosed and degraded by cells (24, 29, 32, 50). To examine whether PAI-1 alone or in a complex with a proteinase is endocytosed and degraded, in vitro clearance studies were performed using MEF cells or embryonic fibroblasts from an LRP null mouse (PEA-13) (35). As shown in Fig. 3, A and B, 32P-PAI-1 complexed with H-uPA, L-uPA, or trypsin was efficiently endocytosed and degraded by MEF cells in a RAP inhibitable process, while little endocytosis or degradation of the complexes was observed with the LRP-deficient PEA-13 cells (Fig. 3, C and D). The enhanced clearance seen with the H-uPA·32P-PAI-1 compared with the other complexes is probably not due to the binding of the H-uPA complex to uPAR, since human H-uPA, which is used in this study, has been reported to not bind to mouse uPAR (51). In contrast to the PAI-1·proteinase complexes, active, latent and cleaved 32P-PAI-1 were not efficiently endocytosed or degraded by MEF or by PEA-13 cells (Fig. 3). Furthermore, the low levels of endocytosis and degradation seen with these forms of PAI-1 were not inhibited by RAP, suggesting that they do not bind to LRP with high affinity.


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Fig. 3.   LRP mediated endocytosis and degradation of 32P-PAI-1 conformers and proteinase·32P-PAI-1 complexes. Panel A, endocytosis by MEF cells of active, latent, and cleaved 32P-PAI-1 and 32P-PAI-1 in complex with H-uPA, L-uPA, or trypsin (10 nM final concentration) was performed in the absence (black bars) or presence (white bars) of RAP (1 µM). Panel B, degradation by MEF cells of the same 32P-PAI-1 conformations and complexes as in panel A. Panels C and D are the same as A and B, respectively, except that LRP-deficient PEA-13 cells were used. The data presented are representative of four experiments. Each plotted value represents the average of duplicate determinations with the range indicated by bars.

To verify whether the active, latent or cleaved forms of HMK-PAI-1 bind to LRP, solid phase binding experiments were performed. These results are seen in Fig. 4 and demonstrate that only H-uPA·32P-PAI-1 or L-uPA·32P-PAI-1 complexes bind efficiently to LRP, and that neither active, latent nor cleaved 32P-PAI-1 bind to LRP with high affinity. In addition, since the HMK-PAI-1 is 32P-labeled at a single specific site on the inhibitor prior to the formation of the uPA complexes and since >95% of the inhibitor forms complexes, we are able to directly compare the binding stoichiometry of H-uPA·PAI-1 to LRP with that of L-uPA·PAI-1. As is seen in Fig. 4, H-uPA·32P-PAI-1 appears to bind LRP with approximately twice the stoichiometry of L-uPA·32P-PAI-1. Consistent with this, the binding of the L-uPA·32P-PAI-1 complex is fit well by a model assuming a single class of binding sites having a Kd of 7.4 ± 1.4 nM. In contrast, the binding data for the H-uPA·32P-PAI-1 complex is poorly fit by a single binding site model (see the dashed line in Fig. 4), but is approximated much better by a model that assumes two classes of binding sites, one with high affinity (1.6 ± 0.4 nM) and one with a moderate affinity (55 ± 14 nM) (Fig. 4). While this model assumes that the two sites act independently we cannot rule out the possibility of cooperativity in binding. These results suggest that both H-uPA and L-uPA PAI-1 complexes have one high affinity binding site similar to that seen with thrombin·PAI-1 complex and that an additional moderate affinity binding site is present on the H-uPA·PAI-1 complex. These data are also consistent with the cellular uptake and degradation shown in Fig. 3, where the clearance of H-uPA·32P-PAI-1 is approximately double that observed with either L-uPA·32P-PAI-1 or trypsin·32P-PAI-1 complexes. Finally, RAP blocked the binding of both forms of uPA·32P-PAI-1 complexes to LRP, but had no effect on the interaction of active, latent and cleaved 32P-PAI-1 (data not shown). Since RAP is thought to block the binding of all known ligands to LRP (52), this result suggests that active, latent and cleaved 32P-PAI-1 do not possess specific high affinity binding for LRP, and is consistent with the poor cellular endocytosis and degradation of these forms seen in Fig. 3. In contrast to the other forms of PAI-1 examined, only active PAI-1 appears to bind to purified LRP with moderate affinity (Fig. 4), but is not efficiently endocytosed and degraded by cells (Fig. 3). This lack of cellular clearance, together with the inability of RAP to block solid phase binding of active PAI-1 to purified LRP, suggest that the moderate affinity binding of active PAI-1 may be an artifact of the solid phase assay. Previous studies have also reported that uncomplexed radioiodinated or plastic adsorbed PAI-1 bound LRP with high affinity and was endocytosed by cells (45). However, since both of these treatments are known to alter the conformation of PAI-1 (46, 47, 53) it is unlikely that these interactions represent the specific binding of native active PAI-1 to LRP.


