(Received for publication, July 12, 1996, and in revised form, December 3, 1996)
From the Department of Pathology and Laboratory
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
19104 and ¶ Abbott Laboratories,
Abbott Park, Illinois 60064-3500
The mechanism by which single-chain urokinase (scuPA) binds to its receptor (uPAR) is incompletely understood. We report that a fragment comprising the first domain of recombinant soluble uPAR (sDI) as well as a fragment comprising the remaining domains (sDII-DIII) competes with the binding of recombinant full-length soluble uPAR (suPAR) to scuPA with an IC50 = 253 nM and an IC50 = 1569, respectively. sDII-III binds directly to scuPA with Kd = 238 nM. Binding of scuPA to each fragment also induces the expression of plasminogen activator activity. sDI and sDII-DIII (200 nM each) induced activity equal to 66 and 36% of the maximum activity induced by full-length suPAR (5 nM), respectively. Each fragment also stimulates the binding of scuPA to cells lacking endogenous uPAR. Although scuPA binds to sDI and to sDII-DIII through its amino-terminal fragment, the fragments act synergistically to inhibit the binding of suPAR and to stimulate plasminogen activator activity. Furthermore, sDII-DIII retards the velocity and alters the pattern of cleavage of sDI by chymotrypsin. These results suggest that binding of scuPA to more than one epitope in suPAR is required for its optimal activation and association with cell membranes.
Binding of urokinase (uPA)1 to its
receptor may play an important role in inflammation (1, 2) and the
development of tumor metastases (3), among other processes. The
urokinase receptor (uPAR) is a single-chain glycoprotein that is
attached to cell membranes by a glycosylphosphatidylinositol-linked
receptor (3, 4). Binding of single chain urokinase (scuPA) to uPAR stimulates its enzymatic activity (5), provides relative protection from plasmin and plasminogen activator type 1 (6), promotes its
association with cell adhesive proteins (7-9), and retards its
internalization by the 2-macroglobulin
receptor/lipoprotein-related receptor (8, 10).
uPAR is comprised of three domains which share notable sequence similarity (3, 11). The exact mechanism by which scuPA binds to these domains and the mechanism by which scuPA is then activated remains unclear. The growth factor domain of uPA plays an important role in the binding to uPAR (12-14). However, whether the growth factor domain binds to uPAR with the same affinity as uPA remains unsettled (15). It has also been reported that the amino-terminal fragment of scuPA (ATF) can bind to the first domain of uPAR, but with a 1500-fold lower affinity than to the full-length receptor (16). These data were obtained using a preparation of sDI and sDII-DIII that contained sufficient residual suPAR to account for the results leading the investigators to conclude that the capacity of isolated domain I to bind to scuPA is negligible (16, 17). This interpretation leaves unresolved the mechanism by which the three domains contribute to the binding energy of scuPA.
The fact that scuPA binds to full-length suPAR with a higher affinity than to sDI can be the result of at least two mechanisms. The second and third domains may stabilize the optimal structure of DI. In other words, DII-DIII may induce conformational changes in DI making it a better ligand for scuPA. An alternative explanation is that DII-DIII participates in the binding of scuPA directly by offering a distinct, low affinity site. The presence of two lower affinity sites on the same molecule may generate a higher affinity interaction between scuPA and suPAR. Either hypothesis is compatible with the observation that the second and the third domains of uPAR promotes its interaction with scuPA (18).
To study these possibilities in greater detail, we analyzed the binding of scuPA to highly purified fragments of suPAR added alone and together as well as the effect of these fragments on several of its biologic activities. The results indicate that sDII-DIII contributes to the activity of suPAR by providing a second binding site for scuPA.
scuPA and its amino-terminal fragment (ATF; amino
acids 1-135) were prepared and isolated as described (14, 19). scuPA, ATF, and soluble urokinase receptor (see below) were radiolabeled with
125I using IODO-BEADS (Pierce Chemical Co.). The
LMTK cell line was obtained from the American Type Tissue
Collection (Rockville, MD). The plasmin substrate Spectrazyme PL
(H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide diacetate salt) and low molecular weight urokinase were provided by
American Diagnostica (Greenwich, CT). EGR-chloromethyl ketone was
purchased from Calbiochem (La Jolla, CA) and fish skin gelatin from
Sigma.
