(Received for publication, February 27, 1995; and in revised form, May 1, 1995)
From the
Integrins bind extracellular matrix and transduce signals
mediating cell adhesion, spreading, and migration. It is unclear how
these distinct responses follow from a common event: integrin
clustering. We examined the relationship between integrin-mediated
signals and the integrin's activation state using a cell line
expressing Integrins comprise a large family of heterodimeric cell surface
receptors involved in cell-matrix and cell-cell interactions. These
receptors mediate adhesion and modulate the cell's responses to
various adhesive ligands (for review, see (1) ). The integrins
role in modulating information flow recently has become a field of
growing interest. Several potentially significant biochemical changes
are known now to be regulated by adhesion events involving integrins,
including changes in intracellular pH, changes and oscillations in
intracellular free calcium, and phosphorylation on tyrosine of a number
of proteins (2, 3, 4, 5, 6) (for
review, see (7) ). One of these latter is itself a protein
tyrosine kinase known as focal adhesion kinase or
FAK In addition to
this ``outside-in'' signaling, the binding affinities and
specificities of several integrins are known to be modulated by events
inside the cell: so-called ``inside-out'' signaling or
activation (10, 11) (for review, see (12) ).
One of the best studied examples of this occurs in the case of the
platelet integrin The role of
The fact that many integrin-mediated biochemical changes can
be induced either partially or fully by clustering of specific
integrins with antibodies has given rise to a paradigm that the
biochemical signals are initiated by clustering of the receptor, as
occurs at the site of adhesion(6, 16) . However, this
simple model does not explain adequately the diverse cellular responses
that can be mediated by a single integrin. For example, focal adhesion
formation and migration would seem to require very different signals.
If a single integrin is capable of directing both, it logical to
propose that it may be able to mediate different signals. Given the
variations in activation state known to exist for
We used Clone B (5) and a panel of activating or
nonactivating mAbs to different epitopes on
Figure 1:
Cells were pretreated
with the primary antibody (9D4) for 5 min at 37 °C (time
``0'' in the blot), followed by addition of the secondary
antiserum for the indicated times. Lysates were subjected to
anti-phosphotyrosine Western blot. Arrows indicate the
prominent doublet between 120 and 130, which can be detected readily by
30 s.
Figure 11:
In a
parallel experiment to that shown in Fig. 10, lysates were
harvested from cells adhered and spread on fibrinogen, 9D4, P9 and CP8,
or from cells adhered on AP5 or LIBS6 (which also does not support
spreading), subjected to SDS-PAGE and anti-phosphotyrosine Western
blot.
Figure 2:
Both
bands of the doublet are immunoprecipitated by the anti-FAK monoclonal
antibody 2A7. Lysates of cells stimulated by antibody-mediated
clustering of
Figure 10:
Adhesion of Clone B to various
substrates. Tissue culture wells were coated with fibrinogen (A), P9 (B), or AP5 (C) and then blocked
with 1.0% BSA. Clone B cells were allowed to adhere in serum-free
medium (RPMI 1640) for 45 min. The cells adhere equally well to all
three substrates, but spread only on fibrinogen and
P9.
