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J. Biol. Chem., Vol. 278, Issue 32, 29863-29872, August 8, 2003
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5
1 in Urokinase (uPA)/Urokinase Receptor (uPAR, CD87) Signaling*
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From the
Department of Cell Biology, The Scripps
Research Institute, La Jolla, California 92037,
Attenuon, LLC, San Diego, California 92121, and
¶Department of Pathology and Laboratory Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104
Received for publication, May 6, 2003 , and in revised form, May 15, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5
1) and supports cell-cell interaction by
binding to integrins on apposing cells (in trans). We studied whether binding
of uPAR to 51 in cis is involved in
adhesion and migration of Chinese hamster ovary cells in response to
immobilized uPA. This process was temperature-sensitive and required
mitogen-activated protein kinase activation. Anti-uPAR antibody or depletion
of uPAR blocked, whereas overexpression of uPAR enhanced, cell adhesion to
uPA. Adhesion to uPA was also blocked by deletion of the growth factor domain
(GFD) of uPA and by anti-GFD antibody, whereas neither the isolated uPA
kringle nor serine protease domain supported adhesion directly. Interestingly,
anti-5 antibody, RGD peptide, and function-blocking
mutations in 51 blocked adhesion to uPA.
uPA-induced cell migration also required GFD, uPAR, and
51, but
51 alone did not support uPA-induced
adhesion and migration. Thus, binding of uPA causes uPAR to act as a ligand
for 51 to induce cell adhesion,
intracellular signaling, and cell migration. We demonstrated that uPA induced
RGD-dependent binding of uPAR to 51 in
solution. These results suggest that uPA-induced adhesion and migration of
Chinese hamster ovary cells occurs as a consequence of (a) uPA
binding to uPAR through GFD, (b) the subsequent binding of a
uPA·uPAR complex to 51 via uPAR, and
(c) signal transduction through
51. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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uPA is composed of three independently folded domain structures, growth factor domain (GFD) (residue 1–43), kringle domain (residue 50–131), and serine protease domain (residue 159–411). Enzymatic digestion of uPA by plasmin generates an amino-terminal fragment (ATF) that consists of the GFD and kringle domains and the low molecular weight fragment (LMW-uPA), possessing serine protease activity. uPA binds with high affinity through GFD (10) to a cell-surface receptor (uPAR/CD87) that has been identified in many cell types (1). uPAR is a glycosylphosphatidylinositol-anchored 35–55-kDa glycoprotein. It is generally accepted that uPA-mediated signaling requires prior binding to uPAR. However, the mechanism by which uPAR mediates signaling events is still to be fully elucidated. A major problem in understanding how uPA signals derives from the fact that uPAR has no transmembrane structure, leading to the proposal that hypothetical transmembrane adapters may be involved in this process (11).
Among the candidate transmembrane adapters are the integrins, a family of
cell adhesion receptor heterodimers that interact with many extracellular
matrix and cell-surface ligands
(12). At least 18 and
8 subunits have been identified. Integrin-ligand interaction is
involved in many biological and pathological situations, including cell
anchorage and migration, cell-cell interaction during immune response,
development, wound healing, vascular remodeling, and cancer metastasis and
invasion (13). Integrins
transduce signals from outside cells through their interaction with specific
ligands. uPAR has been shown to associate with integrins by
co-immunocoprecipitation, immunocolocalization, and resonance energy transfer
approaches
(14–16).
However, it has not been established whether the association of uPAR with
integrins is responsible for uPA-mediated signaling. We have recently reported
that recombinant soluble uPAR is a ligand for several 1 and
3 integrins
(17), and we postulated that
uPAR can transduce signals through the integrin signaling pathway upon binding
to integrin in trans. However, it is still unclear whether uPAR binds to
integrins as a ligand when both are present on the same membrane (in cis). It
has been proposed that integrins "laterally associate" with uPAR
(for review, see Ref. 2) and
play a role in uPA·uPAR-initiated signaling events. However, the role
of the integrin itself in the uPA·uPAR signaling is unclear, since in
the current models other integrin ligands (e.g. fibronectin) appear
to be essential for uPA·uPAR signaling
(8).
