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INTRODUCTION |
Binding of the serine proteinase, urokinase-type plasminogen
activator (uPA),1 to its cell
surface receptor, uPAR, promotes plasminogen activation at the cell
surface and may lead to the activation of metalloproteinases (1).
Because uPAR localizes to the leading edge of migrating cells,
activated proteinases generated downstream of uPA and uPAR may degrade
extracellular matrix proteins, facilitating cell migration through
tissue (2, 3). Indeed, uPA and/or uPAR have been implicated in
physiologic processes requiring cell migration, including inflammation,
scar formation, revascularization of injured tissues, neointima
formation, and cancer metastasis (1, 4-11).
In addition to uPA, uPAR binds directly to the extracellular matrix
protein, vitronectin (12, 13), and may thereby function as an adhesion
receptor. The binding sites for uPA and vitronectin, in the structure
of uPAR, are distinct but interactive because uPA-binding increases the
affinity of uPAR for vitronectin (14, 15). When uPA or vitronectin bind
to uPAR, cell-signaling responses are elicited. Vitronectin-binding
activates a pathway that includes the small GTPase, Rac1, and may drive
new actin polymerization in the migrating cell (16). uPA-binding
activates diverse signaling pathways, and the nature of the response
may be cell type-specific (17, 18). In multiple cells, binding of uPA
to uPAR activates the Ras-extracellular signal-regulated kinase (ERK)
pathway (19-22). Because myosin light chain kinase is activated
downstream of ERK, this signaling pathway also may stimulate cell
migration (20).
uPAR is linked to the plasma membrane only by a
glycosylphosphatidylinositol anchor and thus lacks transmembrane and
intracytoplasmic domains (23). For this reason, it is generally assumed
that the complete uPAR-signaling receptor is a multiprotein complex. In
support of this hypothesis, soluble uPAR binds to the cell surface and
elicits many of the same responses as uPA (24-26). The true function
of uPA may be to alter the conformation of uPAR, promoting interactions
with other proteins in the plasma membrane that are required for
cell-signaling (27).
A question of considerable importance concerns the nature of the
uPAR-containing multiprotein complex involved in cell-signaling. uPAR
associates with
1,
2,
3,
and
5 integrins (15, 28-31), and disrupting these
interactions with synthetic peptides or integrin-neutralizing antibodies inhibits uPAR-signaling to ERK (15, 22, 32). In some cell
types, caveolin may promote association of uPAR with
1
integrin, and this may be necessary for cell signaling (33). The
transmembrane protein, gp130, associates with uPAR and serves as a
critical adaptor protein leading to activation of the JAK/STAT pathway
(34). The G protein-coupled receptor, FPR-like receptor-1/lipoxin A4
receptor (FPRL1/LXA4R), binds soluble uPAR, mediates uPAR-initiated chemotaxis in monocytes, and is necessary for signaling to the tyrosine
kinase, Hck (35). Finally, Liu et al. (36) identified a
complex that includes uPAR,
5
1, and the
epidermal growth factor receptor (EGFR), mainly in cells that express
high levels of uPAR. After uPA stimulation, focal adhesion kinase
operated downstream of
5
1 to activate the
EGFR, and this was necessary for signaling to ERK. Specific antagonists
of EGFR completely blocked ERK phosphorylation. Thus, it was proposed
that EGFR serves as a critical adaptor protein in the pathway that
links uPAR to ERK. The EGFR also may be transactivated by G-protein
coupled receptors and receptor tyrosine kinases such as the
platelet-derived growth factor
receptor (37, 38).
The diversity of adaptor proteins, which associate with uPAR and
promote uPAR signaling, suggests that the uPAR-multiprotein signaling
complex may be large and heterogeneous. Components of the uPAR
signaling complex may be affected by multiple properties of the cell,
including membrane protein expression. We are particularly interested
in signaling pathways leading to activation of Rac1 and ERK, because
these pathways play pivotal roles in cell migration. In this study, we
demonstrate that the EGFR is indeed essential in the pathway that links
uPAR to ERK in cells that express EGFR. However, in cells that lack
EGFR, alternative EGFR-independent pathways are operational, and uPA is
still able to activate ERK. uPAR-dependent Rac1 activation
is EGR-independent.
