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INTRODUCTION |
During development of the retinotectal projection, members of the
Eph family of receptor tyrosine kinases and their "ligands", the
ephrins, are strongly involved in guiding retinal axons to, and in, the
tectum (for review see Refs. 1-3). Besides the graded expression of
Eph receptors on retinal axons and of ephrins in the tectum, the
ephrin-As are also differentially expressed on retinal axons themselves
(4, 5). Gain of function and loss of function analyses suggest that
here the ephrin-As modulate the function of the coexpressed receptors
in that coexpression of ligands and receptors runs in parallel to a
decrease in sensitivity for the repellent activity of the tectally
expressed ephrins (4, 5). These findings fit to subsequent results of
in vivo analyses of ephrin-A5 single and
ephrin-A5;ephrin-A2 double knockout mice (6).
A hint of how ephrin-A functions on retinal axons may be provided by
the concept that these ligands are localized, because of their membrane
attachment by a glycosylphosphatidylinositol (GPI)1 anchor, to so-called
rafts (7-12). rafts are small dynamic microdomains in the membrane
with a special glycosphingolipid and cholesterol composition, which
have been proposed, besides having other functions, to serve as
localized platforms for signal transduction (13).
GPI-anchored proteins are bound to the outer leaflet of the raft
membranes (with no direct contact to the cytosol), whereas signaling
molecules such as members of the src family are localized to the inner
leaflet of rafts. In fact, "activated" GPI-anchored proteins can
transduce signals to the cytosol, leading, for example, to changes in
intracellular Ca2+ concentrations and activation of
specific signaling pathways. This signaling might occur through
formation of larger raft domains triggered by clustering of
GPI-anchored proteins, which allows an interaction of signaling
molecules normally separated through direct interactions between raft
lipids or through (regulated) association of GPI-anchored molecules
with appropriate transmembrane (co) receptors (9, 14). For example, the
GPI-anchored cell adhesion molecule contactin/F11 associates with the
transmembrane protein CASPR (15), representing a coreceptor involved in
signal transduction after contactin/F11 stimulation. Other examples of GPI-anchored proteins capable of mediating signals include Thy-1 (16),
CNTF (17), GDNF (18), and neurturin (19).
Simons and coworkers (7) have put forward the idea that a
fundamental principle guiding the way raft microdomains exert their
function in signal transduction is the (regulated) separation of
different membrane proteins (7, 20). According to this concept,
GPI-anchored molecules and other specific sets of membrane proteins
have a strong tendency to associate with these domains, whereas others,
such as molecules with a transmembrane domain, rarely appear in these areas.
Recently, it was shown that activation of ephrin-A5 results in an
increase in adhesion of the ligand-expressing cells and in the level of
tyrosine phosphorylation of molecules with molecular masses of
75-80 and 60 kDa (21). These changes, however, were not specific for
an ephrin-A activation and were also seen after incubation of these
cells with, for example, basic fibroblast growth factor (bFGF)
(22).
Here we show that binding of the ephA3 receptor to ephrin-A2 and
ephrin-A5 leads to an activation of the integrin system, as seen in a
1-integrin-dependent increase in adhesion of the ephrin-A-expressing cells. Activation of ephrin-As and changes in
adhesion correlate well with the tyrosine phosphorylation of a protein
with a molecular mass of 120 kDa, which is observed only after ephrin-A
activation. p120 thus might represent a component of a new
ephrin-A-dependent signaling pathway functionally linking ephrin-As to integrins.
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EXPERIMENTAL PROCEDURES |
Establishment of ephrin-A-expressing Cell Lines--
HEK293 cell
clones stably expressing chick ephrin-A2 or the long or the short form
of mouse ephrin-A5 were established according to standard protocols. In
brief, about 2 × 106 HEK293 cells were transfected
with 25 µg of the respective pClNeo-based expression plasmids and
were selected for about 2 weeks in standard medium (Dulbecco's
modified Eagle's medium with 10% fetal calf serum and antibiotics)
containing 400 µg/ml G418 (PAA Laboratories). Then individual
clones were isolated, expanded, and analyzed for ephrin-A expression
using ephA3-AP staining and later, Western blots. Those cell
clones most strongly expressing ephrin-As were further expanded and
used in this study.