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Fig. 4.   Binding of 32P-PAI-1 conformers and proteinase·32P-PAI-1 complexes to immobilized LRP. Solid phase binding of increasing concentrations of active (triangle ), latent (diamond ), and cleaved (down-triangle) 32P-PAI-1, or H-uPA·32P-PAI-1 complex (open circle ) or L-uPA·32P-PAI-1 (square ) complex to immobilized LRP. The data presented are representative of at least four experiments. Each plotted value represents the average of duplicate determinations with the range indicated by bars. The fits for H-uPA·32P-PAI-1 and L-uPA·32P-PAI were calculated as described under "Materials and Methods" and the chi 2 values for each fit are: 0.003306 for the single site model (- - - - -) and 0.0004092 the two-site model (------) with H-uPA·32P-PAI-1 (p = 0.01), and 0.0004361 for the single site model (- - - - -) with L-uPA·32P-PAI-1.

Competitive Inhibition of Proteinase·PAI-1 Complexes Binding to LRP-- To determine if proteinase·PAI-1 complexes only show high affinity binding when covalently associated, competitive binding assays were performed. Fig. 5A demonstrates that like thrombin·PAI-1 (32), the binding of H-uPA·32P-PAI-1 to LRP is efficiently competed by increasing concentrations of unlabeled complex. The Ki of this association is 2 nM, which is nearly identical to the calculated Kd of 1.6 nM for H-uPA·32P-PAI-1 binding to LRP (Fig. 4). In contrast, none of the individual components are able to compete for the high affinity binding of the complex to LRP, all of which show minimum estimated Ki values of greater than 200 nM. Fig. 5B shows similar results for L-uPA·32P-PAI-1 complex binding to LRP. In this case the calculated Ki for the L-uPA·32P-PAI-1 complex is 10 nM, which is also very similar to the calculated Kd for this interaction of 7.4 nM (Fig. 4). These results are consistent with our previous observations with the thrombin·PAI-1 complex (32) and with the results of Nykjaer et al. (29). It should also be pointed out that to examine the high affinity interactions of the complexes with LRP, the tracer concentrations used in these experiments were relatively low (5 nM), thus, the majority of the binding in this experiment results from occupancy of only the high affinity binding site.


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Fig. 5.   Competition of proteinase·32P-PAI-1 complexes binding to LRP by either proteinase or PAI-1. Panel A, H-uPA·32P-PAI-1 complex (5 nM) was added to immobilized LRP in the presence of increasing concentrations of H-uPA·wtPAI-1 complex (bullet ), wtPAI-1 (black-square), APMSF treated H-uPA (open circle ) or wtPAI-1 and APMSF treated H-uPA together (square ). Panel B, L-uPA·32P-PAI-1 complex (5 nM) was added to immobilized LRP in the presence of increasing concentrations of L-uPA·wtPAI-1 complex (bullet ), wtPAI-1 (black-square), APMSF-treated L-uPA (open circle ), or wtPAI-1 and APMSF-treated L-uPA together (square ). The data presented are representative of at least five experiments. Each plotted value represents the average of duplicate determinations with the range indicated by bars.