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MATERIALS AND METHODS |
Reagents, Antibodies, and Expression Constructs--
Two-chain
uPA was kindly provided by Drs. Jack Henkin and Andrew Mazar,
previously of Abbott Laboratories (Abbott Park, IL). uPA was treated
with diisopropyl fluorophosphate to generate DIP-uPA, as previously
described (19). DIP-uPA binds to uPAR with unchanged affinity but lacks
proteinase activity. Recombinant human EGF was purchased from R&D
Systems, Inc. (Minneapolis, MN). The mitogen-activated protein kinase
kinase inhibitor, PD098059, was from Calbiochem (San Diego, CA).
Expression constructs encoding human uPAR and green fluorescent protein
(GFP) were previously described (20). The expression construct, which
encodes dominant-negative Rac1 (N17Rac1), was kindly provided by Dr.
Robert Nakamoto (University of Virginia). Constructs encoding wild-type
full-length EGFR and kinase-inactive EGFR (KI-EGFR) were kindly
provided by Dr. Sarah Parsons (University of Virginia). In KI-EGFR,
mutation at residue 721 (Lys
Ala) abolishes kinase activity (39, 40).
Rac/Cdc42 assay reagent (PAK-PBD1), which includes residues 67-150 of
p21-activated kinase (PAK-1) fused to
glutathione-S-transferase and coupled to glutathione-agarose
was from Upstate Biotechnology (Lake Placid, NY). Antibody that
specifically detects phosphorylated ERK1 and ERK2 was from Cell
Signaling Technology (Beverly, MA). Polyclonal antibody that recognizes
total ERK1 and ERK2 was from Zymed Laboratories Inc.
(San Francisco, CA). Rac1-specific monoclonal antibody was from BD
Biosciences. Polyclonal anti-human uPAR antibody was from American
Diagnostica. Horseradish peroxidase-conjugated antibodies specific for
mouse IgG and rabbit IgG were from Amersham Biosciences. Tyrphostin
AG1478, protease inhibitor mixture, sodium orthovanadate, dithiothreitol, G418, and bovine serum albumin were from Sigma.
Cell Culture--
COS-7 cells (ATCC) were maintained in DMEM
(Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Hyclone),
penicillin (100 units/ml), and streptomycin (100 µg/ml). Chinese
hamster ovary cells (CHO-K1) (ATCC) were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% FBS, penicillin (100 units/ml), streptomycin (100 µg/ml), and nonessential amino acids
(0.1 mM). Cells were passaged using Trypsin-EDTA
(Invitrogen) and maintained in culture for 48 h before performing experiments.
COS-7 cells were transfected to overexpress human uPAR by incubation
for 48 h with 2 µg of the uPAR expression construct in the
presence of EffecteneTM (Qiagen). uPAR expression was
demonstrated by immunoblot analysis. An equivalent protocol was used to
transfect CHO-K1 cells to express wild-type EGFR or KI-EGFR. Cultures
were selected in G418 (1 mg/ml) for 30 days and then maintained in DMEM
supplemented with 10% FBS and 0.5 mg/ml G418.
Cell Migration Assays--
Migration of COS-7 and
uPAR-overexpressing COS-7 cells (COS-7/uPAR) was studied using 6.5-mm
Transwell chambers with 8-µm pores (Costar), as previously described
(19). The Transwell membranes were precoated with 20% FBS on the
underside only. Both membrane surfaces were then blocked with 5 mg/ml
bovine serum albumin for 2 h at 37 °C. Cells (105
in 100 µl) in serum-free medium were pretreated with tyrphostin AG1478 (50 nM) or PD098059 (50 µM) for 15 min
in suspension and then with DIP-uPA or EGF for an additional 15 min.
The cells were then added to the upper chamber of each Transwell unit
in the presence of the same agents. The lower chamber was supplemented with DIP-uPA or EGF when these agents were added to the top chamber. Migration was allowed to occur for 6 h at 37 °C. Cell migration was determined by crystal violet staining, as previously described (20).
