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INTRODUCTION |
The receptor for urokinase-type plasminogen activator
(uPA),1 uPAR/CD87, is a
glycoprotein (Mr 35,000-65,000) composed of 283 amino acid residues. It is anchored to the plasma membrane by a
glycosyl phosphatidylinositol (GPI) linkage (reviewed in Refs. 1 and
2). uPAR is the cellular receptor for urokinase, a serine
protease that is constitutively or inducibly secreted by most
uPAR-expressing cells. Receptor-bound uPA can convert plasminogen to
plasmin, which mediates pericellular proteolysis of extracellular matrix proteins in the path of cellular invasion. uPAR is expressed by
activated leukocytes, endothelial cells, fibroblasts, and different types of cancer cells (reviewed in Refs. 2 and 3 and references therein). Expression of uPAR has been shown to correlate with the
prognosis of many human cancers (2). In murine tumor models, expression
or administration of uPAR antagonists has a marked inhibitory effect on
the metastatic ability of cancer cells (4) and on the growth of the
primary tumor (5), and the down-regulation of uPAR leads to dormancy of
carcinoma cells in vivo (6, 7). Thus, uPAR expression has
been implicated in cancer progression. In addition, it appears that
uPAR is up-regulated on cells that are in motion.
Migratory/chemotaxis-inducing stimuli (e.g. vascular endothelial growth factor, fibroblast growth factor, platelet-derived growth factor, and interleukins) up-regulate uPAR in endothelial cells,
smooth muscle cells, and leukocytes in vitro, whereas
unstimulated cells do not have detectable expression of uPAR (reviewed
in Refs. 2 and 3 and references therein). Thus, uPAR may play a role in
leukocyte recruitment, angiogenesis, and tumor metastasis.
uPAR has been reported to associate with many signaling molecules and
to mediate signal transduction (8-10). In recent reports the binding
of uPA to uPAR in tumor or endothelial cells has been shown to activate
the mitogen-activated protein kinases, extracellular regulated kinase 1 and 2 (11-13). However, a major question is how uPAR mediates cellular
signaling, because the molecule has no transmembrane structure. The
existence of one or more hypothetical "transmembrane adapter
molecules" that connects uPAR and signaling molecules inside cells
has been proposed (14).
It has been shown that the
1,
2, and
3 integrin receptor
families interact with uPAR using immunocoprecipitation,
immunocolocalization, and resonance energy transfer approaches
(15-17). The uPAR-integrin interaction may be significant, because
many integrin receptors activate intracellular signals coupled to the
pathways used by both receptor and nonreceptor tyrosine kinases
(18-20). Integrin- and receptor tyrosine kinase-mediated signals may
complement each other to fully activate cell survival and proliferation
pathways (21, 22). It has been proposed that uPAR forms
cis-interactions with integrins on the same cell surface as an
integrin-associated protein (reviewed in Ref. 1). However, it has not
been established that this is the dominant mode of interaction
responsible for signal transduction events.
In the present study, we designed experiments to examine the
uPAR-integrin interaction in detail using isolated domains derived from
recombinant soluble uPAR (suPAR) and cells expressing recombinant
1
or
3 integrins. These studies establish that uPAR binds to integrins
in a manner that is very similar to that of known integrin ligands
(e.g. vascular cellular adhesion molecule-1 (VCAM-1)). Additionally, we have found that uPAR interacts with integrins on
apposing cells (trans-interaction). These unexpected findings may help
to clarify the complex role of uPAR in signal transduction, inflammation, and cancer.
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EXPERIMENTAL PROCEDURES |
Materials
TS2/16, an activating anti-
1 mAb (23), was obtained from
American Type Culture Collection (Manassas, VA). AIIB2, a
function-blocking anti-
1 mAb (24), was obtained from Developmental
Studies Hybridoma Bank (Iowa City). IA. P1B5 (anti-
3) (25)
was a gift from E. Wayner and W. G. Carter (University of
Washington, Seattle, WA). SG73 (anti-
4) (26) and KH72
(anti-
5) were provided by K. Miyake (Saga Medical School, Saga,
Japan). 135-13C (anti-
6) (27) was obtained from S. J. Kennel
(Oak Ridge National Laboratory, Oak Ridge, TN), and Y9A2
(anti-
9) (28) was obtained from D. Sheppard (University of
California, San Francisco, CA). 7E3 (anti-
3) (29) was provided by
B. S. Coller (Mount Sinai Hospital, New York, NY). 8C8
(anti-
2) and 15 (anti-
3) were provided by M. H. Ginsberg (The Scripps Research Institute). Anti-uPAR monoclonal antibody (3B10)
(30) was provided by R. F. Todd III (University of Michigan Medical Center, Ann Arbor, MI).
Recombinant VCAM-1-mouse Ck chain fusion protein was provided by
Novartis (Basel, Switzerland). Recombinant domain 2 and domain 3 forms
of soluble uPAR generated in Chinese hamster ovary (CHO) cells were
prepared as described previously (31). GRGDS and GRGES peptides
were purchased from Advanced ChemTech (Louisville, KY).