If neither component is individually responsible for the high affinity binding to LRP then it may be that moderate affinity sites on both the proteinase and the inhibitor are contributing to the high affinity interaction (29). However, this seems unlikely since active site inhibited uPA-and wtPAI-1 together do not inhibit the high affinity binding of the covalent uPA·32P-PAI-1 complex to LRP, even at 100 fold molar excess (Fig. 5, A and B). A more likely explanation is that the high affinity binding site for LRP is generated on PAI-1 upon reaction with a proteinase. In this case it would be predicted that the same high affinity binding site should be present on all proteinase·PAI-1 complexes independent of the proteinase used and that each complex should be able to cross compete for this high affinity binding site on LRP. To test this possibility competitive inhibition assays for the binding of H-uPA·32P-PAI-1, L-uPA·32P-PAI-1, or trypsin·32P-PAI-1 to LRP were performed. These results are shown in Fig. 6 and demonstrate that unlabeled PAI-1 in complex with each enzyme is able to cross compete for the high affinity binding of 32P-PAI-1 in complex with each of the other enzymes. As expected for competitive inhibition of binding, all of the unlabeled complexes are able to completely inhibit the high affinity binding of the 32P-PAI-1 complexes. Furthermore, the Ki values of L-uPA·PAI-1 and trypsin·PAI-1 are essentially identical in each case (13.9 versus 14.1 nM, respectively) while the Ki of H-uPA·PAI-1 is approximately 10-fold lower with each enzyme·PAI-1 complex. Also consistent with competitive inhibition of binding is the result that the Ki values for each complex are nearly identical to the calculated Kd values for that complex (54). The observation that all the complexes can compete with each of the other complexes in a competitive manner indicates that the high affinity binding site must be very similar on all the enzyme·PAI-1 complexes, and must be interacting with the same site on LRP.


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Fig. 6.   Cross-competition of proteinase·PAI-1 complexes binding to LRP. Panel A, H-uPA·32P-PAI-1 complex (5 nM) was added to immobilized LRP in the presence of increasing concentrations of either H-uPA·wtPAI-1 (bullet ), L-uPA·wtPAI-1 (black-square), or trypsin·wtPAI-1 (black-triangle) Panel B, L-uPA·32P-PAI-1 (7.5 nM) was added to immobilized LRP, and the binding was competed with increasing concentrations of either H-uPA·wtPAI-1 (bullet ), L-uPA·wtPAI-1 (black-square), or trypsin·wtPAI-1 (black-triangle). Panel C, trypsin·32P-PAI-1 complex (7.5 nM) was added to immobilized LRP in the presence of increasing concentrations of H-uPA·wtPAI-1 (bullet ), L-uPA·wtPAI-1 (black-square), or trypsin·wtPAI-1 (black-triangle). The data presented are representative of at least three experiments. Each plotted value represents the average of duplicate determinations with the range indicated by bars.