In some experiments, COS-7 and COS-7/uPAR cells were transiently
co-transfected with the constructs encoding N17Rac1 (2 µg) and with
pEGFP (0.5 µg), which encodes GFP, by incubation with EffecteneTM (Qiagen) for 24 h. Co-transfection
efficiencies were essentially 100%, when determined as previously
described (20). Migration experiments were performed using Biocoat cell
culture inserts (BD Biosciences) instead of Transwell chambers. The
membrane-coating method and protocol for pretreating cells was
unchanged. Cell migration was determined by counting green-fluorescing cells.
Immunoblot Analysis for ERK Activation--
COS-7
cells, COS-7/uPAR cells, CHO-K1 cells, and EGFR-overexpressing CHO-K1
cells were cultured on 60-mm plates until 80-90% confluent and then
were serum-starved for 24 h. When indicated, the cells were
preincubated with tyrphostin AG1478 (50 nM) for 2 h.
DIP-uPA (10 nM) was then added for 10 min. Cell extracts were prepared in 10 mM HEPES, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Nonidet P-40, pH 7.5, containing
protease inhibitor mixture and sodium orthovanadate (1 mM).
The protein concentration in each extract was determined by
bicinchoninic acid assay. Equal amounts of each extract were subjected
to SDS-PAGE on 10% slabs, electrotransferred to polyvinylidene
diflouride membranes, and probed with specific antibodies for
phosphorylated and total ERK.
Affinity Precipitation of GTP-Rac1--
Affinity precipitation
of active Rac1 was performed using the fusion protein, PAK1-PBD, which
specifically recognizes the active GTP-bound forms of Rac1 and Cdc42,
as previously described (16, 41). COS-7 and COS-7/uPAR cells (2 × 105) were cultured in 10-cm plates for 18 h. Some
cultures were pretreated with tyrphostin AG1478 (50 nM) for
2 h. Cultures then were washed with ice-cold phosphate-buffered
saline, and extracted in 1% (v/v) Triton X-100, 0.5% (w/v) sodium
deoxycholate, 0.1% (w/v) SDS, 50 mM Tris-HCl, 0.5 M NaCl, 10 mM MgCl2, pH 7.2, supplemented with protease inhibitor mixture and 1 mM
sodium orthovanadate. The extracts were incubated with 15 µg of
PAK1-PBD coupled to glutathione-Sepharose for 45 min at 4 °C. The
glutathione-Sepharose was washed four times and then treated with
SDS-sample buffer to dissociate the PAK1-PBD and associated proteins.
Immunoblot analysis was performed to detect active Rac1. Samples of
each cell extract were also subjected to immunoblot analysis before incubation with PAK1-PBD to determine total Rac1.
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RESULTS |
EGFR Functions in uPA-stimulated ERK Activation--
In this
study, COS-7 cells, which express EGFR (42), were transfected to
overexpress uPAR. As shown in Fig.
1A, low levels of uPAR were
detected in the parental cells; however, the transfected cells
(COS-7/uPAR) expressed high levels of uPAR. When treated with EGF (10 ng/ml), COS-7 and COS-7/uPAR cells demonstrated significant ERK
activation, as determined by immunoblot analysis (Fig. 1B), confirming that these cells express EGFR. Both cell types also responded to DIP-uPA (10 nM) and demonstrated ERK
activation within 10 min. The major difference observed between the
COS-7 and COS-7/uPAR cells was an increase in the basal level of
activated ERK (in the absence of exogenously added agents) in the
COS-7/uPAR cells. This may reflect autocrine uPAR activation by
endogenously produced uPA, as has been previously described (43). The
ability of uPA to activate ERK in parental COS-7 cells was not
surprising, despite the low level of uPAR, because uPA activates ERK in
MCF-7 cells, which express only 3,000-4,000 copies of
cell-surface uPAR/cell (19).

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Fig. 1.
The EGFR-specific inhibitor, tyrphostin
AG1478, inhibits uPA-mediated ERK phosphorylation in COS-7 and
COS-7/uPAR cells. A, COS-7 cells were stably transfected to
overexpress human uPAR. Expression of uPAR in the parental cells
(COS-7) and transfected cells (COS-7/uPAR) was confirmed by immunoblot
analysis. B, COS-7 and COS-7/uPAR cells were treated with
DIP-uPA (10 nM) for various times (as shown) or with 10 ng/ml EGF (E) for 5 min. Phosphorylated and total ERK were
then determined. C, COS-7 and COS-7/uPAR cells were treated
with tyrphostin AG1478 (50 nM) or vehicle for 2 h and
then with DIP-uPA for 10 min. Phosphorylated and total ERK were
determined.