-Bungarotoxin and rabbit IgG were obtained from Sigma. Human fibrinogen was obtained from Enzyme Research Laboratories (South Bend, IN).
CHO cells and human erythroleukemia K562 cells were obtained from the
American Type Culture Collection. Integrin
5
1-deficinet B2
variant CHO cells (32) were provided by R.L. Juliano (University of
North Carolina, Chapel Hill, NC), and Jurkat human T-cell
leukemic cells were provided by M. H. Ginsberg (The Scripps
Research Institute). CHO cells expressing human
9 (designated
9-CHO) (33) were provided by D. Sheppard (University of California,
San Francisco, CA). CHO cells expressing other human integrin
and/or
subunits (wild type and mutant) have been described (34). B2
cells expressing human
2,
3, and
4 (designated
2-B2,
3-B2, and
4-B2 cells, respectively) were prepared as described
for CHO cells expressing these integrins (34). These cells express
human
/hamster
1 or hamster
v/human
3 hybrid. K562 cells
expressing recombinant human
4 have been described (35). K562 cells
expressing recombinant human
9 (
9-K562) have been described (36).
K562 cells expressing recombinant human
v
3 (
v
3-K562) (37)
are a gift from E. J. Brown (Washington University, St Louis, MO).
CHO cells expressing the three domain forms of human uPAR (designated
HuPAR-CHO) were prepared by transfecting full-length human uPAR
cDNA (provided by L. A. Miles, The Scripps Research Institute)
into a pCDNA3 vector (Invitrogen) together with a plasmid containing a neomycin-resistant gene. Cells were selected with G-418
(0.7 mg/ml medium). Approximately 50% of cells stably expressed uPAR
after selection with mAb 3B10. CHO cells stably expressing uPAR were
sorted by FACStar (Becton-Dickinson) to obtain cells homogeneously
expressing uPAR at a high level.
Methods
Production of suPAR in Drosophila S2 Cells--
cDNAs
encoding soluble wild-type uPAR (amino acids 1-277) and soluble
domains 2 and 3 (amino acids 88-277) were generated by polymerase
chain reaction using pTracer-full-length uPAR as a template. The
fragments were digested with BglII and Xho and sub-cloned into the expression vector (pMT/BiP/V5, Invitrogen). Soluble
suPAR domains were expressed in Drosophila Schneider S2 cells (DES system, Invitrogen) as described by the manufacturer.
Purification of suPAR Expressed in S2 Cells--
Small scale
preparations of suPAR and variants were purified from the medium
using a polyclonal anti-uPAR antibody affinity column. For large scale
preparations, S2 cell culture supernatants were filtered, and the
medium were loaded onto a 40-g hydroxyapatite column
equilibrated with 10 mM K2HPO4, pH
7.0. The column was then eluted with a gradient of 10-200
mM K2HPO4, pH 7.0, and the suPAR-containing fractions were identified by Western blotting. Fractions containing monomeric suPAR eluted between 50 and 120 mM K2HPO4. Fractions containing
monomeric suPAR were pooled, concentrated, and further purified using
C8 reverse-phase HPLC. Crude suPAR (20 mg of total protein) was loaded
in a volume of 2 ml onto a semi-preparative (10 × 250 cm) C8
column and eluted at a flow rate of 4 ml/min with a linear gradient of
0-70% solvent B, where solvent A was 100% H2O,
0.1% trifluoroacetic acid, and solvent B was 100% acetonitrile, 0.1%
trifluoroacetic acid. suPAR eluted as a single broad peak under
these conditions with a retention time of ~27 min. SDS-polyacrylamide
gel electrophoresis analysis of suPAR purified in this manner
demonstrated a single major peak at 35 kDa under nonreducing
conditions. Also observed was a slight laddering effect with three to
four minor higher molecular weight species. These were determined to be
SDS-stable aggregates of suPAR, which disappeared when
SDS-polyacrylamide gel electrophoresis was performed under reducing
conditions. Matrix-assisted laser desorption ionization-time of
flight mass spectrometry revealed a single broad peak with an average
mass of 34,797 Da. The predicted mass based on the amino acid
sequence is 30,672 Da, indicating the presence of about 4 kDa in
glycosylation. Expression levels of suPAR were typically 30 mg/liter
determined by enzyme-linked immunosorbent assay. Purification yielded
about 10-12 mg of pure suPAR protein per liter of culture supernatant.
Enzymatic Digestion of Wild-type suPAR--
suPAR was digested
with chymotrypsin to generate the soluble D1 and D2D3 fragments.