The High Affinity Binding Site on Proteinase·PAI-1 Complexes Is Independent of the Enzyme-- The reason for the apparent difference in the high affinity binding site on the H-uPA·PAI-1 compared with L-uPA·PAI-1 is not clear from these data. It may be that the cryptic site on PAI-1 is exposed slightly differently in the H-uPA·PAI-1 complex compared with that on either the L-uPA·PAI-1 or trypsin·PAI-1 complex, creating a somewhat better binding interaction. Alternatively, cooperativity between the two binding sites in H-uPA·PAI-1 might result in an apparent enhanced affinity of the high affinity binding site in this complex. To distinguish between these two possibilities, direct binding of H-uPA·32P-PAI-1 was performed in the presence of either excess unlabeled L-uPA·PAI-1 complex or excess APMSF treated H-uPA. Fig. 7 demonstrates that in the presence of excess L-uPA·PAI-1 complex only the moderate affinity interaction of the H-uPA·32P-PAI-1 was observed (note the inverted triangles in Fig. 7). The calculated Kd from these data is 58 ± 6 nM which is essentially identical to the calculated moderate affinity interaction of 55 ± 14 nM on the H-uPA·PAI-1 complex (Fig. 4.), and the stoichiometry is also approximately one-half that of the H-uPA·32P-PAI-1 alone. In addition, the predicted plot for the interaction of a molecule having only this moderate affinity binding site (see the small dashed line) is identical to the curve obtained for the binding of H-uPA·32P-PAI-1 in the presence of excess L-uPA·PAI-1. This supports the conclusion above (see Fig. 6) that the high affinity binding site on both uPA·PAI-1 complexes must be very similar and must bind to the same site on LRP. These data also suggest that the moderate affinity binding site does not overlap with the high affinity interaction site. Similarly, the binding of H-uPA·32P-PAI-1 in the presence of excess APMSF inactivated H-uPA demonstrates only the high affinity interaction and like the binding in the presence of excess L-uPA·PAI-1, these data can be fit well with a single binding site model (note the upright triangles in Fig. 7). The calculated Kd from these data is 9.8 ± 1.7 nM, which is essentially identical to the Kd of 7.4 ± 1.4 nM obtained for the high affinity binding of L-uPA·32P-PAI-1 to LRP. This indicates, as suggested above, that the high affinity site present on all the proteinase·PAI-1 complexes is the same. Thus, the apparent difference between the high affinity site on the H-uPA·32P-PAI-1 complex compared with the L-uPA·PAI-1 or trypsin·PAI-1 complex must result from an enhanced "apparent" affinity of the high affinity site due to cooperativity between the moderate affinity site present on H-uPA and the high affinity binding site on PAI-1. Consistent with this interpretation, the binding of H-uPA·32P-PAI-1 in the presence of excess H-uPA is much more similar to the binding of L-uPA·32P-PAI-1 (compare the upright triangles to the thin solid line in Fig. 7) than it is to the hypothetical interaction of a molecule having only the high affinity binding site calculated from the two-site model of H-uPA·32P-PAI-1 (compare the upright triangles to the large dashed line in Fig. 7). Finally, these data also demonstrate that the moderate affinity site must be entirely contained within H-uPA, and that the high and moderate affinity sites must bind to different regions of LRP, since the two sites are not able to cross-compete for binding to LRP. Consistent with this interpretation, the stoichiometry of the H-uPA·32P-PAI-1 binding to LRP in the presence of either excess competitor is approximately half of the maximum binding observed in the absence of any competitor. From these data we suggest a model for the interaction of PAI-1 with LRP, where only PAI-1 in complex with a proteinase expresses the high affinity binding site for LRP, and only PAI-1, and not the proteinase, is contributing significant binding energy to this high affinity interaction. Finally, this model suggests that there is an additional moderate affinity site present only on H-uPA and that the presence of this site on the H-uPA·PAI-1 complex enhances the interaction of the existing high affinity site on PAI-1 with LRP.


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Fig. 7.   Direct binding of H-uPA·PAI-1 complex to LRP in the presence of either excess H-uPA or L-uPA·wtPAI-1 complex. Increasing concentrations of H-uPA·32P-PAI-1 complex were added to immobilized LRP, either alone (open circle ) or in the presence of 2 µM of APMSF inactivated H-uPA (triangle ), or 2 µM of L-uPA·wtPAI-1 complex (down-triangle). The data presented are representative of at least three experiments. Each plotted value represents the average of duplicate determinations with the range indicated by bars. The thin solid line (------) shows the fit for the binding of L-uPA·PAI-1 to LRP in Fig. 4 normalized to these data. The dashed lines show the theoretical plots of molecules having only either the moderate affinity binding site of 55 nM (- - - - -) or only the high affinity binding site of 1.6 nM (- - - -) calculated from the fit of H-uPA·PAI-1 to the two-site model.