In this study we designed experiments to identify the role of integrin
51 in uPA·uPAR signaling using
recombinant uPA fragments and Chinese hamster ovary (CHO) cells overexpressing
uPAR or mutant 51. We found that cells
adhered to immobilized uPA in a signaling-dependent manner. Anti-uPAR
antibody, depletion of uPAR, and deletion of the GFD of uPA effectively
blocked cell adhesion to uPA, suggesting that binding of uPA to uPAR through
GFD is critical for cell adhesion to uPA. Interestingly,
anti-5 antibody, RGD peptide, and function-blocking
51 mutations blocked cell adhesion to uPA,
suggesting that 51 is critical to this
process as well. uPA-induced migration of CHO cells also required GFD of uPA,
uPAR, and 51. We demonstrated that uPA
induced RGD-dependent binding of uPAR to 51
in solution. These results suggest that uPA-induced signaling in CHO cells
involves a process in which (a) uPA binds to uPAR, (b) the
uPA·uPAR complex binds to 51 as a
ligand in cis, and c) signal transduction is initiated through
51.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5) was a
kind gift of K. Miyake (University of Tokyo, Tokyo, Japan). mAb 135-13C
(anti-6)
(18) was a kind gift of S. J.
Kennel (Oak ridge National Laboratory, Oak Ridge, TN). mAbs P1F6
(anti-v5), HA5 (anti-5),
and VC5 (anti-5) were purchased from Chemicon (Temecula,
CA). Anti-uPAR monoclonal antibody (3B10)
(19) was kindly provided by R.
F. Todd III (University of Michigan Medical Center, Ann Arbor, MI). The
polyclonal anti-uPAR has been described previously
(20). The anti-uPA kringle
antibody (Ab963) was a kind gift from J. Henkin (Abbott Laboratories, Abbott
Park, IL). Anti-uPA kringle and anti-LMW uPA mAbs linked to Sepharose 4B were
from IKTEK Ltd. (Moscow, Russia). A mAb against soluble uPAR (clone
D2D3–813, IgG1
) was raised against the soluble uPAR D2D3
fragment. Strategic Biosolutions (Newark, DE) generated the ascites and
purified the antibody using a 50-ml Amersham Biosciences protein A-Sepharose
fast flow column. GRGDS and GRGES peptide were purchased from Advanced
ChemTech (Louisville, KY). Phosphatidylinositol-specific phospholipase C was
obtained from Glyko, Inc. (Novato, CA). PD98059 was purchased from Calbiochem.
Protein G-agarose was from Amersham Biosciences. Na-125I was
purchased from PerkinElmer Life Sciences, and Iodo beads were from Pierce.
CHO cells were obtained from the American Type Culture Collection
(Manassas, VA). CHO cells expressing the three domain forms of human uPAR
(designated uPAR-CHO) have been described
(17). The
5-deficient CHO cells (B2 variant) expressing human
5 (wild type or mutant) have been described
(21).
Methods
Generation of Wild-type uPA and uPA Fragments—cDNA encoding
wild-type single-chain uPA (scuPA) was generated and subcloned into pMT/BiP/V5
(Invitrogen) as described previously
(22). cDNA encoding the
amino-terminal fragment (ATF, amino acids 1–143), kringle (amino acids
47–143), 
GFD-scuPA (amino acids 47–411), and FLAG-LMW-uPA
(amino acids 136–411) were generated by PCR with full-length UK/pUN121
(23) as a template. The PCR
products were digested with BamH1 and XhoI and subcloned
into pMT/BiP/V5 at the BglII and XhoI sites. Recombinant
proteins were expressed using the Drosophila expression system
(Invitrogen) in Schneider S2 cells according to the manufacturer's
recommendations. Wild-type scuPA, GFD-scuPA, and FLAG-LMW-uPA were
purified from S2 medium by affinity chromatography using anti-LMW uPA mAb
immobilized onto Sepharose (IKTEK Ltd.). ATF 1–143 and kringle were
purified from S2 medium by affinity chromatography using an anti-kringle uPA
mAb immobilized onto Sepharose (IKTEK Ltd.). Synthesis of soluble uPAR (D2D3
form) has been described
(17).
Adhesion Assays—Adhesion assays were performed as previously described (24). 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 50–1000 nM and were incubated1hat37 °C. Remaining protein binding sites were blocked by incubating with 0.2% BSA (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% BSA) supplemented with 2 mM MgCl2 were added to the wells and incubated at 37 °C for 1 h unless stated otherwise. After non-bound cells were removed by rinsing the wells with the same buffer, bound cells were quantified by measuring endogenous phosphatase activity (25). Antibodies were used at a 250-fold dilution for ascites (KH72 and 135-13C) and at 10 µg/ml for purified antibodies or IgG. Data are shown as means ± S.D. of three independent experiments. We confirmed that equivalent amounts of the fragments and mutants of uPA were coated on the plate by enzyme-linked immunosorbent assay (data not shown).