FAK phosphorylation was
stimulated equally well by a wide range of mAbs specific to these
integrin subunits or the
Figure 3:
FAK
phosphorylation by antibody-mediated clustering can be induced by
several anti-
All
Figure 4:
The activating, anti-LIBS antibody, AP5,
does not stimulate FAK phosphorylation. Antibody-mediated clustering of
Figure 5:
AP5 binds to
AP5 is a member of a class of
antibodies known as anti-LIBS (for ligand induced binding
site)(25, 26) . As a result, it requires the addition
of a ligand analog to stimulate high levels of binding ( (36) and Fig. 5). Although other low affinity
antibodies, such as CP8 (Table 1), can stimulate FAK
phosphorylation, it was important to exclude AP5's low affinity
as a possible explanation. Accordingly, we tested a range of
concentrations of AP5 in the presence of a cyclic RGD,
G4120(24) . Even under these conditions, AP5 did not stimulate
phosphorylation of FAK (Fig. 4, right-hand panel). The binding of AP5 to clone B was characterized by flow cytometry
with directly labeled AP5 (Fig. 5). FITC-labeled AP5 was exposed
to the cells at the indicated concentrations either in the absence (upper panel) or the presence (lower panel) of the
soluble RGD analog G4120. The inset depicts graphically the
mean fluorescence intensity for 10,000 cells, extracted from the
histograms, for each concentration of AP5. AP5 showed saturation
binding at a concentration of about 100 µg/ml in the absence of
G4120 (see inset). In the presence of G4120, saturation
occurred between 25 and 50 µg/ml. As we found for
platelets(36) , treatment of the cells with an RGD analog both
decreased the amount of the antibody required for saturation and
increased its saturation binding level. This is consistent with the RGD
analog increasing both the affinity of the interaction as well as the
number of sites detected on the surface of the cell. Therefore, in the
absence of G4120, AP5 recognized only a subset of the total
We next determined that AP5 mediated cluster formation of
Figure 6:
AP5
supports clustering of
Fig. 7shows an
example of cluster formation when a different antibody, P9, was used.
P9 is a very effective stimulator of FAK phosphorylation (Fig. 3). The treatment was carried out exactly as in Fig. 6. We have observed no differences between clusters
stimulated by AP5 and those stimulate by P9 at this gross level. Note
that the level of fluorescence in Fig. 7and Fig. 8is
similar, indicating that the binding of the two antibodies as detected
by the indirect fluorescence is similar. When cells are fixed prior to
exposure to the clustering antibodies, or exposed them at 0 °C, the
pattern of
Figure 7:
This
figure is essentially identical to Fig. 6, except that the mAb
P9 was used instead of AP5. Note that P9 stimulated phosphorylation of
FAK very efficiently (see Fig. 3).
Figure 8:
AP5 activates
We performed Western blot analysis of lysates from
cells stimulated by antibodies while adhered on polylysine and found
that the antibodies behaved identically as in solution. That is, P9
stimulated FAK phosphorylation and AP5 did not (data not shown). For
those experiments, cells were plated below confluence, such that
minimal cell-cell contact occurred. Thus, antibody-mediated clustering
of
Adhesion to solid-phase fibrinogen, which occurs
via basally active
Figure 9:
Binding of soluble fibrinogen to
antibody-activated
Our results indicate that in our transfected model system,
Clone B, clustering of The
failure of the activating antibodies to stimulate FAK phosphorylation
was not a dose effect based on three observations: 1) the low affinity,
nonactivating antibody, CP8, stimulated FAK phosphorylation ( Table 1and Fig. 11); 2) in the presence of G4120, the
affinity of the activating anti-LIBS is good; and 3) we have tested
levels of AP5 and LIBS6 binding that equal or exceed those of the
nonactivating antibodies at their optimal concentration for stimulation
of FAK phosphorylation. Although the maximal binding of all the
antibodies is similar (within 10%), we cannot exclude the possibility
that some small, but critical, population of
Finally, we report that activating antibodies adsorbed to plastic
will support adhesion of Clone B, but not spreading or FAK
phosphorylation. In contrast, nonactivating antibodies will support
adhesion, spreading, and FAK phosphorylation. Therefore, we have
established a system in which the adhesion of cells via an integrin can
be separated from spreading of cells mediated by the same integrin. We
conclude from these data that integrin-mediated cell spreading is the
result of specific signals that are not initiated following adhesion
via the antibody-activated form of Interestingly,
Clone B expresses two distinct electrophoretic variants of FAK, one at
approximately 125 kDa and another slightly greater than 130 kDa. We
have studied the upper band and found that by partial peptide mapping,
it is nearly identical to the M Shattil and
co-workers (3) have studied
Our results are consistent with those of
Shattil and co-workers(3, 15) in that adhesion to
solid-phase fibrinogen does stimulate FAK phosphorylation whereas
binding of soluble fibrinogen to antibody-activated receptor does not.