The High Affinity LRP Binding Site on PAI-1 Overlaps with the Heparin Binding Domain-- Previously, van Zonneveld and co-workers had suggested that basic residues within the heparin binding domain of PAI-1 may be involved in PAI-1's association with LRP (55). Therefore, to see if the PAI-1 heparin binding domain contained the cryptic high affinity LRP binding site, a PAI-1 mutant was constructed with the arginine at position 76 mutated to a glutamic acid. The purified mutant was fully active toward uPA, tPA, and trypsin (data not shown) but no longer competed well for the binding of 125I-trypsin·PAI-1 to LRP (Fig. 8). Analysis of these data indicated that the trypsin·R76E-PAI-1 complex competes poorly for LRP binding, having an estimated Ki greater than 100 nM compared with 11 nM for the trypsin·PAI-1 complex. The R76E mutant in complex with L-uPA was also a poor competitor for the binding of the 125I-L-uPA·PAI-1 complex, yielding a similar Ki (data not shown). These data indicate that mutation of Arg76 right-arrow Glu reduces the affinity of PAI-1·proteinase complexes by approximately 10-fold. Consistent with this, the LRP-dependent endocytosis and degradation of the 125I-trypsin·R76E-PAI-1 by MEF cells was also significantly reduced (Fig. 9, A and B), as was the endocytosis and degradation of 125I-L-uPA·R76E-PAI-1 complexes (data not shown). Together, these data suggest that the cryptic high affinity LRP binding site on PAI-1 may overlap with the heparin binding domain. It should be noted, though, that these results do not unequivocally identify the LRP binding site, since it is possible that the mutation may induce conformational changes elsewhere in PAI-1 that result in reduced LRP binding. However, given that the R76E mutant is fully functional as an inhibitor toward all enzymes examined suggests that any generalized structural changes must be relatively minor. Finally, studies with other serpins have alternatively suggested that LRP binding is mediated through sequences located either near the serpin-reactive center loop (56) or to a region near the amino terminus (50). The location of Arg 76 in PAI-1 is distant form the reactive center loop suggesting that this latter region is not important for the high affinity interaction of PAI-1 with LRP. However, it is near to the region of PAI-1 that is homologous to the proposed LRP binding site in protease nexin-1 (50). Consistent with these data, recent analysis by Knauer et al. (57) of proteinase nexin-1 demonstrated that simultaneous mutation of all seven lysine residues within the heparin binding domain resulted in an approximately 10-fold reduction in cellular degradation of proteinase nexin-1-thrombin complexes. The authors of this study suggested that the results were due to loss of cell surface glycosaminoglycan binding, and not due to a direct effect on LRP binding. However, they did not examine the effects of these mutations on interaction with purified LRP as we have done in Fig. 8, and our results indicate that at least for PAI-1 the effect is due to loss of affinity for LRP.


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Fig. 8.   Competition of 125I-trypsin·PAI-1 complexes binding to LRP. 125I-Trypsin (10 nM) in complex with active PAI-1 was added to immobilized LRP in the presence of increasing concentrations of trypsin·14-1b-PAI-1 complex (bullet ), trypsin·R76E-14-1b-PAI-1 complex (open circle ), or trypsin alone (black-square). Each plotted value represents the average of duplicate determinations with the range indicated by bars. The inset shows an autoradiogram of 125I-trypsin alone (lane 1) in the presence of R76E-14-1b-PAI-1 (lane 2) or active 14-1b-PAI-1 (lane 3).


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Fig. 9.   LRP mediated endocytosis and degradation of 125I-trypsin·PAI-1 complexes by cells. Panel A, endocytosis of 125I-trypsin in complex with either 14-1b-PAI-1 or R76E-14-1b-PAI-1 (10 nM final concentration) was performed in the presence or absence of RAP (1 µM). Data shows RAP inhibitable endocytosis. Panel B, degradation of the same 125I-trypsin complexes as in panel A. Data show RAP-inhibitable degradation. Each plotted value represents the average of duplicate determinations with the range indicated by bars.