Monoclonal Antibodies and Reagents--
ephA3-AP and AP were
produced as described previously (23, 24); protein G-agarose is from
Roche Molecular Biochemicals. Antibodies against chicken
ephrin-A2 were described by Hornberger et al. (4),
monoclonal antibodies to phosphotyrosine (clone 4G10), the
1-integrin-neutralizing antibody (clone DE9), and anti-human fyn
(rabbit whole serum, used for immunoprecipitation) were from Upstate
Biotechnologies. Monoclonal antibodies to fyn and caveolin-2 were from
Transduction Laboratories, and the horseradish peroxidase-conjugated
secondary antibodies were from Dianova.
Activation of ephrin-As by ephA3--
About 75% confluent
HEK293 cell clones expressing the indicated ephrin-As were starved for
36 h in serum-free RPMI medium, detached from the dishes with
prewarmed PUCKS-EDTA (5 mM KCl, 130 mM
NaCl, 3 mM NaHCO3, 5 mM
D-glucose, 10 mM HEPES, pH 7.0, 0.1 mM EDTA), pelleted at 1000 rpm for 5 min, washed once with serum-free RPMI medium, pelleted again, and resuspended in serum-free RPMI medium containing either 10 nM ephA3-AP or 10 nM AP. Then the cells were incubated at 37 °C for the
indicated times, placed immediately on ice, and washed once with
ice-cold PBS containing 1 mM
Na3VO4. Subsequently the cells were solubilized
at 4 °C for 10 min in Triton X-100 lysis buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM iodoacetamide, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 0.5% Triton X-100, 20 µM aprotinin, 50 microunits of leupeptin, 2 µM pepstatin, 1 mM
Na3VO4). This lysate was used for Optiprep
density gradient ultracentrifugation.
Purification of Detergent-insoluble Glycolipid-enriched Domains
by Optiprep Density Gradient Ultracentrifugation--
The cell lysate
was adjusted to 35% (v/v) in Optiprep (Nycomed, Oslo, Norway).
0.96 ml of this solution was overlaid with 3 ml of 30% Optiprep in
lysis buffer, 0.5 ml of 10% Optiprep in lysis buffer, 0.4 ml of lysis
buffer and was centrifuged at 42 krpm for 16 h at 4 °C in an SW
50.1 rotor. Subsequently, 0.6-ml fractions were taken from the top. The
second fraction containing the white blur band between 30 and 10%
Optiprep was collected as caveolin fraction, and the last fraction was
collected as the soluble fraction (see also Ref. 21). The various
fractions were extracted with 1% Nonidet P-40 for 5 to 10 min at room
temperature and then precipitated with 10% trichloroacetic acid on ice
for 15 min and spun down at 14 krpm for 15 min at 4 °C. The pellet was washed with 80% acetone at room temperature for 10 min and spun
down again at 14 krpm for 15 min at 4 °C. The supernatant was
discarded, and pellets were air-dried for further experiments such as
Western blot analysis.
Immunoprecipitation and Immunoblotting--
The "caveolin"
fraction was diluted 1:3 with 0.5% Triton X-100 lysis buffer
containing protease inhibitors and was then precleared with appropriate
amounts of protein G-agarose for 2 h at 4 °C under rotating
conditions. Immunoprecipitations were done by incubating the precleared
lysate with antibody-conjugated protein G-agarose for at least 4 h
at 4 °C under rotating conditions. Subsequently the
immunoprecipitate was centrifuged and washed three times with lysis
buffer. SDS polyacrylamide gel electrophoresis and immunoblotting were
done according to standard protocols.