The most consistent interpretation of our data is that
Other authors have suggested that
ligand-induced binding sites are specific sites involved in post-ligand
binding interactions(26) . Since our activating antibodies are
also anti-LIBS antibodies, it is possible that they block a site
involved in interactions required to signal FAK phosphorylation. While
this model is attractive and remains tenable, we believe that it is not
the explanation for the phenomenon we report here. The antibodies that
do stimulate FAK phosphorylation do not stimulate presentation of
ligand-induced binding sites, which argues against the idea that these
sites are required for FAK phosphorylation. However, at this time it is
still possible that the three activating anti-LIBS antibodies we have
used all block some interaction required for FAK phosphorylation. We
have not detected tyrosine phosphorylation of any proteins induced by
clustering of activated A more interesting possibility, however, is
that the failure of Clone B to respond to the active form of the
receptor is related to its highly transformed nature. It has been
reported that over expression of an integrin can revert partially the
transformed phenotype of Chinese hamster ovary cells(28) . It
is possible that Clone B has lost the ability to respond to the
activated integrin as part of the progression to tumorigenesis. That
is, the activated integrin may exert the tumor-suppressive activity,
and Clone B may have overcome this suppression by losing that signaling
pathway.
There is direct evidence that on a given cell, a single
integrin can be found in discrete subpopulations. For example, AP5
detects only 5-20% of the total Other investigators have reported data that suggest that
conformation changes in the extracellular domain of
Phosphorylation of FAK appears to be a general effect of
Our data demonstrate clearly that clustering of
Very recently, Miyamoto et al.(35) have reported that for It is likely that other integrins
exist in different activation states to account for the cycles of
binding and release required for migration. We are exploring currently
the role of different activation states in other
(Clone B) and a panel
of monoclonal antibodies against this integrin. Nonactivating
antibodies used to cluster
stimulated focal adhesion kinase (FAK) phosphorylation,
regardless of affinity, subunit specificity, or ligand-blocking
phenotype. Coated on plastic, these antibodies supported cell adhesion,
spreading, and FAK phosphorylation. In contrast, clustering of
induced with activating
antibodies, or binding of soluble fibrinogen to antibody-activated
, did not induce FAK
phosphorylation. Thus, clustering of
on Clone B does not necessarily result in FAK phosphorylation.
Coated on plastic, activating antibodies supported cell adhesion, but
not spreading or FAK phosphorylation. Therefore, it appears the
resting, not the active form of
,
induces cell spreading and FAK phosphorylation in Clone B. These data
indicate that ``inside-out'' signals may alter not only the
binding specificity of an integrin, but the ``outside-in''
biochemical signals that integrin initiates as well. This activation
state-linked signaling represents a novel mechanism, which may explain
how diverse cellular responses are induced by integrin-matrix
interactions.
(
)(8, 9) .
(GPIIbIIIa).
This integrin can exist in different activation states that can be
distinguished by differences in ligand binding as well as the binding
of monoclonal antibodies (mAbs) specific for these states(13) .
In the so-called ``resting'' state,
binds solid-phase fibrinogen, but
does not bind the soluble form of fibrinogen, which is found in
abundance in plasma. A wide variety of stimuli can induce platelet
activation, which in turn results in an apparent conformation change or
``activation'' of
. This
``active'' state can bind soluble fibrinogen and other
molecules important to thrombus formation, such as von Willebrand
factor(14) . Since the resting form of the receptor is active
for binding some ligands, it may be more appropriate to describe it as
expressing ``basal'' activity.
in outside-in signaling has been
examined in platelets (see (3) and (15) , and
references therein) and in a model system in which
is expressed in 293 cells (ATCC
1573). In platelets, stimulation of
either by adhesion to fibrinogen or by antibody-mediated
clustering is associated with increases in protein-tyrosine
phosphorylation(3, 15) . Likewise, adhesion of the
transfected 293 cells, designated ``Clone B''(5) , to
solid-phase fibrinogen via
or
clustering of
with antibodies
induces very rapid protein-tyrosine phosphorylation. We have identified
FAK as one of the rapidly phosphorylated proteins resulting from
cross-linking in Clone B (see
below).