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To determine the role of the EGFR in uPA-stimulated ERK activation, we
pretreated COS-7 and COS-7/uPAR cells with the EGFR-specific inhibitor,
tyrphostin AG1478 (50 nM). This drug completely blocked ERK
activation in response to DIP-uPA in both cell types (Fig. 1C). A slight decrease in the basal level of activated ERK
was also observed. These results suggest that the EGFR plays an
essential role in the pathway by which uPA activates ERK in COS-7 cells and that the level of uPAR expression is not critical in determining whether the EGFR participates in this signaling event.
EGFR Does Not Function in uPAR-induced Rac1 Activation--
Kjoller et al. (16) demonstrated that uPAR
overexpression is associated with an increase in Rac1 activation and
increased cell migration. This response requires vitronectin binding to uPAR and not uPA. Because of the role of the EGFR in uPA-induced ERK
activation in COS-7 cells, we performed experiments to determine whether the EGFR is necessary in the pathway that links uPAR to Rac1.
As shown in Fig. 2, GTP-bound Rac1 was
increased in the uPAR-overexpressing COS-7 cells (p < 0.05, n = 3), confirming the results of Kjoller
et al. (16). Tyrphostin AG1478 had no effect on the level of
GTP-Rac1 in COS-7 or COS-7/uPAR cells. These results suggest that the
adaptor protein interactions, which facilitate uPAR signaling either to
ERK or Rac1, are distinct.

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Fig. 2.
Rac1 activation by uPAR overexpression in
COS-7 cells does not depend on the EGFR. A, COS-7 and
COS-7/uPAR cells were treated with tyrphostin AG1478 (50 nM) or vehicle for 2 h. GTP-bound Rac1 was
affinity-precipitated with PAK1-PBD and quantitated by immunoblot
analysis. The original cell extracts were also studied by immunoblot
analysis using the same antibody to determine total Rac1. B,
immunoblots were analyzed by densitometry. The results of three
separate experiments were then averaged to generate the bar graph
(mean ± S.E.).
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Role of EGFR in uPAR-stimulated COS-7 Cell Migration--
uPAR
overexpression was associated with a modest but significant increase
(p < 0.01, n = 9) in COS-7 cell
migration through serum-coated Transwell membranes (Fig.
3A). In the absence of uPA,
tyrphostin AG1478 had no effect on the migration rate of COS-7/uPAR
cells. uPA further stimulated the migration of COS-7/uPAR cells
(1.9-fold; p < 0.01, n = 5), and this
response was blocked by tyrphostin AG1478. Tyrphostin AG1478 also
blocked the effects of EGF on COS-7/uPAR cell migration, as
anticipated.

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Fig. 3.
Cooperation between uPAR and the EGFR in
COS-7 cell migration. A, COS-7 cells (uPAR ) and
COS-7/uPAR cells (uPAR+) were pretreated with tyrphostin
AG1478 or vehicle and then with DIP-uPA (10 nM) or EGF (10 ng/ml) for 15 min. The cells were then allowed to migrate through
serum-coated Transwell membranes for 6 h. Migration is expressed
as the percentage of that observed with untreated COS-7 cells
(mean ± S.E.). B, COS-7 and COS-7/uPAR cells were
treated first with PD098059 or vehicle and then with DIP-uPA (10 nM) for 15 min. Cells were allowed to migrate through
serum-coated Transwell membranes for 6 h. Migration is expressed
as the percentage of that observed with untreated COS-7 cells
(mean ± S.E., n = 4). C, COS-7 and
COS-7/uPAR cells were transfected to express N17Rac1 or with empty
vector. All cells were co-transfected with pEGFP to express GFP. After
24 h, cells were pre-incubated with DIP-uPA, as indicated, and
allowed to migrate through serum-coated Biocoat cell-culture inserts.
Cell migration was quantitated by counting green-fluorescing cells, and
is expressed as the percentage of that observed with COS-7 cells that
were transfected to express GFP only (mean ± S.E.,
n = 4).