Adhesion Assay--
96-Well microtiter plates were coated with
serially diluted (from 20 to 0.625 µg/ml in PBS) laminin or
poly-L-lysine overnight at 4 °C and washed once with
PBS. Nonspecific binding sites were blocked at room temperature for
1 h using 1% bovine serum albumin/PBS. The starved cells (36 h)
were detached from the dishes with prewarmed PUCKS-EDTA, washed once,
and resuspended in serum-free RPMI medium. ephA3-AP or AP was added to
a final concentration of 10 nM. Subsequently, about 2 × 105 cells in 100 µl were plated per well (in
triplicates) and incubated at 37 °C for 30 min. Then the medium was
discarded, and remaining cells were washed once with PBS. Subsequently
the cells were fixed with 0.5% paraformaldehyde/0.5% glutaraldehyde
at room temperature for 30 min and stained with 0.5% crystal violet in
20% methanol at room temperature for 10 min. The cells were washed
three times with H2O and were extracted with 50%
ethanol/50 mM sodium citrate, pH 4.5 (50 µl/well).
Absorbance was measured at 550 nm. Data shown in Figs. 1 and 6 were
calculated on the basis of two to three independently performed experiments.
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RESULTS |
Increased Adhesion of ephrin-A2-expressing Cells after Activation
by Its Receptor--
To characterize a possible receptor or signaling
function of the GPI-anchored ephrin-A ligands, we established a HEK293
cell line stably expressing ephrin-A2. As the ephA system has been shown to be involved for example in the regulation of cell migration (25), we investigated whether treatment of these cells with their
receptor changes the adhesive properties of these cells to laminin. For
an activation we used ephA3-AP or AP (the latter serving as a negative
control). ephA3-AP is a fusion protein of the extracellular part of the
ephA3 receptor (known to bind strongly to ephrin-A2 (23)) and alkaline
phosphatase. As alkaline phosphatase itself oligomerises, ephA3-AP,
too, is in an oligomeric form (24) and thus might represent the
"active" form of the receptor.
As shown in Fig. 1A,
incubating HEK293-ephrin-A2 cells with ephA3-AP, but not AP, led to a
significant increase in adhesion to laminin but not to
poly-L-lysine, which indicates an activation of the
integrin system. To confirm that the increase in adhesion is indeed
dependent on the presence of ephrin-A ligands, we performed this assay
also using the parental cell line HEK293. Here we did not observe any
increase in cell adhesion after ephA3-AP treatment (data not shown). To
investigate the role of integrins directly, we performed the adhesion
assay in the presence of a function-blocking monoclonal antibody to
1-integrin, used at a concentration of 10 µg/ml (see
"Experimental Procedures"). Here we observed a strong reduction in
the ephA3-induced increase in adhesion, whereas a control antibody used
at the same concentration did not affect this increase (Fig.
1B). Thus, activation of ephrin-As by their receptors leads
to an activation of the integrin system.

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Fig. 1.
Increased adhesion to laminin after
stimulating HEK293-ephrin-A2 cells with ephA3.
ephrin-A2-expressing cells were starved for 36 h, detached from
the culture dishes, and plated in the presence of 10 nM
ephA3-AP or 10 nM AP on dishes coated with increasing
concentrations of laminin (0.625-20 µg/ml) or
poly-L-lysine (0.625-20 µg/ml). 30 min later unbound
cells were washed away. The number of adherent cells was determined
using a colorimetric assay (see "Experimental Procedures").
y axis, number of adherent cells in arbitrary units.
A, activation of ephrin-A2 with ephA3-AP results in a
specific increase in adhesion to laminin, but not to
poly-L-lysine (PLL), when compared with cells
incubated with AP. B, the increase in ephA3-AP-mediated
adhesiveness can be blocked by incubation with a neutralizing antibody
to 1-integrin. Here ephrin-A2-expressing cells were incubated in the
presence of ephA3-AP either with an anti- 1-integrin antibody (10 µg/ml) or with a control antibody (anti-human Fc; 10 µg/ml).
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ephrin-A2 Is Associated with raft Microdomains--
To understand
the signaling function of ephrin-A ligands more closely, we were
interested in identifying those proteins that link stimulated ephrin-A
ligands to the integrin system. As ephrin-A molecules are GPI-anchored,
they possess no direct link to the cytosol and thus might require a
"coreceptor" for exerting such a signaling function. GPI-anchored
molecules are found in so called rafts, which are believed to represent
platforms in which molecules involved in signal transduction are
concentrated, thus a coreceptor might colocalize here with
ephrin-As.