, we hypothesized that activation
state affects the outside-in signals mediated by this receptor. To test
this, we took advantage of a class of
anti-
antibodies that activate
(17) . That is, when one of
these antibodies binds, it induces a change in the receptor that is
characterized by binding soluble fibrinogen and activation-specific
antibodies. These activating antibodies provide a method for examining
the active form of the integrin without activating inside-out pathways,
which could complicate interpretation of the results(18) .
to test our hypothesis. We found
that clustering of
in the active
state, whether mediated by antibodies or by soluble fibrinogen, did not
stimulate phosphorylation of FAK. In contrast, clustering with
nonactivating antibodies did. Moreover, when plated in solid phase,
antibodies that induce the active state of
supported adhesion, but did not
induce spreading or FAK phosphorylation. In contrast, those antibodies
that did not induce the active state supported adhesion and induced
spreading and FAK phosphorylation. We therefore establish a link
between the integrin's activity state and its signaling ability.
It may be important to consider this link when defining how integrins
regulate cellular processes and when designing means of intervention
into these processes.
Cell Lines and Media
Clone B has been described
elsewhere (5) . Briefly, it is a derivative of the transformed
human kidney epithelial cell line ``293'' (ATCC 1573) that
has been stably transfected with cDNAs for the human and
integrin subunits. The parent cell line
expresses neither of these integrins. Cells were grown in
Dulbecco's modified Eagle's medium (BioWhittaker,
Walkersville, MD) supplemented with glutamine and 10% fetal calf serum
and including a penicillin/streptomycin antibiotic mix. The clone was
selected from a three-plasmid cotransfection including a neo
plasmid by growth in G418 (Life Technologies, Inc.). However, the
transfection is stable, and cells are no longer grown in the presence
of G418. Instead, cells are monitored for changes in morphology and
screened periodically for expression of
and
. The cell line has been re-cloned by A. J. Pelletier
since its original isolation, and the current strain is designated B.1.
Medium for antibody-mediated clustering (see below) was RPMI 1640
(BioWhittaker) with 10 mM HEPES, pH 7.2 (ICN Biomedicals,
Costa Mesa, CA). RPMI 1640 was chosen for its moderate levels of
divalent cations (0.2 mM Mg
and 0.4 mM Ca
).
Other Buffers and Solutions
Lysis solution: 1%
Nonidet P-40 (Sigma) in Tris-buffered normal saline (20 mM Tris, pH 7.5) containing 100 µM sodium metavanadate
(Sigma) and 1 mM phenylmethylsulfonyl fluoride; Western
blocking solution: 1% Tween 20 (polyoxyethelenesorbitan monolaurate,
Sigma) in phosphate-buffered saline (PBS) plus 1% gelatin (from bovine
skin, Sigma); Western wash solution: same as block without gelatin;
cell wash buffer for flow cytometry: PBS without divalent cations plus
1% BSA (Sigma) and 0.1% sodium azide (Sigma). Immune-precipitation
buffer contains 50 mM Tris, pH 7.4, 0.1% SDS, 1.0% Nonidet
P-40, 0.5% deoxycholic acid, and 150 mM NaCl, to which 1
mM phenylmethylsulfonyl fluoride and 100 µM sodium metavanadate were added immediately prior to use.Antibody- and Fibrinogen-mediated
Stimulation
Antibody 9D4 was generated at Genentech Inc. and was
the kind gift of Dr. Jin Kim and Genentech(19) . LIBS6 was the
kind gift of Dr. Mark Ginsberg. AP5 was described
elsewhere(36) . All other anti and
anti-
complex antibodies were
generated in the lab of Dr. Z. Ruggeri and have been described
elsewhere(20, 21, 22) . Control antibodies
for clustering were P3 (an anti-GP1b(23) ) and P10 (which binds
an unidentified protein on the cell surface).