Chymotrypsin was added to suPAR (1 mg/ml in phosphate-buffered saline)
at a final molar ratio of suPAR:chymotrypsin of 1000:1, and the digest
was allowed to proceed for 2 h at room temperature. The digest was
quenched by addition of Pefablock (100 µM final
concentration). The D1 and D2D3 fragments were separated using C8
reverse phase-HPLC. The D1 and D2D3 fragments eluted with a
retention time of ~27 and 25 min, respectively. The purified D1
fragment was sequenced, and a single N terminus was observed beginning with RS (representing the two extra amino acids present in
this construct) followed by LR, amino acids 1 and 2 in the mature suPAR
sequence. Sequencing of the D2D3 fragment revealed a single N
terminus as well, beginning with amino acids SRS, corresponding to
amino acids 88-90 in the mature suPAR sequence. Matrix-assisted laser
desorption ionization-time of flight mass spectral analysis revealed a single peak for the D1 fragment (mass = 11,041 Da) and
several peaks for the D2D3 fragment (mass = 23,846; 23,691; 22,936; 22,800). These were presumed to be glycosylation isoforms, although additional digestion of the D2D3 fragment from the C terminus
could not be excluded. However, no consensus chymotryptic cleavage site
that could result in the observed mass differences is present at the C
terminus of the D2D3 fragment.
Affinity-purified Anti-suPAR--
A polyclonal antibody against
suPAR was prepared as described (38). Briefly, recombinant suPAR was
expressed in SP2/0 cells and purified using a single-chain
uPA-Sepharose column. Serum was collected from rabbits immunized with
purified suPAR, and the IgG was obtained by a 50% ammonium sulfate
precipitation. This material was dialyzed (10,000×) against
phosphate-buffered saline and further purified using a suPAR-Sepharose
column to generate affinity-purified anti-suPAR IgG.
Adhesion Assays--
Adhesion assays were performed as described
previously (34). Briefly, wells in 96-well Immulon-2 microtiter plates
(Dynatech Laboratories, Chantilly, VA) were coated with 100 µl of
phosphate-buffered saline (10 mM phosphate buffer, 0.15 M NaCl, pH 7.4) containing substrates at a concentration of
0.2-5 µg/ml and were incubated overnight at 4 °C. Remaining
protein-binding sites were blocked by incubating with 0.2% bovine
serum albumin (Calbiochem) for 1 h at room temperature. Cells
(105 cells/well) in 100 µl of Hepes-Tyrode buffer (10 mM HEPES, 150 mM NaCl, 12 mM
NaHCO3, 0.4 mM NaH2PO4,
2.5 mM KCl, 0.1% glucose, 0.02% bovine serum albumin)
supplemented with 2 mM MgCl2 were added to the
wells and incubated at 37 °C for 1 h, unless stated otherwise.
After nonbound cells were removed by rinsing the wells with the same
buffer, bound cells were quantified by measuring endogenous phosphatase
activity (39). Antibodies were used at 250× dilution of ascites
(TS2/16, 8C8, P1B5, SG73, KH72, and 135-13C), at 4 µg/ml (AIIB2,
7E3, and Y9A2), and at 3.4 µg/ml (anti-uPAR and rabbit IgG). Data are
shown as means ± S.D. of three independent experiments.
Cell-Cell Binding Assay--
Integrin-transfected,
mock-transfected (vector only), or parental K562 cells were labeled
with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl
ester (Molecular Probes, Eugene, OR) according to the manufacturer's
instructions. The labeled cells (105 cells/well)
were added to the monolayer of parent, mock-transfected, or HuPAR-CHO
cells and incubated for 30 min at 37 °C. After the wells were rinsed
with medium to remove unbound cells, bound cells were quantified by
assaying fluorescence (excitation 485 nm, emission 530 nm) using an
FL500 microplate fluorescence reader (Bio Tek Instruments, Winooski,
VT). Antibodies were used at the same concentrations as described
above. Data are shown as means ± S.D. of three independent experiments.
Other Methods--
Site-directed mutagenesis (40), flow
cytometric analysis (41), and transfection (41) were performed as
described in the cited references.
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RESULTS |
Binding of Recombinant suPAR to
1 and
3 Integrins--
To
study whether and how integrins are involved in uPAR-mediated signal
transduction, we first asked whether recombinant soluble uPAR fragments
interact with
1 and
3 integrins. It has been proposed that the
activity of suPAR requires chymotrypsin cleavage between the N-terminal
domains 1 and 2 (14). We used several different suPAR fragments that
were expressed in Drosophila S2 cells (domain 1, domains 2 and 3, or full-length suPAR containing domains 1, 2, and 3, designated
D1, D2D3, D1D2D3, respectively) and domains 2 and 3 expressed in CHO
cells. Unexpectedly, these suPAR fragments supported adhesion of both
4- and
3-CHO cells expressing recombinant human
4/hamster
1
(
4
1) and hamster
v/human
3 hybrid (
v
3), respectively,
but not CHO cells (Fig. 1A).
Parental CHO cells express endogenous
5
1,
v
1, and
v
5 but not
4
1 or
v
3 integrins (42). It appears that suPAR
expressed in Drosophila and CHO cells supports adhesion of
4- and
3-CHO cells at comparable levels. We used suPAR D2D3
expressed in CHO cells (43) for further characterization of
suPAR-integrin interaction throughout this study. The rationale of
using D2D3 is that removal of D1 leaves uPAR D2D3 that is capable of
signal transduction without uPA (14), and thus suPAR D2D3-integrin
interaction is likely to be biologically relevant.