In initial experiments we investigated whether ephrin-A2 is indeed
associated with rafts. A criterion for the association of a particular
protein with rafts is the presence of that protein in
detergent-insoluble glycolipid-enriched domains, which can be isolated
because of their high lipid content by sucrose gradient centrifugation
(26). Thus ephrin-A2-expressing cells were subjected to an Optiprep
(sucrose) step gradient centrifugation, and individual fractions were
subjected to Western blot analysis using monoclonal antibodies to
ephrin-A2 and caveolin-2. Caveolins (27) are proteins that are often
found associated with these rafts and might function in stabilizing the
raft domains (13). These analyses showed that ephrin-A2 is contained in
the same fractions as caveolin-2, indicating that ephrin-A2 is indeed
localized to rafts. Fractions containing soluble proteins neither
contained ephrin-A2 nor caveolin-2 (Fig.
2, A and C). This
purification step considerably increased the sensitivity of our
analyses, as the majority of cell proteins was contained in the nonraft
fractions (data not shown).

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Fig. 2.
Analysis of the tyrosine phosphorylation
pattern of raft fractions after activating ephrin-A2 by ephA3. A
fibroblast cell line stably expressing ephrin-A2 (HEK293-ephrin-A2) was
serum-starved for 36 h, followed by an incubation with 10 nM ephA3-AP or 10 nM AP for the indicated times
(0, 30, and 60 min). Subsequently raft fractions (C), as
well as fractions containing soluble proteins (S), were
isolated by Optiprep centrifugation and subjected to Western blot
analysis using the anti-phosphotyrosine-specific antibody 4G10
(A). Then the filter was stripped and reprobed with
antibodies specific for (B) fyn and (C)
caveolin-2 (cav-2). -Cav-2 antibodies were used to show a
separation of raft-associated proteins versus soluble
proteins and also to verify that similar amounts of the raft fractions
had been loaded. Molecular mass markers (in kDa) are indicated to the
left. Labelings to the right highlight the
tyrosine phosphorylation patterns of p120, p80, and src family kinases
at roughly 60 kDa. Whereas the tyrosine phosphorylation of p120 is
transiently up-regulated, the phosphorylation of p80 is strongly
reduced after ephrin-A activation. Apparently neither the concentration
nor the phosphorylation level of fyn is changed (A and
B). D, to confirm the identity of fyn, the raft
fraction was immunoprecipitated (IP) using an anti-fyn
antibody and was then probed with the anti-phosphotyrosine antibody
4G10 (left) and, after stripping, with a different fyn
antibody (right). The left lanes in each figure
show the raft fraction before immunoprecipitation.
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Activation of ephrin-A2 Leads to Changes in the Tyrosine
Phosphorylation Pattern of raft Proteins--
To identify proteins
that might be components of an ephrin-A2 signaling pathway, we compared
the raft fractions from HEK293-ephrin-A2 cells after incubation with
either ephA3-AP or with AP. Using Western blot analysis we focused in
particular on changes in tyrosine phosphorylation levels using
phosphotyrosine-specific monoclonal antibodies. Before activation,
cells were serum-starved for 36 h to reduce the considerable level
of tyrosine phosphorylation of membrane proteins, which is evident for
cells grown under standard conditions with serum.
As shown in Fig. 2, we indeed found such changes in the tyrosine
phosphorylation pattern of proteins contained in the raft fractions
after activating ephrin-A2-expressing cells with ephA3-AP but not with
AP. In particular, a protein of roughly 120 kDa, which is only slightly
tyrosine phosphorylated in the uninduced state, becomes strongly
phosphorylated after 30 min, and later, after about 60 min, returns to
a close to initial phosphorylation level. This protein was not detected
in the fractions containing soluble proteins but in the same fraction
as caveolin-2, suggesting that it represents a molecule specifically
associated with raft fractions (Fig. 2). Additionally, proteins with a
molecular mass of 70-80 kDa, which were highly tyrosine-phosphorylated
before induction, became dephosphorylated after ephA3-AP treatment,
with an almost complete dephosphorylation after 60 min (for simplicity, this set of proteins will be referred to in the following
paragraphs as p80). These proteins also were specifically
contained in the raft fraction. Thus, there are (at least) two proteins
whose tyrosine phosphorylation levels were changed, in opposite
directions, after activation of ephrin-A2. However, we can not exclude
the possibility that the increase in p120 tyrosine phosphorylation is
because of a recruitment of p120 to the raft domains after ephrin-A2
stimulation. Similarly, the down-regulation of p80 phosphorylation
might be because of a lower concentration of p80 in rafts after
ephrin-A2 activation.