(
)Both were raised in the laboratory of Z. M. Ruggeri.
For all of the experiments presented here, the secondary antiserum was
an affinity-purified polyclonal goat anti-mouse IgG (Jackson
ImunoResearch, West Grove, PA). In other experiments, goat anti-mouse
Fc (Jackson), and rabbit anti-mouse (raised at Genentech, Inc.) were
used with comparable results. Human fibrinogen was the kind gift of Dr.
Brunhilde Felding-Habermann. Cells were grown for assay in 10-cm tissue
culture-treated Petri dishes (various). Subconfluent plates were
harvested by washing twice with Ca
- and
Mg
-free PBS and treating with 2 mM EDTA (in
PBS) at room temperature for 10 min. Cells were re-suspended in
HEPES-buffered RPMI 1640 (10 mM HEPES) and triturated to
obtain single cells. They then were centrifuged at low speed at room
temperature and re-suspended in fresh RPMI/HEPES a total of three times
and brought to a final density of approximately 1-2
10
cells/ml. The basic assay was as follows: 100 µl of
cells in 1.6-ml microcentrifuge tubes were treated with primary and
secondary (where appropriate) for times indicated at 37 °C.
Following incubation, cells were quenched on ice, pelleted at low speed
for 20 s, and transferred back to ice. Medium was aspirated and cells
were lysed in 30-50 µl of lysis solution. Following 15 min on
ice, lysates were cleared by centrifugation in a microcentrifuge at
15,000 rpm for 15 min at 4 °C. In the case of adhered cells, they
were lysed in the wells with lysis buffer, transferred to
microcentrifuge tubes, and cleared as above. Relative protein
concentrations were determined with the use of a modified Bradford
assay (Bio-Rad). In experiments designated ``plus RGD'' cells
were treated at 40 µM with the synthetic cyclic peptide
G4120 (24) (kind gift of Dr. Thomas Gadek and Genentech Inc.)
for 5 min prior to or simultaneously with addition of the antibodies.
G4120 has a high affinity for
and
inhibits platelet adhesion to fibrinogen with an IC
of
0.15 µM(24) . Adhesion of Clone B to fibrinogen
is completely abolished at 20 µM(5) . The dose of
40 µM was chosen because, by all functional criteria, it
appeared to be well in excess of saturation. For fibrinogen-mediated
stimulation, cells were pretreated with AP5 at 100 µg/ml for 15 min
on ice in HEPES-buffered RPMI 1640. Fibrinogen was added at the
indicated concentrations, and incubations were carried out at 37 °C
for 10 min. In the past, I have found that the issue of stimulation by
soluble fibrinogen is complicated by the binding of fibrinogen to the
sides of the tube and subsequent binding of the cells to the
solid-phase fibrinogen, even under these short incubations. This
problem was avoided by carefully pipetting only those cells in
suspension into a new, ice-cold tube and collecting and lysing them as
described above.
Immunoprecipitations, SDS-PAGE, and Western
Blots
Immunoprecipitations from precleared lysates were
performed using standard protocols in lysis buffer. Anti-FAK mAb, 2A7,
was purchased from Upstate Biotechnology, Inc., Lake Placid, NY and
used at the recommended dilution. Immune complexes were precipitated
with anti-mouse IgG-agarose (Sigma). For Western analysis, equal
protein amounts of each lysate were diluted 1:1 with 2 loading
dye and electrophoresed on 7.5% polyacrylamide gels (37.5:1
acrylamide:bis) at 60 volts for 15 h. Transfer to Immobilon-P
(Millipore, Bedford, MA) was performed in CAPS (Sigma) buffer as
described(5) . All incubations below were performed at room
temperature on a rocking or orbital platform. Blots were air-dried,
re-wetted in methanol, blocked for 2 h in blocking buffer, exposed to
primary antibody (anti-phosphotyrosine mAb 4G10, Upstate Biotechnology,
Inc.) at 1:2000 dilution in blocking buffer for 45 min, washed three
times, 15 min each, with wash solution, exposed to the secondary
horseradish peroxidase-conjugated anti-mouse IgG (Jackson
Immunological) in blocking buffer for 30 min, and finally washed
several times for 15 min each in wash solution. Visualization was
achieved with the Renaissance® chemiluminescence reagent (DuPont
NEN).