Recombinant soluble urokinase receptor
(suPAR; amino acids 1-281) was expressed and purified as described
previously (6). Domain 1 (amino acids 1-87) was separated from a
fragment containing domains II and III (amino acids 88-281) by
proteolytic digestion and gel filtration. Briefly, suPAR (1 mg/ml) in
phosphate-buffered saline (PBS) containing 10 mM EDTA was
digested with chymotrypsin (5000:1 mol/mol ratio) for 24 h at
4 °C. The reaction was quenched by adding phenylmethylsulfonyl
fluoride (final concentration 100 µM). Soluble domain 1 (sDI) was separated from soluble domains II+III (sDII-DIII) by reverse
phase high performance liquid chromatography using a Vydac C8
analytical column (Phenomenex, Torrance CA). Typically, 1.5 ml of the
reaction mixture was injected per run and the column was developed
using a linear gradient (0-70% acetonitrile containing 0.1%
trifluoroacetic acid) over 1 h at a flow rate of 1 ml/min. sDI and
sDII-DIII eluted with retention times of approximately 36 and 34 min,
respectively. Peaks from repetitive runs were collected, lyophilized,
and analyzed by SDS-PAGE. Neither sDI nor sDII-DIII contained
full-length suPAR detected by SDS-PAGE. Trace amounts of sDII-DIII were
detected in sDI (Fig. 1). sDII-DIII was free of sDI by
SDS-PAGE and NH2-terminal sequence analysis of the peptide
showed a single, expected sequence starting at amino acid 88. Laser
desorption mass spectral analysis of sDI showed at least 12 peaks
ranging from 11,000 to 18,000 mass units. This heterogeneity was
completely abolished when the protein was deglycosylated using
N-glycanase. Mass spectral analysis of the resultant
deglycosylated sDI yielded a single peak of 9759 mass units, consistent
with its predicted molecular weight calculated from the primary amino
acid sequence (9761 mass units).
Binding of suPAR Fragments to scuPA
Several methods were employed to measure the binding of suPAR and suPAR fragments to uPA. In the first set of experiments, the binding of 125I-suPAR to a stably transfected CHO cell line expressing GPI-anchored scuPA was measured in the presence or absence of increasing concentrations of unlabeled suPAR, sDI or sDII-III, as described (20). In a second set of experiments, binding was measured using a solid phase enzyme-linked immunsorbent assay. Briefly, two-chain urokinase (tcuPA) was catalytically inactivated using Biotin-EGR-chloromethyl ketone. 96-Well microtiter plates were coated overnight at 4 °C with full-length suPAR (500 ng in 100 µl of PBS). The plates were then washed, unreactive sites were blocked with 3% fish skin gelatin at 37 °C, and the plates were washed again. Various concentrations of competitor (sDI, sDII-DIII, or scuPA) were added in a final volume of 100 µl. Biotin-EGR-tcuPA (0.5 nM) and avidin-horseradish peroxidase were then added to all wells simultaneously and the incubation was continued for 3 h at room temperature. The plate was washed and the color development at 490 nm was measured using the appropriate substrate.
Measurement of Plasminogen Activation ActivityIn one set of experiments, scuPA (5 nM) was added either alone or together with various concentrations of suPAR, sDI, or sDII-DIII to a reaction mixture containing plasminogen (5 nM) and the plasmin chromogenic substrate (900 µM) in PBS at 37 °C, and the light absorbance at 405 nm was measured continuously over time. In another set of experiments, the plasmin-insensitive scuPA variant (scuPA Glu158) (5, 21, 22) was used instead of the wild-type protein.
Effect of sDII-DIII on the Proteolysis of sDIThe
susceptibility of suPAR, sDI, and an unrelated protein (heat shock
protein-70) to proteolytic digestion in the presence of sDII-DIII was
tested. Each protein was dialyzed against PBS and adjusted to a final
concentration of 70 µM. Chymotrypsin (1:500 mol/mol ratio
of enzyme:substrate) was added at 22 °C for various periods of time.
The samples were diluted 1:1 in SDS sample buffer containing 2%
2-mercaptoethanol and were either flash frozen overnight at 80 °C
or processed immediately with identical results. Before analysis, the
samples were boiled at 100 °C for 5 min, cooled briefly, and
analyzed by SDS-PAGE using either a 10-18% or a 10-27% gradient.