In addition, we observed a doublet of two strongly
tyrosine-phosphorylated proteins, whose phosphorylation pattern did not change after ephA3-AP treatment (Fig. 2). Reprobing filters with an
anti-fyn antibody (Fig. 2) resulted in the appearance of a band at
exactly the same position as after probing the filter with the
phosphotyrosine-specific antibody, suggesting that one of these bands
corresponds to the nonreceptor tyrosine kinase fyn (Fig.
2B). This was confirmed in an additional set of experiments. Here the raft fraction of ephA3-AP-stimulated cells was
immunoprecipitated using an anti-fyn polyclonal serum and subsequently
analyzed in a Western blot using firstly an anti-phosphotyrosine
antibody and secondly, after stripping of the filter, an anti-fyn
monoclonal antibody (Fig. 2D). Also, our analyses indicated
that neither the amount nor the phosphorylation level of fyn was
changed after activation of ephrin-A2 (Fig. 2, A and
C). A rehybridization of the filters with an antibody
against caveolin-2, as shown in Fig. 2B, demonstrated that
similar amounts of raft proteins have been loaded.
Subsequently, we investigated in more detail the temporal changes that
took place in the level of tyrosine phosphorylations of p120 and p80
after treating cells with ephA3-AP. As shown in Fig.
3, p120 becomes significantly
tyrosine-phosphorylated only after about 15 min, thus with an apparent
lag phase of about 10 min. The tyrosine phosphorylation remains at a
high level for about 30 min and then decreases, leading to a faint
signal 60 min after the start of ephrin-A2 activation. The
dephosphorylation of p80 started earlier, that is already 5 min after
ephA3-AP treatment, a significant drop in the level of tyrosine
phosphorylation is apparent and with time, leads to an almost complete
dephosphorylation. The expression level and tyrosine phosphorylation
level of fyn was not affected within the time window investigated (Fig.
3C). We also verified that, even after a 60-min incubation
with ephA3-AP, the concentration of ephrin-A2 associated with the raft
fraction is not changed (Fig. 3D).

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Fig. 3.
Temporal changes in the tyrosine
phosphorylation of proteins localized in raft fractions after
activation of ephrin-A2 by ephA3. Serum-starved
HEK293-ephrin-A2 cells were incubated for the indicated times with
ephA3-AP. Subsequently raft fractions were isolated and subjected to
Western blot analysis. A, tyrosine phosphorylation patterns
using the anti-phosphotyrosine-specific monoclonal antibody 4G10,
(B) fyn, (C) cav-2, and (D) ephrin-A2.
15 min after start of activation with ephA3, there is a marked increase
in the tyrosine phosphorylation of p120, which, however, is again
reduced after 60 min. The tyrosine phosphorylation of p80 is
significantly decreased already after 5 min. The decrease in the
tyrosine phosphorylation of a protein of roughly 45 kDa is observed
also after incubation with AP alone (see Fig. 2). E, cell
adhesion of HEK293-ephrin-A2 cells measured at different times of
activation by ephA3-AP (right bars) or AP (left
bars).
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When analyzing the temporal changes in cell adhesion after ephrin-A2
activation (Fig. 3E), we found that the increase in adhesion was observed only after a lag phase of at least 20 min, that is the
increase in adhesion is seen clearly only after the increase in p120
tyrosine phosphorylation has occurred.
In an attempt to determine the identity of p120, we have probed the
cells using ephrin-A5-AP or ephrin-A2-AP, and we have analyzed the raft
fraction with selected antibodies. However, results from these
experiments ruled out the possibility that p120 corresponds to an ephA
receptor or focal adhesion kinase (data not shown).