Mitogen-activated Protein Kinases (MAPK) Activation Assay—uPAR-CHO cells were plated into 6-ell tissue culture plates at 2 x 106 cells/ml in Dulbecco's modified Eagle's medium supplemented with 0.5% fetal calf serum, 1x penicillin-streptomycin-glutamine solution and incubated for 2 days at 37 °C in a 95% air, 5% CO2 humidified atmosphere. The cell culture media was removed, and the cells were washed once with prewarmed serum-free Dulbecco's modified Eagle's medium. The cells were incubated for 3 h with serum-free Dulbecco's modified Eagle's medium with or without the MEK inhibitor PD98059 (500 µM) at 37 °C in a 95% air, 5% CO2 humidified atmosphere. The cells were stimulated with different concentrations of soluble scuPA for 5 min at 37 °C. The reaction was terminated by removing the stimulation media and washing the cells with 1 ml of ice-cold phosphate-buffered saline containing 1 mM Na3PO4 followed by incubation with 100 µl of ice-cold radioimmune precipitation assay buffer for 20 min on ice. The whole cell lysate was collected, and the nuclear material was pelleted by centrifugation at 14,000 x g for 10 min. The supernatant from each treatment was retained and stored at –20 °C until required. Whole cell lysates (40 µg of protein) were fractionated using 4–20% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose for Western blotting. The membranes were blocked for 1 h at room temperature with blocking buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.5, supplemented with 0.1% Tween 20 and 5% BLOTTO; Biorad, Hercules, CA). To determine the phosphorylation changes in MAPK the membranes were washed 3 times for 5 min with wash buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.5, supplemented with 0.1% Tween 20) and incubated overnight at 4 °C with a 1:1000 dilution of rabbit anti-phospho-p44/42 MAPK (Thr-202/Tyr-204) antibody (Cell Signaling Technology, Beverly, MA) in blocking buffer. The blots were washed 3 times for 5 min with wash buffer and probed with a 1:2000 dilution of anti-rabbit horseradish peroxide-conjugated secondary antibody (Cell Signaling Technology) in blocking buffer at room temperature for 1 h. The blots were washed 3 times for 5 min and developed using the Immun-Star horseradish peroxide chemiluminescence substrate kit (Bio-Rad). The blots were stripped by incubating with stripping buffer (0.1 M glycine, pH 2.6, and 2% SDS) for 30 min at 50 °C. The blots were washed 3 times with 10 mM Tris-HCl, 150 mM NaCl, pH 7.5, re-blocked for 1 h at room temperature with blocking buffer, washed 3 times for 5 min, incubated with a 1:1000 dilution of rabbit anti-p44/42 MAPK (Cell Signaling Technology) overnight at 4 °C in blocking buffer, and processed as above to determine the total MAPK levels of each lane.
Migration Assays—Cell migration was analyzed using tissue culture-treated 24-well Transwell plates (Costar, Cambridge, MA) with polycarbonate membranes of pore size 8 µm. The lower side of the filter was coated with various concentrations (20–200 nM) of substrates. Coated filters were placed into a serum-free migration buffer (Dulbecco's modified Eagle's medium supplemented with 10 mM Hepes, 0.5% bovine serum albumin, and 1x penicillin-streptomycin), and cells (100 µl) suspended in the same buffer (8 x 105 cells/ml) were added to the upper chamber. The cells were incubated at 37 °C in 5% CO2 for 20 h. Cells in the upper chamber were removed by wiping, and those that migrated to the lower surface of the filters were fixed and stained with 0.5% crystal violet in 20% ethanol and counted. The result in each well is the mean cell number of 4 randomly selected high magnification microscopic fields from triplicate experiments. In some experiments, anti-integrin antibodies (10 µg/ml) were incubated with cells for 15 min before to the assay.