Cells were grown to confluency in 48-well Falcon Multiwell tissue culture dishes (Becton Dickinson, Lincoln Park, NJ) at a final density of approximately 5 × 104 cells/well. The cells were prechilled to 4 °C for 30 min and then washed 2 × with prechilled binding buffer. A 150-µl aliquot was taken from a mixture containing either 10 nM 125I-labeled scuPA in PBS containing 1% bovine serum albumin or 10 nM labeled scuPA in the presence of various concentrations of sDI or sDII-DIII or 10 nM suPAR. These aliquots were then added to the cells in the presence or absence of 100-fold molar excess unlabeled low molecular weight uPA. The incubation was continued for 2 h at 4 °C, buffer containing the unbound ligand was removed, the cells were washed four times with binding buffer, 0.1 N NaOH was added for 10 min, and the radioactivity in each well was measured. Nonspecific binding was defined as cell-associated radioactivity not inhibited by excess unlabeled ligand or by low molecular weight urokinase (8). Specific binding was defined as the difference between total and nonspecific binding.
Our
initial goal was to study the contribution of the first domain of
soluble urokinase receptor, sDI, and the other two domains, sDII-DIII,
to the binding of scuPA. To do this, we measured the capacity of sDI
and sDII-III individually to compete with the binding of radiolabeled
suPAR to a stably transfected CHO cell line expressing GPI-anchored
scuPA. The data in Fig. 2 show that suPAR binds to these
cells as previously reported (20). The binding of 1 nM
125I-suPAR was inhibited in dose-dependent
manner by unlabeled suPAR. Half-maximal inhibition occurred at a
concentration of unlabeled suPAR of 3.7 nM and binding was
totally inhibited at 17 nM suPAR, indicating that all of
the binding of labeled suPAR to the cell-bound ligand was specific. sDI
inhibited the binding of 125I-suPAR with an
IC50 of 253 nM, and sDII-DIII inhibited with an IC50 of 1569 nM (Fig. 2). Of note, neither sDI
nor sDII-DIII completely inhibited the binding of suPAR at the
concentrations studied. Similar results were obtained in a cell-free
system in which the binding of biotinylated scuPA to immobilized suPAR
was measured by enzyme-linked immunosorbent assay. suPAR inhibited the
binding of labeled scuPA by 85%. Each fragment was also able to
compete with the binding of scuPA to immobilized suPAR with
half-maximal inhibition measured at concentrations of approximately 100 nM for sDI and 1000 nM for sDII-DIII (not
shown).
These results indicate that sDII-III as well as sDI can compete with
suPAR for binding to scuPA. This inhibition could occur because
sDII-DIII binds to scuPA directly, as reported previously for sDI (23),
thereby competing with the binding of suPAR, or the fragment could
interact with suPAR inhibiting the capacity of the full-length receptor
to bind to scuPA. To distinguish between these possibilities, we next
examined the capacity of 125I-sDII-DIII to bind directly to
the uPA-expressing CHO cells in the presence and absence of unlabeled
sDII-DIII, suPAR, or scuPA. sDII-DIII bound specifically to
cell-associated scuPA. Half-maximal binding occurred at a ligand
concentration of 238 nM. Specific binding of
125I-sDII-DIII was totally inhibited by 100-fold
M excess soluble scuPA, by full-length suPAR, and by sDI
(not shown), confirming that binding of sDII/DIII to the CHO cell line
was mediated by cell-associated scuPA. To be certain that the bound
radioactivity did not represent contaminating full-length suPAR or sDI,
an eluate from these cells was prepared and analyzed using SDS-PAGE
(Fig. 3). These results show the labeled protein eluted
from CHO-scuPA migrated exclusively with the mobility expected of
sDII-DIII. Thus, sDII-DIII as well as sDI can bind to scuPA, albeit
with lower avidity than does the full-length receptor.
sDI and sDII-DIII Interact to Promote the Binding of scuPA
The fact that sDII-DIII and sDI contain distinct binding
sites for scuPA does not establish whether or how these sites function within the intact receptor. Therefore, experiments were performed to
determine whether the interaction between the binding epitopes in sDI
and sDII-DIII can generate the higher binding affinity of the
full-length receptor. To examine this possibility, the capacity of
subinhibitory concentrations of sDI and sDII-DIII to compete with the
binding of 125I-suPAR was examined. The results shown in
Fig. 4 indicate that the simultaneous addition of both
fragments exerts a synergistic inhibitory effect on the binding of
suPAR.
This result also suggests that sDI interacts with sDII-DIII. This interaction may approximate the two binding epitopes for scuPA, thereby increasing its net affinity for the ligand without either domain undergoing a conformational change. However, it is also possible that sDI undergoes a conformational change in the presence of sDII-DIII that facilitates its interaction with scuPA or that sDI induces a conformational change in scuPA that facilitates its binding to sDII-DIII.