Specificity of the Activated Signaling Pathway--
Next we asked
whether the changes in phosphorylation observed were specific for
ephrin/Eph interactions or whether other proteins such as growth
factors also induce these or similar changes in tyrosine
phosphorylation. For this purpose, we analyzed a number of different
signaling molecules known to affect the tyrosine phosphorylation
patterns of cellular proteins, such as epidermal growth factor (EGF),
insulin growth factor (IGF), and bFGF. We also included fetal calf
serum (FCS) in our analysis, which contains a large variety of
different growth factors. We observed a decrease in the phosphorylation
of p80 after FCS treatment; however, none of these factors led to
changes in the tyrosine phosphorylation pattern of p120 comparable with
those caused by ephA3-AP (Fig. 4).

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Fig. 4.
The tyrosine phosphorylation of p120 is
specifically induced by an ephrin-A2/ephA receptor interaction.
After serum starvation, ephrin-A2 expressing cells were incubated for
30 min with bFGF (20 ng/ml), EGF (20 ng/ml), IGF (50 ng/ml), 10% FCS,
or 10 nM ephA3-AP (the bFGF, EGF, and IGF concentrations
used are at the upper limit of working concentrations according to the
instructions of the suppliers). Subsequently, raft fractions from these
cells were isolated and analyzed by Western blot using an
anti-phosphotyrosine-specific antibody. Only ephA3-AP induced the
tyrosine phosphorylation of p120, whereas both ephA3-AP and FCS
treatment result in a down-regulation of p80 tyrosine phosphorylation.
In a control the tyrosine phosphorylation pattern before incubation
with ephA3-AP treatment is shown.
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Activation of ephrin-A5 Leads to an Activation of the Same
Signaling Pathway--
We then wanted to know whether the observed
changes in tyrosine phosphorylation can be observed also for other
ephrin-A ligands. We established HEK293 cells stably expressing either
the long or the short splice form of ephrin-A5 (28) and performed the same experiments as described above for ephrin-A2. Indeed, after activation by ephA3-AP, both ligands induced the down-regulation of p80
phosphorylation and the transient up-regulation of p120 phosphorylation (Fig. 5).

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Fig. 5.
The long and short splice forms of ephrin-A5
induce the same changes in tyrosine phosphorylation of raft proteins as
ephrin-A2. HEK293 cell lines stably expressing either the long or
the short splice form of ephrin-A5 were serum-starved for 36 h and
were subsequently treated with 10 nM ephA3-AP for the
indicated times. raft fractions were isolated and subjected to Western
blot analysis using the phosphotyrosine-specific antibody 4G10. Similar
to the results obtained with the HEK293-ephrin-A2 cell line (see Figs.
2 and 3), there is a transient increase in the amount of
tyrosine-phosphorylated p120 and a rapid decrease in the tyrosine
phosphorylation of p80. There is no apparent difference between the two
different ephrin-A5 splice forms with respect to tyrosine
phosphorylation of raft proteins.
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Blocking Tyrosine Phosphorylation of p120 Abolishes the Increase in
Cell Adhesion--
Our next step was to investigate more closely the
inter-relationship between ephrin-As and integrins. A number of
molecules are known to be involved in the regulation of integrin
signaling, and we were interested in whether signaling pathways used in
ephrin-A and integrin signaling share common members. We concentrated
on src family kinases, which are known to be involved in integrin signaling and are required for an optimal adhesion efficiency but
appear not to be essential for these events (29-33). We used the
pyrazolopyrimidine PP2, which selectively inhibits src family kinases
(34). This inhibitor and a closely related molecule, PP1, have both
been used in a number of investigations to show an involvement of src
family kinases in different developmental processes (21, 35, 36).
Treating the ephrin-A2-expressing cells with 10 µM PP2
alone, thus without stimulation by ephA3-AP, led to only a small
reduction in the adhesiveness of these cells (Fig.
6). However, the strong increase in
adhesion typically seen after ephA3-stimulation (see Figs. 1 and 6) was
prevented by PP2, i.e. by blocking src family kinases. The
incubation in the presence of 10 µM PP3, which is a
structurally closely related but functionally inactive compound to PP2,
had no effect on the increase in cell adhesion (Fig. 6).