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We hypothesized that uPAR overexpression promotes cell migration by its
effects on Rac1 activation, which is independent of EGFR, and that uPA
and EGF stimulate cell migration by their effects on ERK activation,
which is EGFR-dependent. To test this hypothesis, we
examined COS-7/uPAR cell migration after treatment with the mitogen-activated protein kinase kinase inhibitor, PD098053 (50 µM). As shown in Fig. 3B, PD098059 blocked the
effects of uPA on COS-7/uPAR cell migration without affecting the basal
level of migration of these cells. To examine the role of Rac1, we
transfected COS-7/uPAR cells to express dominant-negative Rac1
(N17Rac1). The cells were co-transfected with pEGFP, so that migration
of N17Rac1-expressing cells could be determined by counting fluorescent cells. As shown in Fig. 3C, N17Rac1 neutralized the increase
in cell migration that was associated with uPAR overexpression in COS-7/uPAR cells, consistent with our model. uPA failed to stimulate migration of N17Rac1-expressing cells, suggesting that ERK activation may not promote cell migration when the constitutive activity of Rac1
is neutralized. A similar relationship was recently defined, regarding
ERK and RhoA in uPA-stimulated MCF-7 cell migration (44).
An EGFR-independent Pathway for ERK Activation by uPA--
CHO-K1
cells express cell-surface uPAR but not EGFR (45). Thus, we chose this
cell line to further probe the dependence on EGFR for uPAR-signaling to
ERK. CHO-K1 cells were transfected to express wild-type EGFR or a
kinase-inactive mutant of the EGFR. Expression was confirmed by
immunoblot analysis (results not shown). As shown in Fig.
4A, EGF did not stimulate ERK
activation in the parental cells, as anticipated because of the lack of
EGFR. Cells that were transfected to express KI-EGFR also failed to
respond to EGF. By contrast, CHO-K1 cells that expressed wild-type EGFR demonstrated significant ERK phosphorylation in response to EGF.

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Fig. 4.
uPA activates ERK by an EGFR-independent
pathway in CHO-K1 cells. A, CHO-K1 cells were transfected to
express wild-type EGFR (WT-EGFR) or KI-EGFR.
These cells were treated with EGF (10 ng/ml) for 5 min. Phosphorylated
and total ERK were then determined. B, CHO-K1 cells were
treated with tyrphostin AG1478 or vehicle for 2 h and then with
DIP-uPA (10 nM) for 10 min, as indicated. Phosphorylated
and total ERK were then determined. C, CHO-K1 cells that
expressed wild-type EGFR (WT) or KI-EGFR (KI)
were treated with tyrphostin AG1478 for 2 h and then with DIP-uPA
for 10 min, as indicated. Phosphorylated and total ERK were then
determined.
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DIP-uPA stimulated ERK activation in the parental CHO-K1 cells, despite
the lack of EGFR (Fig. 4B). Thus, we hypothesized that these
cells use an EGFR-independent pathway to couple uPAR to ERK. To rule
out the possibility that trace levels of EGFR are responsible for the
activation of ERK in uPA-treated CHO-K1 cells, we pretreated these
cells with tyrphostin AG1478; however, the EGFR kinase antagonist was
without effect.
DIP-uPA promoted ERK activation equally well in cells that were
transfected to express wild-type EGFR or KI-EGFR (Fig. 4C). Tyrphostin AG1478 inhibited ERK activation in response to uPA only in
the cells that express wild-type EGFR. Thus, whereas CHO-K1 cells
apparently have an alternative EGFR-independent pathway to couple uPAR
to ERK, this pathway is not operational when EGFR is expressed.
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DISCUSSION |
uPA-binding to uPAR activates multiple cell-signaling proteins and
systems, including focal adhesion kinase, ERK, protein kinase C
, the
Src-family tyronine kinase Hck, and the JAK/STAT pathway (17, 18).
Vitronectin binding to uPAR activates Rac1 (16). Various adaptor
proteins that associate with uPAR in the plasma membrane have been
implicated as transducers of uPAR-signaling across the plasma membrane.