PAI-1 Is a Molecular Switch-- Taken together, these data indicate that PAI-1 contains a high affinity binding site for LRP. However, in uncomplexed PAI-1 this binding site is cryptic, and thus only following inhibition of a proteinase will the proteinase·PAI-1 complex be cleared. This conformational control of PAI-1's interaction with LRP is remarkably similar to PAI-1's association with the matrix protein vitronectin. Recently, we demonstrated that PAI-1 binding to vitronectin was also conformationally controlled (14). However, in the case of vitronectin only the active form of PAI-1 binds with high affinity. This suggests that under normal physiological conditions deposition of PAI-1 at sites of tissue injury will result in the association of active PAI-1 with vitronectin in the subcellular matrix. However, as proteinase concentration increases locally, as would be expected during wound healing, PAI-1 present in the matrix will inhibit these active enzymes and in doing so loose its affinity for the matrix while simultaneously gaining affinity for the clearance receptor. This should result in rapid removal of the PAI-1 from the matrix and equally rapid internalization and degradation of the complex by local cells. To see if such a scenario can occur, the metabolic fate of 32P-PAI-1 in cell culture was monitored in the presence or absence of proteinase (Fig. 10). These data indicate that as expected in the absence of proteinase PAI-1 is primarily associated with the extracellular matrix. However, if H-uPA is added to the culture medium, there is a rapid release of PAI-1 from the matrix and a concomitant increase in endocytosis of PAI-1 by cells. This suggests that PAI-1 may act as a molecular switch that either localizes to the extracellular matrix or to the endocytic compartment depending on its conformation, which in turn is regulated by its activity state. That this switch in affinity is most likely to occur at sites of active wound healing is also remarkable given the recent observations that active PAI-1 in the matrix is able to inhibit cellular migration by blocking both integrin and uPAR association with the extracellular matrix (18-20). Finally, the ability of PAI-1 to switch its affinity in response to a variety of proteinases including thrombin and plasminogen activators suggests that PAI-1 acting as a molecular switch may be an important factor controlling the orderly progression of cellular migration during wound healing in a variety of different tissues and that LRP may also be an important factor in this process.


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Fig. 10.   Metabolic fate of PAI-1. Panel A, matrix-associated 32P-PAI-1 (5 nM) either alone or in the presence of RAP (1 µM), H-uPA (200 nM) or H-uPA and RAP (200 nM and 1 µM, respectively). Panel B, endocytosis of 32P-PAI-1 (5 nM) either alone or in the presence of RAP (1 µM), H-uPA (200 nM), or H-uPA and RAP (200 nM and 1 µM, respectively). The data presented are representative of at least two experiments. Each plotted value represents the average of duplicate determinations with the range indicated by bars.

    ACKNOWLEDGEMENTS

We thank N. Ulbrandt and M. Sandkvist for helpful discussions.

    Note added in Proof

Since the submission of this work, Rodenburg et al. (Rodenburg, K. W., Kjoller, L., Petersen, H. H., and Andreasen, P. A. (1998) Biochem. J. 329, 55-63) have also demonstrated that residues within the heparin-binding domain of PAI-1 are important for its interaction with LRP.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL55374 and HL55747 (to D. A. L.) and GM42581 and HL50784 (to D. K. S.), and by The American Heart Association Grant in Aid 96007350 (to D. A. L.).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 American Heart Association, Maryland Affiliate Inc. Research Fellowship MDFW 0195.

To whom correspondence should be addressed: Biochemistry Dept., J. H. Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD, 20855. Tel.: 301-517-0356; Fax: 301-738-0794; E-mail: Lawrencd{at}hlnt.redcross.org.

1 The abbreviations use are: serpin, serine proteinase inhibitor; PAI-1, plasminogen activator inhibitor type 1, LRP, low density lipoprotein receptor-related protein, HMK, heart muscle kinase; uPA, urokinase plasminogen activator; uPAR, uPAR receptor; tPA; tissue-type plasminogen activator; RCL, reactive center loop; TBS, tris-buffered saline; PIPES, 1,4-piperazindiethanesulfonic acid; BSA, bovine serum albumin; APMSF, aminophenylmethylsulfonyl fluoride; PMSF, phenylmethylsulfonyl fluoride; L-uPA, low molecular weight uPA; H-uPA, high molecular weight uPA; RAP, receptor associated protein, MEF, mouse embryo fibroblasts; wt, wild type.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

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