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Fig. 6.
Blocking src family kinases greatly reduces
the increase in cell adhesion seen after stimulating
ephrin-A2-expressing cells with ephA3-AP. Serum-starved ephrin-A2
expressing cells were treated for 4 h with PP2 or PP3. Then the
cells were detached from the culture dishes and plated in the presence
of ephA3-AP or AP on dishes coated with increasing concentrations of
laminin (0.625-20 µg/ml). 30 min later unbound cells were washed
away. The number of adherent cells was determined using a colorimetric
assay (see "Experimental Procedures"). y axis, number of
adherent cells in arbitrary units. The increase in ephA3-AP mediated
adhesiveness of HEK293-ephrin-A2 cells can be inhibited by treating
cells with 10 µM PP2 (a selective src family kinase
inhibitor) but not with PP3, a very similar but functionally inactive
compound to PP2. For details see Fig. 1 and "Experimental
Procedures."
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In parallel to these binding assays, we performed Western blot analyses
of raft fractions from ephA3-AP-treated cells, which showed that PP2
pretreatment of these cells almost completely abolished the increase in
tyrosine phosphorylation of p120, whereas PP3 did not affect this
pattern (Fig. 7).

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Fig. 7.
The tyrosine phosphorylation of p120 is
abolished after blocking src family kinases. A,
Serum-starved HEK293-ephrin-A2 cells were treated for 4 h with 10 µM PP2, a selective src family kinase inhibitor.
Subsequently cells were incubated with 10 nM ephA3-AP for
the indicated periods of time. For control, a second batch of cells was
treated identically but with added PP3, a very similar but functionally
inactive compound to PP2. Then cells were lysed, and raft fractions
were isolated and subjected to Western blot analysis. It is shown here
that in the presence of PP2, but not PP3, the increase in tyrosine
phosphorylation of p120 is almost completely abolished. B,
to indicate that similar amounts of raft proteins have been loaded, the
stripped filter was reprobed with an anti-caveolin-2 antibody.
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DISCUSSION |
In this report we have shown that binding of ephA3 to
ephrin-A-expressing cells leads to an activation of the integrin system and an increased tyrosine phosphorylation of a 120-kDa raft protein. Both the increase in adhesion and in tyrosine phosphorylation of p120
can be abolished by inhibiting the function of src family kinases.
Besides p120, we identified a set of proteins having a
molecular mass of about 80 kDa, whose tyrosine phosphorylation was
down-regulated after ephrin-A stimulation.
Signaling Complexes in raft Microdomains--
It has been proposed
that raft microdomains serve as platforms in the membrane, within which
molecules involved in signaling to the cytosol are organized (7, 12,
13). Both GPI-anchored ephrin-A ligands and B-class ephrins, which are
transmembrane-anchored, are found in these structures. Whereas
A-ligands appear to be constitutively associated with these rafts, the
incorporation of B-ligands in these structures might be regulated, as
ephrin-Bs without their cytoplasmic tail no longer associate with rafts (37), suggesting an active (regulated) transport of B-ephrins into and
out of rafts. Interaction of ephB receptors with their ligands results
in the phosphorylation of highly conserved tyrosines in the
intracellular domain of the ligands, which then might serve as binding
sites for other signaling molecules (37-41).
After stimulation of ephrin-A2-expressing cells with ephA3-AP, we
observed an increase in cell adhesion, which could be abolished by
using a neutralizing monoclonal antibody to
1-integrin, indicating the existence of an ephrin-A-dependent signaling pathway
controlling integrin function. The mechanism by which ephrin-As exert
this function, e.g. signal into the cytosol, is poorly
understood. It is quite possible that a communication between ephrin-As
(bound to the outer leaflet of the membrane) and intracellular
signaling molecules such as lipidated src family kinases (bound to the
inner leaflet of the membranes) is mediated by (conformational changes of) transmembrane proteins. The proteins we have identified on the
basis of their presence in raft membranes and their differential tyrosine phosphorylation pattern in response to ephrin/Eph interactions represent candidates for such a role. p120 is only slightly
phosphorylated in the unstimulated state but becomes strongly
tyrosine-phosphorylated after ephrin-A activation. We cannot exclude
the possibility that the increase in tyrosine phosphorylation of p120
is because of a recruitment of already tyrosine-phosphorylated p120 to
the raft domains. However, we think this is unlikely, because the
increase in p120 tyrosine phosphorylation can be blocked by inhibiting src family kinases. It remains to be investigated whether a
similar modification of p120 can be observed also after clustering of other GPI-anchored proteins expressed at similar levels in HEK293 cells.