Flow Cytometry
FITC (fluorescein
isothiocyanate)-labeled AP5 was generated by incubation of 1 mg of AP5
with 0.1 mg FITC-Cellite (Sigma) in the dark at room temperature for 45
min (in 100 mM sodium carbonate, pH 9.0). Cellite was removed
from suspension by centrifugation, and labeled antibody was separated
from free FITC by chromatography on a PD-10 column (Pharmacia Biotech,
Uppsala, Sweden). Relative incorporation was determined by comparing A to A
. Cells were
removed from growth plates with 2 mM EDTA in PBS as above,
washed twice with cell wash buffer, and exposed to appropriate control
or experimental antibodies for 30 min on ice in cell wash buffer
(concentrations as indicated in figures). When RGD is specified, G4120
was used at 40 µM. Cells were treated with propidium
iodide (Sigma, 500 ng/ml, final) just prior to flow cytometry, which
was performed on a Becton-Dickinson FACScan with ``Lysys II''
software. Appropriate forward and side scatter gates were set for these
cells, and only those cells excluding propidium iodide were counted.
Confocal Immunohistochemistry
Cells were prepared
for antibody-mediated clustering as above except that they were plated
on polylysine-coated coverslips following the washes (polylysine
(Sigma)-coated at 0.01% overnight at 4 °C). Cells were treated with
primary and secondary antibodies exactly as described for suspension
assays above. The concentration of AP5 used was 100 µg/ml and G4120
(40 µM) was included to enhance binding. P9 was used at 4
µg/ml. After 5 min, cells were fixed with 2.5% paraformaldehyde
(Malinkrodt Specialty Chemicals, Paris, KY), for 15 min at 4 °C.
Following washes with PBS and blocking with PBS plus 0.5% BSA, cells
were treated with a FITC-labeled donkey anti-goat tertiary antibody for
visualization of the integrin-immune clusters. Cells were mounted in
Mowiol® (Calbiochem) and visualized on a Zeiss LSM-4 confocal
imaging system.Binding of Soluble
Fibrinogen
I-Fibrinogen was prepared in the
laboratory of Z. M. Ruggeri as described(20) . Cells were
removed from plates and washed exactly as described above and incubated
on ice for 15 min either with 100 µg/ml AP5 or P4 (a nonblocking,
nonactivating anti-
) as a control.
Assays were performed in 0.8-ml microcentrifuge tubes that had been
blocked for 2 h with 1.0% heat-denatured BSA (in PBS). Aliquots of
10
treated cells were transferred to assay tubes either
with or without 40 µM RGD, and
I-fibrinogen
at the indicated concentrations was added (assays all in duplicate).
Incubation was carried out for 30 min on ice. Cells were washed with
500 µl of ice-cold PBS containing 1.0% BSA, collected by low speed
centrifugation, and re-suspended in cold PBS/BSA by gentle vortexing. A
total of three washes were performed. After the final wash, the cells
were re-suspended in 200 µl of PBS/BSA and transferred to
scintillation vials, followed by the addition of 5 ml of
``Safety-Solve'' (Research Products International, Mount
Prospect, IL) and gentle vortexing. Counts were measured on a Beckman
Gamma 8000 scintillation counter for 5 min each tube. For each
concentration of fibrinogen, the amount of bound
I
detected in the assays containing RGD was considered nonspecific
binding and was subtracted from total counts to. The remaining
RGD-inhibitable counts were considered specific binding of
I-fibrinogen. Both the raw counts, and the RGD-sensitive,
or specific, counts are presented in the figure.