To test the latter possibility, we examined the effect of sDII-DIII on
the susceptibility of sDI to proteolytic cleavage by chymotrypsin. sDI
was incubated with chymotrypsin in the presence and absence of
sDII-DIII. As shown in Fig. 5, the addition of sDII-DIII
not only inhibited the extent to which sDI was cleaved, but also
changed the pattern of its cleavage. Identical results were obtained
when the effect of sDII-DIII on chymotrypsin-mediated cleavage of
deglycosylated sDI was studied (not shown). These results suggest that
the cleavage site in sDI becomes less accessible to the active site of
the enzyme in the presence of sDII-DIII, but do not exclude a direct
effect of sDII-DIII on chymotrypsin itself.
To explore the mechanism of this effect in more detail, we next asked
whether sDII-DIII altered the susceptibility of suPAR to proteolysis in
a similar manner. The results shown in Fig. 6 indicate
several points. First, sDI is released from suPAR prior to further
proteolytic cleavage. Second, the velocity of cleavage of nascent sDI
released from suPAR is slower than the cleavage of isolated sDI
(compare Figs. 5A and 6A). Third, the pattern of
cleavage of sDI newly released from suPAR is similar to that of
isolated sDI. Fourth, exogenous sDII-DIII further slowed the velocity
and altered the pattern of cleavage of sDI released from suPAR (Figs.
6A and 5A). Finally, sDII/DIII had no effect on
the chymotryptic cleavage of an unrelated protein substrate, HSP-70, in
the presence and absence of sDII-DIII, studied in parallel (not shown).
These latter two results exclude a direct inhibitory effect of
sDII-DIII on the enzyme itself.
We next examined the last possibility, i.e. that sDI induced a conformational change in scuPA that facilitates the binding of sDII-DIII. To do this, we measured the binding of 125I-sDII-DIII to scuPA in the presence of varying concentrations of sDI. Although sDI at high concentrations inhibited the binding of labeled sDII-DIII to CHO-scuPA, binding of sDII-DIII to scuPA was not augmented at any concentration of sDI tested, including those below which its inhibitory effect was evident (not shown). This outcome argues against the possibility that sDI induces a conformational change in scuPA that facilitates its binding to sDII-III.
Expression of Plasminogen Activator ActivityWe previously
reported that suPAR stimulates the plasminogen activator activity of
scuPA (5). Therefore, we asked whether the isolated domains of suPAR
have a similar effect. As shown in Fig. 7, both sDI and
sDII-DIII stimulated the plasminogen activator activity of scuPA. sDI
was more potent. At optimal concentrations, sDI and sDII-DIII (200 nM each) stimulated scuPA activity by 66 and 36% of that
attained with 5 nM suPAR, respectively. A similar induction
of activity was seen using the plasmin-insensitive scuPA variant
(scuPA-Glu158). The induction of scuPA activity by sDI and
sDII-DIII were both completely abolished by 100-fold M
excess ATF, suggesting that each fragment exerts its effect through a
similar site in scuPA. We then asked whether the two fragments of scuPA
when combined would exert a synergistic effect on the activation of
scuPA. Plasminogen and scuPA were incubated in the presence both
fragments, each added at substimulatory concentrations. The data shown
in Fig. 8 indicate that the combination of both
fragments exert a synergistic effect on the plasminogen activator
activity of scuPA, an effect similar to their ability to interact and
compete with the binding of suPAR shown earlier (Fig. 2).
Binding of scuPA to Cells
We have also previously reported
that the suPAR promotes the binding of scuPA to certain cell-associated
integrin ligands expressed by LMTK cells
which lack uPAR (8). Increased binding occurred as a consequence of induced conformational changes in both the scuPA and
uPAR components of the complex. Therefore, we examined the capacity of
each fragment of suPAR to induce the binding of scuPA to
LMTK
cells. The data shown in Fig. 9
indicate that sDI as well as sDII-DIII stimulated the binding of scuPA
to these cells. The maximal effects of sDI and sDII-DIII were almost
comparable to that of full-length suPAR.