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Fig. 1.
Integrin-mediated cell adhesion to
recombinant soluble human uPAR. A, 4-CHO cells
(closed columns), 3-CHO cells (open columns),
and CHO cells (shaded columns) were tested for their ability
to adhere to immobilized recombinant suPAR (at the coating
concentration of 5 µg/ml). suPAR domain 1 (D1), domains 2 and 3 (D2D3), and domains 1, 2, and 3 (D1D2D3) were expressed in
Drosophila S2 cells or in CHO cells. B and
C, CHO cells expressing different human integrins or CHO B2
variant cells lacking endogenous 5 1 were tested for their ability
to adhere to immobilized recombinant suPAR D2D3 synthesized in CHO
cells (up to 5 µg/ml). D, the effect of function-blocking
anti-integrin antibodies on adhesion to suPAR D2D3 was studied. The
concentration of suPAR D2D3 used for coating was 5 µg/ml. Bovine
serum albumin, closed columns; suPAR D2D3, open
columns; and suPAR D2D3 plus function-blocking antibodies,
shaded columns. The antibodies used were SG73 (anti- 4)
for 4-CHO cells, KH72 (anti- 5) for CHO cells, 135-13C
(anti- 6) for 6-CHO cells, Y9A2 (anti- 9) for 9-CHO cells,
and 7E3 (anti- 3) for 3-CHO cells. E, the effect of
affinity-purified anti-uPAR antibody on v 3-suPAR D2D3 interaction
was studied. Polyclonal anti-uPAR or control rabbit IgG was included in
the assay medium at 3.4 µg/ml. SuPAR D2D3 was used at a coating
concentration of 5 µg/ml. F, the capability of
-bungarotoxin, a protein structurally similar to uPAR, to support
v 3-mediated cell adhesion was studied as a function of substrate
concentration. SuPAR D2D3 and -bungarotoxin were coated up to 5 µg/ml.
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We next studied the specificity of suPAR D2D3 binding to
1 and
3
integrins. We used CHO cells expressing different human
subunits
(
2,
3,
6, and
9, designated
2-,
3-,
6-, and
9-CHO cells, respectively) in addition to
4- and
3-CHO cells. These transfectants express human
/hamster
1 hybrids (
2
1,
3
1,
6
1, or
9
1, respectively). We found that
3-,
4-,
9-, and
6-CHO cells adhered to uPAR in a
dose-dependent manner (Fig. 1, B and
C). However, other transfectants (
2- and
3-CHO cells), parental CHO cells, CHO B2 variant cells lacking endogenous
5
1 (32) (Fig. 1, B and C), and mock-transfected CHO
cells (data not shown) adhered only weakly under the conditions used.
The adhesion of
4-,
6-,
9-, and
3-CHO cells to uPAR was
blocked by specific function-blocking antibodies (SG73 to
4,
135-13C to
6, Y9A2 to
9, and 7E3 to
3) (Fig. 1D).
These results suggest that suPAR D2D3 specifically binds to several
integrins (
4
1,
6
1,
9
1, and
v
3).
We further tested the specificity of interaction using antibodies
against uPAR. We found that antibodies against uPAR effectively blocked
suPAR binding to
v
3 (Fig. 1E). We then tested whether
-bungarotoxin, which has a "three-finger protein" structure
similar to the uPAR structure (44), "nonspecifically" binds to
v
3. The toxin showed only weak, if any, affinity to
v
3
(Fig. 1F). These results suggest that suPAR binding to
integrins is specific to the uPAR sequence and structure.
Effect of Cations and
1 Integrin Activation and Inactivation on
uPAR Binding--
Ligand binding to integrins is tightly regulated by
activation/inactivation of integrins through inside-out signal
transduction (45).
3-CHO cells significantly adhere to suPAR D2D3 in
the presence of Mg2+ but not in the presence of
Ca2+ (Fig. 2A). CHO cells do not adhere to suPAR
in either condition. These results suggest that
v
3 requires
Mg2+ for binding to suPAR D2D3 under the conditions
employed. Although
2-CHO,
3-CHO,
and CHO cells do not strongly adhere to suPAR D2D3 under the assay
conditions used, it is still possible that binding of
2
1,
3
1, and
5
1 may require more activation. Therefore, we
studied whether Mn2+ (0.1 mM), which
universally activates integrins, facilitates uPAR binding to these
integrins (Fig. 2A). We found that
5
1-deficient B2
variant CHO cells did not significantly adhere to suPAR D2D3 in the
presence of Mn2+, but parental CHO cells did. We found that
B2 cells expressing human
3 or
4 (designated
3- or
4-B2
cells, respectively) also adhered to suPAR D2D3 in the presence of
Mn2+.
2-B2 cells did not adhere to suPAR D2D3 under any
conditions tested. Ca2+ did not significantly support
adhesion of these cells. These results suggest that
3
1,
4
1,
and
5
1 but not
2
1 bind to suPAR D2D3 in an
activation-dependent manner.