Co-precipitation of uPAR and Integrin
511—Soluble
uPAR was radioiodinated with Na-125I using Iodo beads (specific
activity 14,500 cpm/ng). Purified human 51
integrin was obtained from Chemicon International. Purified
51 (6 µg/ml), mAb HA5 (9 µg/ml),
125I-labeled soluble uPAR (6 µg/ml), and uPA (12 µg/ml) were
incubated with protein G-agarose beads either in the presence or absence of
RGD peptide (150 µg/ml) in serum-free RPMI 1640 medium supplemented with 10
mM HEPES, pH 7.4, 0.02% bovine serum albumin at 4 °C for 4 h.
As a control, experiments were performed in the absence of uPA. Beads were
washed 3 times in RPMI supplemented with 10 mM HEPES, 0.02% bovine
serum albumin. Bound materials were extracted into reducing SDS-PAGE sample
buffer and analyzed by SDS-PAGE and autoradiography.
Other Methods—Flow cytometric analysis and stress-fiber staining were performed as described before (17, 26).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We also found that adhesion of uPAR-CHO cells to immobilized uPA occurred at 37 °C, but not at 4 °C, suggesting that this process is temperature-dependent and requires signal transduction (Fig. 2a). It has been reported that uPA induces activation of MAPK (27). An inhibitor to MEK-1 (PD98059) that prevents the activation of MAPK (extracellular signal-regulated kinases1/2) (Fig. 2b) consistently blocked adhesion of uPAR-CHO cells to uPA in a dose-dependent manner (Fig. 2c). These results suggest that this process requires extracellular signal-regulated kinase 1/2 activation (most likely induced by immobilized uPA). We found that immobilized uPA did not induce spreading or stress-fiber formation in uPAR-CHO cells, in contrast to fibronectin used as a positive control (Fig. 2d).
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Role of Integrins in uPAR-mediated Adhesion to uPA—It has
been proposed that integrins may be critically involved in the uPA·uPAR
signaling (2). To examine this
hypothesis we first determined whether integrins contribute to cell adhesion
to uPA. To do so, we tested the effects of anti-integrin mAbs on adhesion of
uPAR-CHO cells to uPA. CHO cells have endogenous hamster integrins
51, v1,
and v5 (data not shown). We found that RGD
peptide (100 µM) blocked adhesion of uPAR-CHO cells to uPA, but
control RGE peptide did not (Fig.
3a). Consistent with the findings that RGD-dependent
integrin(s) is involved in this process, anti-5 mAb (KH72)
completely blocked adhesion of uPAR-CHO to uPA, whereas
anti-v5 mAb (P1F6) or control ascites did
not (Fig. 3a and data
not shown). These results suggest that adhesion of uPAR-CHO cells to
immobilized uPA is 51-dependent.
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Then we tested whether the level of 51
expression affects cell adhesion to uPA using the B2 variant of CHO cells,
which expresses 
2% of 51 compared with
parental CHO cells (28). The
B2 cells adhered to uPA at a level lower than CHO cells
(Fig 3b). This
adhesion was completely blocked by anti-uPAR polyclonal antibodies and
anti-integrin 5 (data not shown). These results suggest that
cell adhesion to uPA is dependent on the level of
51 but that a small amount of
51 on the B2 cells still supports the
adhesion to uPA to some extent. Another possibility is that integrin
v1 may also be involved in this process,
although we were not able to test this hypothesis since function-blocking
anti-hamster v mAb is not currently available. To test the
specific contribution of 51 to
uPA·uPAR-dependent cell adhesion, we used immobilized anti-uPAR mAb as
a uPAR ligand. We found that uPAR-CHO cells adhered to anti-uPAR and that this
adhesion was not inhibited by anti-integrin 5 mAb
(Fig. 3c), suggesting
that 51 was not required for this process.
Taken together, adhesion of CHO cells to uPA requires uPAR and
51, and
51 is specifically involved in this process
only when uPA is used as a ligand.
The Domains of uPA Required for Cell Adhesion to uPA—To
identify which uPA domains are involved in
uPAR/51-dependent cell adhesion to uPA, we
used several uPA fragments including the ATF, the kringle domain, the LMW-uPA,
GFD-uPA, which lacks GFD, and Kringle-uPA, which lacks the
kringle domain (Fig
4a). We found that ATF and Kringle-uPA supported
cell adhesion at levels comparable with that of wild-type uPA. Kringle,
LMW-uPA, or GFD-uPA did not support the adhesion at all
(Fig 4b). These
results suggest that GFD is primarily involved in cell adhesion to uPA, but
other domains are not.