The results of this study indicate that soluble human urokinase
receptor is composed of at least two active units. Both soluble domain
I and a fragment containing domains II and III have the capacity to
bind scuPA, to stimulate its enzymatic activity, and to promote its
association with cell surfaces. These conclusions are based on the
capacity of sDI and sDII-DIII to compete with the binding of
full-length suPAR to scuPA, the capacity of each fragment to stimulate
the plasminogen activator activity of scuPA and to promote the binding
of scuPA to LMTK cells which lack endogenous uPAR, and by
direct measurement of the binding of each fragment to scuPA. The
binding and activation of scuPA by sDI and sDII-DIII, like suPAR,
requires ATF since this fragment blocked both activities.
The possibility that scuPA bound to sDII-DIII has been noted by others but was attributed to contaminating full-length suPAR (16). The reagents used in the present study were essentially free of suPAR by several independent criteria. No full-length receptor was evident on gels overloaded with sDI or sDII-DIII nor by amino acid sequencing of the DII-DIII fragment. No suPAR was detected in the 125I-sDII-DIII eluted from cell-bound scuPA. Furthermore, the fact that neither sDI nor sDII-DIII alone or together activated scuPA to the same extent as suPAR, even when present at saturating concentrations, excludes a role for contaminating full-length receptor as a suitable explanation for the observed results.
The observation that sDI and sDII-DIII both bind to scuPA is consistent either with the possibility that each recognizes a different epitope within a single binding site or that each recognizes a distinct binding site. The discrepancy between the affinity with which radiolabeled sDII-DIII binds and activates scuPA and its capacity to compete with full-length suPAR suggests the existence of two binding epitopes in scuPA. One of these sites may be occupied preferentially by DI within the native receptor, the other by DII-DIII, but sDII-DIII can occupy both sites when present alone at higher concentrations. If scuPA binds to suPAR through two epitopes, it is expected that each would contribute independently to the final interaction between full-length receptor and scuPA and that the affinity of the final interaction would best be described by the multiple of the individual binding affinities (24). Another plausible explanation is that two sites in suPAR bind to the same epitope in scuPA and cooperate to maintain a high local concentration of ligand. Thus, the results of the present study suggest that the net affinity of scuPA for full-length receptor can be explained by the presence of two or more lower affinity binding sites in the native receptor.
The results of these experiments do not exclude the possibility that sDII-DIII facilitates the binding of scuPA by inducing a conformational change in sDI or by stabilizing a conformation in which its binding site in sDI is more accessible to the ligand. This notion is consistent with the synergistic effect of sDI and sDII-DIII on scuPA activity, with the synergistic inhibition of suPAR binding to cell-associated scuPA when the two fragments are added together, and with the observation that sDII-DIII modulates the velocity and the pattern of the enzymatic cleavage of sDI and suPAR by chymotrypsin. The observation that sDII-DIII interferes with the cleavage of sDI more completely than it does in the context of full-length suPAR suggests that isolated sDI has a somewhat higher affinity for the remaining fragment than does nascent sDI released from suPAR, a difference that is overcome at a somewhat higher concentration of sDII-DIII. However, additional experiments will be required to determine whether such conformational changes actually occur with the first domain of uPAR or whether such changes modulate the binding of scuPA or other proteins that bind to the uPA receptor (7-9, 25).
The finding that sDI stimulates the binding of scuPA to
LMTK cells supports our previous suggestion that the
capacity of suPAR to increase the binding of scuPA to cells is due in
part to changes in scuPA, specifically within the low molecular weight
portion of the molecule (8). This contention is consistent with the observations of others that tcuPA has a biphasic effect on the binding
of suPAR, while ATF has only a stimulatory effect and by the
observation that the addition of scuPA to suPAR increases the total
amount of ligand bound without an increase in its affinity (9). A
contribution of the second and third domains of the receptor to the
cell binding induced by full-length receptor cannot be excluded by our
data (7), since the maximal effect of sDI required 5-fold more protein
than did full-length suPAR. Nor do our results exclude the possible
contribution of conformational changes in DI induced by scuPA (9).
However, the reported ability of a monoclonal anti-uPAR antibody
presumed to recognize DI exclusively to block scuPA binding to cells
(9) could be due to cross-reactivity with
sDII-DIII.2 Nevertheless, the observation
that complexes composed of scuPA and sDI, sDII-DIII, or full-length
suPAR bind to LMTK
cells to the same extent provides
additional support for the involvement of urokinase in this
interaction. However, the exact mechanism by which the binding of scuPA
to suPAR changes the capacity of the resultant complex to bind to other
proteins requires further study.
The presence of multiple binding sites in suPAR has recently been reported (Behrendt, N., Ronne, E., and Dano, K. (1996) J. Biol. Chem. 271, 22888-22894).