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Fig. 2.
Effects of cations and anti-integrin
activating or function-blocking antibodies on suPAR binding.
A, the effect of cations on adhesion to suPAR D2D3 was
studied. Ca2+ (2 mM), Mg2+ (2 mM), or Mn2+ (0.1 mM) was added to
Hepes-Tyrode buffer. The concentration of suPAR D2D3 used for coating
was 5 µg/ml. Antibodies used were KH72 (anti- 5) for CHO, 8C8
(anti- 2) for 2-B2, P1B5 (anti- 3) for 3-B2, and SG73
(anti- 4) for 4-B2. B, Jurkat T-cell leukemic cells
were tested for adhesion to suPAR D2D3. The concentration of suPAR D2D3
used for coating was 2 µg/ml. Antibodies used were TS2/16 (anti- 1,
activating), AIIB2 (anti- 1, function-blocking), SG73 (anti- 4),
and KH72 (anti- 5).
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We then asked whether the activation status of
1 integrins that is
required for uPAR binding to natural integrins is similar to the
requirements for the recombinant integrins that we studied. To answer
this question, we studied uPAR binding to nonrecombinant integrins on Jurkat human T-cell leukemia cells (
4
1+,
5
1+). We can stimulate or suppress
1 integrin-ligand interaction from outside cells using anti-
1 antibodies (e.g. TS2/16,
activating; AIIB2, inhibiting) (reviewed in Ref. 46). We found that
adhesion of Jurkat cells to suPAR D2D3 is stimulated by TS2/16 and
blocked completely by AIIB2 and SG73 (anti-
4 mAb) and partially by
KH72 (anti-
5 mAb) (Fig. 2B). These results suggest that
nonrecombinant
4
1 and
5
1 in Jurkat cells may interact with
suPAR D2D3 in an activation-dependent manner.
Does uPAR Share Common Binding Sites in Integrins with Known
Integrin Ligands?--
We then asked whether uPAR competes with
VCAM-1, a known
4
1 ligand, for binding to
4
1. To address
this question, we examined the adhesion of
4-B2 cells to suPAR D2D3
in the presence of soluble VCAM-1. We used B2 cells to completely
eliminate the contribution of
5
1 in uPAR binding. We found that
adhesion of
4-B2 cells to suPAR D2D3 was blocked by VCAM-1 in a
dose-dependent manner (Fig.
3A) but not by irrelevant
ligand fibrinogen. These results suggest that the inhibitory effect of
VCAM-1 is specific to
4
1-suPAR D2D3 interaction, and thus
suPAR and VCAM-1 compete for binding to
4
1. We also studied the
effect of the GRGDS peptide, a widely distributed integrin-binding
motif, on uPAR-
v
3 interaction (Fig. 3B). We found that
GRGDS peptide completely blocked adhesion of
3-CHO cells to suPAR
D2D3, but control GRGES peptide did not, suggesting that uPAR and the
ligand-derived RGD motif compete for binding to
v
3. Thus it is
highly likely that the uPAR-binding sites in these integrins may
overlap with those of known integrin ligands.

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Fig. 3.
Competition between uPAR and VCAM-1 for
binding to 4 1
(A) or between uPAR and RGD peptide for binding
to v 3
(B). A, the effect of soluble VCAM-1
on 4 1-suPAR D2D3 interaction was studied. The concentration of
suPAR D2D3 used for coating was 5 µg/ml. The 5 1-deficient CHO
B2 variant cells expressing human 4/hamster 1 (a receptor for
VCAM-1) ( 4-B2) were used instead of 4-CHO cells. Fibrinogen, an
irrelevant ligand, was used as a negative control. VCAM-1 and
fibrinogen were added at the final concentrations of 1, 5, or 10 µg/ml in the incubation medium. The numbers in parentheses
are concentrations of proteins (µg/ml). Fg,
fibrinogen. B, the effect of GRGDS or GRGES peptide on
v 3-suPAR interaction was studied with 3-CHO cells. The
concentration of suPAR D2D3 used for coating was 5 µg/ml. GRGDS or
GRGES peptide was added at 10 or 100 µM final
concentration in the incubation medium. The numbers in
parentheses are concentrations of proteins
(µM).
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We next studied whether uPAR binding is inhibited by mutations in these
integrins that block binding of known ligands. We have previously
reported that the mutation to Ala of several amino acid residues,
Tyr-187, Trp-188, and Gly-190 in
4 (designated Y187A, W188A, and
G190A mutations, respectively), blocks VCAM-1 and fibronectin
connecting segment-1 (CS-1) peptide binding to
4
1 (47). These
residues are located within the putative ligand-binding site in
4,
and the corresponding residues in
IIb or
5 have also been
reported to be critical for ligand binding to
IIb
3 or
5
1
(47, 48). We studied whether these
4 mutations block adhesion of
4
1 to suPAR D2D3. We found that these mutations blocked
uPAR-
4
1 interaction, but several other mutations did not (Fig.