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As a second approach, we tested whether mAbs to different domains of uPA
block cell adhesion to uPA. We found that anti-GFD (AD3471) and anti-kringle
mAb (Ab963) blocked adhesion of uPAR-CHO cells to uPA, whereas anti-LMW
(UNG-5) did not (Fig
4c). Ab963 has been observed to inhibit the binding of
uPA to whole cells despite the fact that its epitope has been mapped to the
kringle domain. These results are consistent with the results with uPA
fragments (Fig. 4b)
with the exception of the effect of the anti-kringle antibody. GFD is required
for uPA to bind to uPAR (10).
Taken together these studies suggest that
uPAR/51-mediated cell adhesion to uPA is
also critically dependent on the interaction with GFD.
Mutations in Integrin 5 Affect Cell
Adhesion to uPA—We have recently reported that uPAR is a ligand for
several integrins (17).
Specifically, soluble uPAR supports integrin-mediated cell adhesion, and
glycosylphosphatidylinositol-linked uPAR binds to integrins in apposing cells
in trans and supports cell-cell interaction. We have reported that mutations
in the ligand binding region of integrin 4 subunit blocked
adhesion of 41-transfected CHO cells to
immobilized soluble uPAR (17),
suggesting that uPAR binds to 41 as a
ligand. These critical residues are located within the ligand binding site in
integrins based on the crystal structure of integrin
v3
(29,
30). We suspected that cell
adhesion to uPA in the present study involves interaction between uPAR and
51 on the same cell surface (in cis). We
have reported that similar mutations (the Tyr-186 to Ala (Y186A), F187A, and
W188A) in the ligand binding site of 5 blocked fibronectin
binding to 51
(21). We therefore studied
whether uPAR binds to 51 as a ligand during
adhesion to uPA using these 5 mutants.
We first tested the effects of these function-blocking mutations of
5 (21) on
adhesion to soluble uPAR. We used B2 cells expressing wild type and Y186A,
F187A, and W188A mutants of integrin 5 (designated
5-B2, 5/Y186A-B2,
5/F187A-B2, and 5/W188A-B2, respectively).
Expression levels of integrin 51 among
those transfectants were comparable as measured by flow cytometry with
non-function blocking anti-human 5 mAb (VC-5)
(21). CHO cells, but not B2
cells, bind to coated soluble uPAR upon Mn2+ activation
(17), indicating that
51 requires activation to bind to uPAR. We
found that 5-B2 cells adhered to soluble uPAR if activated
with Mn2+ (Fig
5a) and that the Y186A and W188A mutations completely,
and the F187A mutation partially, blocked the adhesion to soluble uPAR.
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We then tested the effect of these 5 mutations on cell
adhesion to uPA. In this experiment we used ATF instead of wild-type uPA to
exclude the possible contribution of the serine protease domain because we
found that both B2 and 5-B2 cells bind weakly to LMW-uPA
when 51 is activated with
Mn2+.2
We found that 5-B2 cells adhered to ATF more than parental
B2 cells, but that 5/Y186A-B2 and
5/W188A-B2 cells adhered at the level comparable with or
lower than that of parental B2 cells (Fig
5b). The adhesion of parent B2 cells to ATF may be due to
endogenous low levels of 51 on B2 cells or
due to endogenous v1, as noted above. These
results suggest that Tyr-186 and Trp-188 of 5 are critical
for cell adhesion to both soluble uPAR and ATF.
Taken together these results suggest that uPAR-mediated cell adhesion to
uPA requires the intact ligand binding function of 5. This
is consistent with the observation that RGD peptide blocked adhesion of
uPAR-CHO cells to uPA in the present study
(Fig. 3a) and that the
anti-5 mAb we used in the present study (KH/72) binds to the
ligand binding site of
5.3
We propose that uPAR binds to 51 in cis as
a ligand upon cell adhesion to uPA.
uPA-induced Cell Migration Also Depends on Both uPAR and Integrin
51—It has
been reported that physiological concentrations of uPA stimulated a
chemotactic response in human monocytic THP-1 through binding to uPAR
(11). We tested whether uPAR
and 51 are involved in uPA-induced cell
migration. We found that wild-type uPA was chemotactic for uPAR-CHO cells,
whereas GFD-uPA or kringle was not
(Fig 6a), indicating
that GFD is required for this process. We found that anti-human uPAR
polyclonal antibodies, anti-uPAR mAb (anti-D2D3), and anti-5
mAb (KH72) effectively blocked uPA-induced migration
(Fig 6b). Neither
control rabbit IgG nor anti-integrin 6 mAb blocked
uPA-induced migration. Taken together these results suggest that GFD, uPAR,
and integrin 51 are critically involved in
uPA-induced cell migration.
