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.
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INTRODUCTION |
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
-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
-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
-sheet A is an essential step in the
serpin inhibitory mechanism and results in a conversion of
-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.
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MATERIALS AND METHODS |
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
[
-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
-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
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 [
-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
|
(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
|
(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)
|
(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 |
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
[
-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
[ -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.
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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 ( ) or unlabeled HMK-PAI-1. ( ). 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 ( ) or unlabeled H-uPA·HMK-PAI-1
complexes ( ). 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.
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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.
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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
( ), latent ( ), and cleaved ( ) 32P-PAI-1, or
H-uPA·32P-PAI-1 complex ( ) or
L-uPA·32P-PAI-1 ( ) 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
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.
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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 ( ), wtPAI-1 ( ), APMSF treated
H-uPA ( ) or wtPAI-1 and APMSF treated H-uPA together ( ).
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 ( ), wtPAI-1 ( ), APMSF-treated L-uPA ( ), or wtPAI-1 and APMSF-treated L-uPA together ( ). 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.
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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 ( ), L-uPA·wtPAI-1 ( ), or trypsin·wtPAI-1
( ) 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 ( ), L-uPA·wtPAI-1 ( ), or trypsin·wtPAI-1 ( ). 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 ( ), L-uPA·wtPAI-1 ( ), or
trypsin·wtPAI-1 ( ). 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.
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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 ( ) or in the presence of 2 µM of APMSF
inactivated H-uPA ( ), or 2 µM of L-uPA·wtPAI-1
complex ( ). 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.
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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
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 ( ),
trypsin·R76E-14-1b-PAI-1 complex ( ), or trypsin alone ( ). 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.
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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.
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