It is noteworthy that the strong increase in p120 tyrosine
phosphorylation occurs only after a lag phase of about 10 min, suggesting the involvement of other signaling components. Possibly the
lag phase is correlated with changes in the structure or composition of
ephrin-A-containing raft microdomains. Thus treatment with oligomeric
ephA3-AP might lead to a clustering of ephrin-A2 and a concomitant
fusion of initially smaller raft microdomains, which results, by an
unknown mechanism, in an activation of src family kinases, p120
tyrosine phosphorylation, and integrin activation. In good agreement
with p120 playing a role in coupling ephrin-A activation to integrin
activation, we have shown here (using a specific src family kinase
inhibitor) that blocking p120 tyrosine phosphorylation correlates with
an abolishment of the increase in cell adhesion. In further support,
the increase in cell adhesion occurs subsequent to the increase in p120
tyrosine phosphorylation, which thus supports the concept that p120
phosphorylation is involved in the control of cell adhesion rather than
its being a consequence. The p80 protein might not be involved in this
process, as its dephosphorylation was observed for example also after
FCS treatment, which on the other hand does not affect cell adhesion.
Interestingly, activation of ephA receptors leads to a suppression of
integrin function and to an inhibition of cell spreading and migration
(of fibroblast cells) (42-45). Therefore, the unexpected picture
arises that activation of ephAs and ephrin-As might
contribute to the regulation of a similar intracellular signaling
pathway but in opposite directions. It appears that interaction between ephA-expressing and ephrin-A-expressing cells results in a decrease in
adhesion of the ephA-expressing cells and an increase in adhesion of
the ephrin-A-expressing cells. It will be of interest to investigate the behavior of cells expressing both ephrin-As and ephAs
simultaneously and to study the responses of an activation of ephAs or
ephrin-As or both. The physiological meaning of these regulations
in vivo is not known, but understanding this process might
contribute to an understanding of the development of the retinotectal
projection, where on subpopulations of retinal axons ephrin-As and
ephAs are coexpressed.
Specificity of the Tyrosine Phosphorylation of p120 and
p80--
Recently, in an approach similar to the one used here, Davy
et al. (21) identified a number of proteins whose tyrosine
phosphorylation level increased after binding of ephA5-Fc to an
ephrin-A5-expressing cell line. They found most prominently a
75-80-kDa protein, which became highly phosphorylated after ephrin-A2
treatment. However, stimulation with bFGF induced the tyrosine
phosphorylation of the same protein, which indicates the activation of
a more general, but not ephrin-A-specific, signaling pathway. The
tyrosine phosphorylation level of the p70-80 protein we identified was
quickly down-regulated after ephrin-A activation and was also not
specific for an ephrin-A signaling pathway. Thus, it is unclear whether
these two sets of proteins are the same, albeit of a rather similar,
molecular weight. Overall, although the approaches of Davy et
al. (21) and our own group appear to be similar, there are some
differences in the experimental set-up, like the use of serum-starved
cells, and these might explain the different results observed. A
molecular identification of, for example, the 80-kDa protein/s will
help answer this puzzle.
In contrast, the up-regulation of tyrosine phosphorylation of p120 was
specific for the ephrin/ephA receptor interaction within the limits of
molecules tested here. Thus, it was not seen after treatment of cells
with growth factors such as bFGF, IGF, or EGF or with fetal calf serum,
which contains a large variety of different growth factors. A
characterization of a 120-kDa protein was not reported by Davy et
al. (21). The precise role of p120 in the process linking ephrin-A
activation to integrin activation is presently unclear, thus further
experiments will be directed toward a more detailed characterization of
this molecule.