4). The overall effect of these mutations
on uPAR binding to
4
1 is similar to their effect on VCAM-1 or
CS-1 binding to
4
1. These results suggest that the uPAR-binding
site in
4 overlaps with those for VCAM-1 and CS-1.

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Fig. 4.
Critical residues in
4 for uPAR binding. CHO cells expressing
different 4 mutants were tested for their ability to adhere to suPAR
D2D3 (closed columns) and VCAM-1 (open columns).
Mutation to Ala at positions 187 (Tyr), 188 (Trp), and 190 (Gly) within
the putative ligand-binding sites of 4 blocks 4 1-VCAM-1
binding; the data on VCAM-1 binding are from a previous publication
(47) and are shown for comparison. The concentration of substrates used
for coating was 3 µg/ml for suPAR D2D3 and 0.25 µg/ml for VCAM-1-Ck
fusion protein. Goat anti-mouse Ck antibody was used to facilitate
VCAM-1-Ck coating (47). wt, wild type.
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The Asp-130 residue of
1 (42, 49) and the corresponding Asp residues
in
2 (50),
3 (Asp-119) (51), and
6 (52) have been reported to
be critical for ligand binding. We tested whether mutation of Asp-119
to Ala in
3 affected suPAR D2D3 binding to
v
3. The wild type
or Asp-119 to Ala (D119A) dominant-negative mutant of human
3 was
transiently expressed in CHO cells, and the ability of cells to adhere
to suPAR D2D3 was determined. Wild-type or mutant
3 is expressed as
hamster
v/human
3 hybrid. We found that cells transiently
expressing the
v
3(D119A) mutant did not adhere to suPAR D2D3,
although cells expressing wild-type
v
3 did (Fig.
5A). The levels of adhesion
(~22%) of wild-type
3 are lower than in Fig. 1 but are
substantial, considering that only 35% of added cells express human
3. Note that the nonfunctional
3(D119A) mutant was expressed on
the surface of cells at a comparable level.

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Fig. 5.
Conserved Asp residues in the
subunits are critical for uPAR binding.
A, the wild type or Asp-119 to Ala (D119A) dominant-negative
mutant of human 3 was transiently expressed in CHO cells. Expression
vector (pBJ-1) was transiently transfected in CHO cells as control.
Transfected wild-type or mutant 3 is expressed as hamster v/human
3 hybrid. Forty-eight h after transfection, the ability of cells to
adhere to suPAR D2D3 was determined. The level of 3 expression is
35.8% in cells expressing wild-type 3, 43.8% in cells expressing
mutant 3, and 8.8% (background) in mock-transfected cells with
anti-human 3 antibody (mAb 15). The concentration of suPAR D2D3 used
for coating was 5 µg/ml. B, the wild type or Asp-130 to
Ala (D130A) dominant-negative mutant of human 1 was expressed in
clonal CHO cells together with human 4. The resulting 4 1- and
4 1(D130A)-CHO cells were tested for their capacity to adhere to
suPAR D2D3. The expression levels of human 4 and 1 are
comparable. 4 1(D130A)-CHO cells show lower adhesion to suPAR D2D3
than do 4 1- or 4-CHO cells (dominant-negative effect). It
should be noted that the remaining binding in 4 1(D130A)-CHO cells
to suPAR D2D3 is due to endogenous intact hamster 1.
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We also studied the effect of the corresponding Asp-130 to Ala (D130A)
mutation in
1 on suPAR D2D3 binding to
4
1. For this purpose we
transfected wild-type human
1 or
1(D130A) mutant into cells
stably expressing human
4 (49). Cells stably expressing both human
4 and
1 or
1(D130A) were cloned to obtain cells expressing
1 or
1(D130A) at high levels (49) (designated
4
1- and
4
1(D130A)-CHO cells, respectively). The expression levels of
wild-type and mutant
1 in
4
1 and
4
1(D130A) are
comparable, and the
1(D130A) mutant shows a dominant-negative effect
on VCAM-1 and CS-1 binding to
4
1 (49). We found that the
4
1(D130A) mutant bound far less well to uPAR than did
4
1-CHO (dominant-negative effect) (Fig. 5B). The
residual adhesion of
4
1(D130A)-CHO cells to suPAR D2D3 may be due
to endogenous intact hamster
1/human
4 complex. These results
suggest that Asp-130 of
1 is critical for uPAR binding to
4
1
and that the conserved Asp residues in
1 and
3, which are
required for known integrin ligand binding, are critical for uPAR
binding as well. Taken together, these results strongly suggest that
uPAR is an integrin ligand.