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uPA-induced Binding of uPAR to Integrin
51—The
results in the present study predict that binding of uPA to uPAR through GFD
induces uPAR binding to 51. We tested
whether uPA directly induces binding of uPAR and
51 in a cell-free system. We incubated
labeled soluble uPAR with isolated 51 in
the presence and absence of uPA. We isolated
51-uPAR complex with
anti-5 mAb HA5 (non-function blocking). We found that uPAR
co-precipitated with 51 in the presence of
uPA but not in the absence of uPA (Fig.
7). Consistent with the results in the present study, RGD peptide
reduced the co-precipitation of uPAR and
51. These results suggest that uPA markedly
increases binding of uPAR to 51 in solution
and probably on the cell surface.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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A major finding of the present study is that cell adhesion and migration to
uPA required the ligand binding function of
51. We have previously reported that uPAR
binds (as a ligand) to integrins in trans and that trans interaction between
uPAR and integrins supported cell adhesion and cell-cell interaction
(17). In the present study we
have shown that function-blocking anti-5 mAb blocked
uPA-induced cell adhesion and migration and that RGD peptide and the
5 mutations that block ligand binding effectively blocked
cell adhesion to uPA. The crystal structure of integrin
v3 has only a single RGD binding site
between v and 3 subunits
(30) but did not show the
position or existence of allosteric RGD binding sites. The integrin mutations
we used in the present study (Y186A, F187A, and W188A in 5)
are very close to the RGD peptide in the
v3·RGD complex
(30) (the distances between
their backbones are within 10 Å), suggesting that these mutations
directly block ligand binding to 51. Thus,
it is highly likely that RGD peptide and the integrin mutations directly
blocked uPAR binding to 51 and thereby
blocked cell adhesion to uPA. We do not, however, preclude the possibility
that RGD peptide binds to allosteric RGD binding sites and affects uPAR
binding to 51.
Because anti-uPAR antibodies effectively blocked adhesion of uPAR-CHO cells
to uPA, 51 alone cannot directly support
adhesion to uPA. Consistent with this observation, no
51 ligand other than uPAR was required for
uPA·uPAR signaling in the present study. It is highly likely that uPAR
is a primary ligand for 51 upon uPA-induced
cell adhesion and migration in the present study and that uPAR binds to
51 as a ligand even when uPAR and
51 interact in cis. Thus uPA-induced cell
adhesion and migration involves the following sequence. 1) uPA binds to uPAR,
2) uPAR then binds to 51 in cis, and 3)
signal transduction is mediated through 51
(Fig. 8). CHO and B2 cells
express v1 and
v5, and we have shown that
anti-v5 (P1B6) does not block adhesion to
uPA. However, we do not rule out the possibility that
v1 is involved in uPAR binding in cis. We
are not able to test whether v1 is involved
because anti-v antibody that cross-reacts with hamster
v is not available.
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It has previously been reported that the avidity of
51 in uPAR-rich human carcinoma Hep3 cells
for fibronectin was higher than that of uPAR-poor dormant Hep3 cells
(8). The levels of MAPK
activation by fibronectin were much higher in uPAR-rich cells than uPAR-poor
cells. It is unclear, however, how fibronectin, uPAR, and
51 are involved because both fibronectin
and uPAR are ligands for 51. We suspect
that 1) the initial uPAR binding to a limited number of
51 in cis induces the increased signaling
through 51 and activated MAPK, 2)
51 is activated by inside-out signaling,
and 3) fibronectin binds to activated unoccupied
51, leading to more outside-in signaling in
uPAR-rich cells. In uPAR-poor cells, in contrast, the initial
uPAR-51 binding may not be enough to induce
51 signaling. This would be a possible
reason that reduction in the level of uPAR induced a protracted state of
dormancy in tumor cells. We were not able to detect stress-fiber formation
upon adhesion of uPAR-CHO or mock-transfected CHO cells to immobilized uPA in
the absence of fibronectin (Fig.
2d). It is unclear whether signal transduction through
51 on adhesion to uPA stabilized the
interaction between uPAR and immobilized uPA and/or induced re-organization of
cytoskeletal proteins in the present study.