FPRL1/LXA4R has been implicated in signaling to Hck (35), gp130 in
signaling to JAK1 (34), and EGFR in signaling to ERK (36). An important
question that arises is whether these adaptor proteins are truly
compartmentalized in function, transmitting uPAR signals to some
signaling pathways but not to others. In this case, how a cell responds
to uPA may be determined by the composition of the uPAR-signaling
receptor complex. Furthermore, the composition of the uPAR-signaling
receptor complex may be dynamic and thus altered by changes in membrane protein expression or catabolism.
In this study, our goal was to elucidate the function of the EGFR in
uPAR-initiated cell signaling. Our results provide evidence for
compartmentalized function of uPAR-associated adaptor proteins in cell
signaling. In a single cell type (COS-7), the EGFR was essential for
transmitting uPAR signals to ERK but not Rac1. The branch point remains
to be determined. In epidermoid carcinoma cells, EGFR was activated
downstream of uPAR-integrin complex and focal adhesion kinase; however,
EGFR was recovered in complex with uPAR and
5
1, so these reactions probably occurred
within the context of a single multicomponent complex (36). Rac1
activation may occur downstream of tyrosine kinases and G
protein-coupled receptors and require the activity of
phosphatidylinositol 3-kinase or p130Cas (46-49). Rac1 is also
activated downstream of integrins (50). Thus, the integrin-uPAR complex
may signal directly to Rac1.
In CHO-K1 cells, EGFR is not expressed (45). We demonstrated that EGF
does not activate ERK in CHO-K1 cells, as anticipated because of the
lack of receptor; however, uPA did activate ERK. Thus, an
EGFR-independent pathway exists for transmitting signals from uPAR to
ERK in CHO-K1 cells and possibly in other cells as well. In MCF-7
cells, focal adhesion kinase, c-Src and Shc function upstream of Ras in
the pathway that couples uPAR to ERK (26). These same factors may
directly couple integrins to ERK, without the EGFR as a necessary
intermediate (51, 52). Thus, uPAR and integrins may form an adequate
signaling complex to activate the Ras-ERK pathway in the absence of
EGFR. It is also possible that when EGFR is not expressed, an
alternative adaptor protein associates with the uPAR multiprotein
signaling-receptor complex. This alternative adaptor protein could be
another member of the EGF receptor family or an unrelated membrane
protein. In this regard, tyrphostin AG1478 is specific for EGFR
(53).
An interesting and potentially important observation concerns the
effects of EGFR expression on the response of CHO-K1 cells to uPA. An
obvious change in the magnitude or kinetics of ERK activation in
response to DIP-uPA was not observed; however, the response became
sensitive to tyrphostin AG1478. This result suggests that when EGFR is
expressed, it associates with uPAR in the plasma membrane and assumes a
dominant role in the uPA-dependent signaling complex that
activates ERK. The inability of uPA to activate ERK, in EGFR-expressing
CHO-K1 cells, when these cells are pretreated with tyrphostin AG1478,
suggests that EGFR silences the alternative pathway. One explanation
for these data is that EGFR displaces an alternative adaptor protein
from the uPAR signaling receptor complex; however, if this model is
correct, we would expect KI-EGFR to inhibit uPAR-signaling to ERK,
which was not observed. The inability of KI-EGFR to silence the
alternative pathway may reflect conformational variation in the mutant
receptor so that it does not associate with uPAR, or possibly
insufficient expression in our experiments.
From these studies, we propose that cell signaling initiated from the
uPAR-containing multiprotein complex is compartmentalized. By this we
mean that different proteins that are associated with uPAR may be
responsible for triggering different signaling pathways. Furthermore,
we hypothesize that the uPAR signaling-receptor complex is dynamic.
When a protein like EGFR is expressed in cells, it may enter the
complex and alter its properties. This model provides an explanation
for the diversity of adaptor proteins shown by others to facilitate
uPAR signaling. This model also provides a possible explanation for
differences in signaling responses observed in different cell types.
In processes such as breast cancer, the EGFR and uPAR are both
important determinants of disease progression. Biochemical and
functional interactions between these two receptors, described here and
elsewhere (36), raise the possibility that extensive cross-talk may
occur in cancer cells. Understanding this cross-talk is an important
goal for the future.