uPAR-Integrin Trans-interaction Supports Cell-Cell
Adhesion--
uPAR has been proposed to interact with integrins on the
same cells (cis-interaction) (reviewed in Ref. 1). Because the present
results suggest that uPAR may interact with integrins as a ligand, it
is possible that uPAR may interact with integrins on apposing cells
(trans-interaction), analogous to the interaction between VCAM-1 and
4
1. To determine whether uPAR interacts in-trans with integrins,
we tested whether K562 erythroleukemic cells expressing recombinant
4
1 (designated
4-K562 cells) interact with unsorted CHO cells
expressing human uPAR (the three-domain form, designated HuPAR-CHO
cells) (Fig. 6A). We found
that
4-K562 cells showed significantly greater adhesion to HuPAR-CHO
cells than to parental CHO cells and that this adhesion was blocked by
SG73 (anti-
4). Mock-transfected and parental K562 or CHO cells
generated essentially the same results, and the data with parental K562
or CHO cells are shown. Endogenous
5
1 in K562 cells and
endogenous hamster uPAR in CHO cells (53) may explain the background
binding of K562 cells to CHO cells. We obtained essentially the same
results with K562 cells expressing recombinant
9
1 and
v
3
(designated
9- and
v
3-K562 cells, respectively) and cloned
HuPAR-CHO cells (Fig. 6, B and C). Y9A2 and 7E3
blocked binding of
9- and
v
3-K562 cells to cloned HuPAR-CHO
cells, respectively, suggesting that these interactions are specific to
the respective integrins. These results suggest that GPI-anchored uPAR
specifically interacts with several integrins on the apposing cells and
supports cell-cell interaction.

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Fig. 6.
uPAR-mediated cell-cell interaction.
A, fluorescence-labeled parent K562 cells or K562 cells
expressing recombinant human 4 ( 4-K562) were incubated with a
monolayer of CHO cells expressing human uPAR (HuPAR-CHO) or CHO cells
in RPMI 1640 medium. Labeled 4-K562 cells were incubated with
HuPAR-CHO cells in the presence of mAbs against integrin 4 (SG73).
The HuPAR cells used were not clonal (~50% of the population was
positive in human uPAR expression). B and C,
fluorescence-labeled parent K562 cells or K562 cells expressing
recombinant human 9 ( 9-K562) (B) or v 3
( v 3-K562) (C) were incubated with a monolayer of
HuPAR-CHO or CHO cells in Hepes-Tyrode buffer supplemented with 2 mM Mg2+. Labeled 9- or v 3-K562 cells
were incubated with HuPAR-CHO cells in the presence of mAbs (Y9A2 to
9, or 7E3 to 3), respectively. HuPAR-CHO cells used in
B and C are clonal. Very similar results were
obtained with parent or mock-transfected CHO or K562 cells in
A, B, and C.
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DISCUSSION |
uPAR as a Ligand for Several Non-I Domain Integrins--
The
present study for the first time establishes that uPAR is a ligand for
several
1 and
3 integrins. We demonstrated that 1) these
integrins adhere to suPAR D2D3 in a dose-dependent and cation-dependent manner and 2) specific antibodies against
these integrins or uPAR block these interactions. Another structurally related protein,
-bungarotoxin, showed only weak integrin binding relative to suPAR D2D3, suggesting that binding to integrins is a
specific property of uPAR. We also demonstrated that the uPAR-binding sites in
4
1 and
v
3 overlap with the previously reported
putative ligand-binding site in these integrins. This is based on the
observations that 3) soluble ligands for
4
1 and
v
3 compete
with suPAR D2D3 for binding to these integrins and 4) the known
integrin mutations that block ligand binding to
4
1 and
v
3
block binding of suPAR D2D3 to these integrins as well. These results
suggest that uPAR may directly compete with other ligands for binding
to these integrins, rather than only operating indirectly by regulating
the binding affinity of integrins to other ligands as an
integrin-associated protein. These unexpected results are consistent
with the observation that soluble uPAR blocks fibronectin binding to
5
1 (17) but do not necessarily agree with the current view that
uPAR interacts with integrins exclusively as an associated protein
rather than as a ligand (reviewed in Ref. 1). Integrin specificity
experiments were performed using suPAR D2D3. It is possible that if we
had used suPAR D1 or full-length suPAR (D1D2D3), differences in
integrin specificity or avidity would have become apparent. Although
all of the recombinant suPAR fragments used supported the
4
1- and
v
3-mediated cell adhesion to the same extent, it is not yet conclusive whether each of the domains contains similar or distinct integrin-binding sites. Detailed binding kinetic studies using individual soluble domains would be required to address this question. Also, there are known differences in uPAR glycosylation among mammalian
cells and between some mammalian tumor cells and S2 cells. It would be
interesting to study the influence of glycosylation, if any, on avidity
or specificity in future experiments.
We also demonstrated that uPAR may mediate cell-cell adhesion by
trans-interaction with integrins (Figs. 6 and
7B). This broadens the
currently held concept of uPAR-integrin interactions, in which uPAR is
proposed to interact exclusively with integrins residing on the same
cell (cis-interaction) as an "associated protein" that mediates
signal transduction directly or through the mediation of a distinct
transmembrane adapter protein. Thus, the current model holds that all
of the molecules that are involved in the uPAR signaling pathway are
located on the same cell surface as uPAR (Fig.
7A) (reviewed in Refs. 1 and
54). The present results do not rule out such uPAR-integrin
cis-interactions. However, the present results predict that if uPAR
interacts with integrins on the same cell surface (cis-interaction),
uPAR may bind as a ligand rather than as an associated protein (Fig.