Several recent papers suggest that uPAR binds to unique "non-ligand
binding sites" in repeat 4 of the subunits in several integrins
(31,
32). Several integrin peptides
from the predicted 2–3 loop in repeat 4 (e.g.
PRHRHMGAVFLLSQEAG, residues 240–257 of 3) have been
reported to block uPAR-integrin interaction. Several laboratories including
ours have previously identified many amino acid residues within repeats
2–4 of the integrin subunits that are critical for binding to
integrin ligands (21,
33–38).
A recent crystal structure of integrin v3
(29) verified these mapping
results; the critical amino acid residues that have been identified by
mutagenesis are located within the ligand binding site of integrins and
exposed to the surface. We suspect that the PRHRHMGAVFLLSQEAG peptide of
3 may be located within the ligand binding site of
3 because mutations of the predicted 2–3 loop in
repeat 4 of IIb effectively blocked fibrinogen binding to
IIb3
(33). We have previously shown
that mutations in the predicted 2–3 loop of repeat 3 of
4 (which is clearly within the ligand binding site of
4) effectively blocked
uPAR-41 interaction
(17). In the present study we
demonstrated that the corresponding mutations in 5 also
blocked uPAR-51 interaction. It is, thus,
highly likely that the uPAR binding site may actually overlap the binding
sites for other integrin ligands. If uPAR and other integrin ligands bind to
the overlapping region within repeat 2–4 in non-I-domain integrins, it
is possible that the integrin-derived peptides described above might block the
binding of other ligands to integrins as well as uPAR.
We recently reported that the angiostatin, a plasminogen fragment that
contains the amino-terminal three or four kringles, binds to several integrins
(including v3 and
91 but not
51)
(39). We found that
51 did not bind to the kringle domain of
uPA in the present study. It is, thus, not likely that uPA may cross-link uPAR
and 51 through GFD and kringle. We found
that deletion of the kringle domain (kringle-uPA) did not block the
uPAR/51-mediated cell adhesion to uPA in
the present study. This indicates that uPA does not bind to
51 through the kringle. The finding that an
anti-kringle antibody effectively blocked cell adhesion to uPA may be due to
steric hindrance of GFD function by the antibody or by causing the kringle to
block access of GFD for uPAR. We have observed that that Ab963 and other mAbs
whose epitopes map to the kringle can block the binding of uPA to uPAR on
cells.4 However, these
studies do not preclude the possibility that uPA may cross-link uPAR and other
integrins through GFD and kringle, forming a uPA·uPAR·integrin
signaling complex on the cell surface.
It has been reported that uPA increases association of uPAR with integrins
using co-immunoprecipitation
(8,
32,
40,
41) although to date the
mechanism underlying this phenomenon has remained unclear. Consistent with
these reports, we demonstrated that uPA induces RGD-dependent binding of
soluble uPAR to isolated 51 in solution in
the present study (Fig. 7).
These results predict that uPA binding to uPAR induces uPAR binding to
51 in cis on the cell surface. What is the
mechanism of uPA-induced uPAR binding to integrins? It has been reported that
soluble uPAR forms dimers and oligomers
(42). Interestingly, the
addition of an equimolar concentration of uPA leads to the dissociation of
these dimers and oligomers. It is tempting to speculate that the ability of
uPAR to binding to integrins is related to dissociation of uPAR, i.e.
that GFD binding to uPAR may induce dissociation of uPAR dimers and oligomers,
exposing the integrin binding sites within uPAR. Additional studies to examine
the role of uPA and uPAR in 51 activation
are currently under way.
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FOOTNOTES |
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|| To whom correspondence should be addressed: Dept. of Cell Biology, VB-6, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7636; Fax: 858-784-7645; E-mail: takada{at}scripps.edu.
1 The abbreviations used are: uPA, urokinase-type plasminogen activator;
uPAR, uPA receptor; scuPA, single-chain uPA; CHO, Chinese hamster ovary; GFD,
the growth factor domain; LMW, low molecular weight; mAb, monoclonal antibody:
MAPK, mitogen-activated protein kinase; scuPA; ATF, amino-terminal fragment;
BSA, bovine serum albumin.
2 T. Tarui and Y. Takada, unpublished data.
3 T. Tarui and Y. Takada, unpublished observation.
4 G. Parry and A. Mazar, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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