7C) and that signal transduction events are mediated through
the engaged integrin.

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Fig. 7.
Models of interaction between uPAR and
integrins. The current view of uPAR signaling is based on
association of uPAR with integrins in-cis as an associated protein
(A). We have shown that uPAR may interact with integrins
in-trans (B). If uPAR interacts with integrins in-cis, uPAR
may bind to the ligand-binding site of integrins (C). It has
been shown that soluble uPAR may bind to integrins (D). The
present study suggests that uPAR is a ligand but not an associate
protein of integrins, suggesting that uPAR (GPI-anchored or soluble)
binding to integrins may induce signal transduction through integrin
pathways.
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It has been reported that association of uPAR with integrins induces
tyrosine phosphorylation of focal adhesion kinase, paxillin, p130(cas),
and mitogen-activated protein kinase (13). These signaling molecules
have also been identified as being involved in signaling through
integrins. If uPAR binds to integrins as a ligand, it is not difficult
to imagine that GPI-anchored uPAR may transduce signals through the
integrin pathway without the help of an additional putative
transmembrane adapter molecule. Phosphorylation of these proteins may
be induced through integrins on uPAR-integrin trans-interaction. The
trans-interaction model (Fig. 7B) predicts that tyrosine
phosphorylation of proteins and the resulting induction of gene
expression occur in the cells that have integrin receptors but not in
the cells in which uPAR alone is expressed. In
uPAR-integrin-mediated cell-cell interaction, both of the
apposing cells may express uPAR and integrins, and uPAR-integrin interaction may occur reciprocally; thus tyrosine phosphorylation might occur on both sides.
Implication of uPAR as an Integrin Ligand in Pathological
Situations--
It has been reported that down-regulation of uPAR
makes a human epidermoid carcinoma Hep3 dormant in vivo (6,
7) as a result of a reduced proliferation rate rather than an increased apoptotic rate (7). It has been proposed that reduction of uPAR
expression makes cells either incapable of responding to a
signal, or unable to generate a sufficient signal, to propel them through G0/G1 in vivo (10).
However, cells expressing less than 50% of the normal level of uPAR
grow indistinguishably from parental cells in culture (10). It is
unclear why the altered uPAR expression level affects the proliferation
of cancer cells in vivo but not in culture. We suspect that
uPAR-integrin trans-interaction, which occurs during
three-dimensional growth in tumor mass in vivo but may not
occur in culture, may contribute to this discrepancy. Trans-interactions between uPAR and integrins as described in this
study have the potential to transduce proliferative signals within
tumor masses through integrin-dependent pathways. It is possible that uPAR may also function as a ligand for integrin through
cis-interactions (Fig. 7C), thereby providing autocrine-type proliferative signals. If this is the case, cancer cells that express
high levels of uPAR may have the potential to grow faster than cells
that express uPAR at lower levels.
Soluble uPAR is present in ascites of ovarian cancer patients (55).
Soluble uPAR levels in plasma increase in patients with rheumatoid
arthritis (56), ovarian cancer (57), and leukemia (58). Soluble uPAR
levels correlate with resistance to chemotherapy in leukemia (58) and
with tumor volume in animal models (59). It has also been shown that
soluble uPAR induces extracellular regulated kinase activation when
added to uPAR-low Hep3 cells (10). The present study suggests that
soluble uPAR might also interact as a ligand with integrins on leukemic
cells, solid tumor cells, or inflammatory leukocytes and transduce
proliferative signals (Fig. 7D). Thus binding of
GPI-anchored uPAR (trans-interaction) or soluble uPAR to integrins as a
ligand is a potential therapeutic target in cancer, inflammation, or
other pathological situations.
It has been reported that leukocyte rolling and adhesion dramatically
increase in mesenteric postcapillary vessels in adjuvant-induced chronic vasculitis in rats (60). An anti-integrin
4 antibody significantly blocks this leukocyte rolling and adhesion.
Interestingly, this
4-dependent interaction is not
dependent on VCAM-1 or the CS-1 region of fibronectin. It has also been
reported that platelet and endothelial uPAR is involved in the survival
of platelets in the circulation in mice (61). Injection of tumor
necrosis factor increases the number of platelets in the lung
alveolar capillaries in wild-type mice, but platelet trapping is
insignificant in mice deficient in uPAR. uPAR may be a candidate
integrin ligand in these cases. It is possible that uPAR-integrin
trans-interaction may be involved in these leukocyte-endothelium and
platelet-endothelium interactions during inflammation.
In summary, we have shown that uPAR is a ligand for several integrins
and mediates cell-cell adhesion through trans-interactions with these
integrins. These findings also help to clarify how uPAR binding to
integrins as a ligand transduces signals through integrin pathways. In
addition, the present findings predict that interaction of uPAR with
integrin as a ligand (cis or trans) may be involved in transduction of
proliferative or activating signals in cancer and inflammatory cells.
Additional studies will be required to clarify the role of
uPAR-integrin interactions in these processes.