Adhesion Assays
Antibodies (50 µg/ml in PBS)
or fibrinogen (20 µg/ml in PBS) or BSA as a negative control (1% in
PBS) were adsorbed to 48-well, non-tissue culture-treated plates for 2
h at room temperature. The plates were washed twice with PBS and
blocked for 2 h at room temperature with 1% BSA in PBS. Cells were
prepared for adhesion assays in the same way as for antibody-mediated
clustering. Approximately 5 10
cells were plated
per well, either in the presence or absence of G4120. Following a
30-min incubation at 37 °, plates were washed three times by
aspirating medium and gently adding 200 µl of warm medium. Finally,
medium was aspirated and cells were fixed with 2.5% paraformaldehyde
(Sigma) in PBS containing 1% sucrose (Sigma) for 10 min at room
temperature and photographed.
Antibody-mediated Clustering of
Anti-phosphotyrosine Western blots showed
that treatment of Clone B cells with a combination of a mAb to
Stimulates FAK
Phosphorylation
(9D4 at 10 µg/ml) (19) and a secondary
(anti-mouse IgG at 10 µg/ml) antiserum resulted in stimulation of
phosphorylation of two major bands in the 120-130-kDa range,
while treatment with either antibody alone did not (data not shown).
The time course of induction was rapid; phosphorylation of these
proteins can be detected within 30 s and reached a maximum by 5 min (Fig. 1). The major phosphotyrosine-containing proteins detected
comprised a pair of bands of approximate molecular weights 122,000 and
130,000. These bands correspond exactly to those induced by adhesion of
Clone B to fibrinogen reported previously ( (5) and see Fig. 11). Anti-FAK Western blots of Clone B lysates showed two
bands that comigrated with the phosphotyrosine-containing proteins seen
in Fig. 1(not shown). Immunoprecipitation with the anti-FAK
monoclonal antibody, 2A7, but not a control mAb, precipitated two bands
that were phosphorylated on tyrosine following antibody-mediated
clustering of
(Fig. 2).
These bands were not detected in nonstimulated cells, and the time
course of appearance roughly parallels that seen in Fig. 1.
Moreover, they comigrated with the two major phosphotyrosine-containing
bands in the whole lysate (Fig. 2). In vitro kinase
activity experiments on, and peptide mapping of, anti-FAK
immunoprecipitated material all support the conclusion that the two
bands represent two forms of FAK.
We conclude that this
cell line expresses two electrophoretic variants of FAK and that both
are phosphorylated on tyrosine following antibody-mediated clustering
of
. The cause and significance of
these electrophoretic variants is not known.
for the indicated
times were divided in two and precipitated either with anti-FAK or a
control, isotype-matched mAb (anti-beta
). The
immunoprecipitates were subject to SDS-PAGE and anti-phosphotyrosine
Western blot, as above. Numbers at left represent the
positions of the indicated molecular mass standards.
``L'' is the whole cell lysate from the 5 min time
point.
complex.
Several examples are shown in Fig. 3. Cells were treated either
with the secondary antibody alone (``neg.''), 9D4
(10 µg/ml) plus secondary, or one of five mAbs (also 10 µg/ml)
with or without the clustering secondary antibody (20 µg/ml), as
indicated. The mAbs P4, P5, and P9 (20, 21, 22) all stimulated phosphorylation,
but only in the presence of the secondary antibody. In contrast, the
anti-GP1b mAb P3 (23) , which does not bind to clone B, and
P10, which binds an unidentified cell surface protein on Clone B
(details under ``Materials and Methods''), were not capable
of stimulating FAK phosphorylation. These controls indicate that FAK
phosphorylation is not the result of interactions of IgG molecules with
the surface or a general effect of capping surface proteins.
or anti-
mAbs. Cells were treated with several mAbs, with or without
secondary antiserum, for 5 min. Lysates were subject to SDS-PAGE and
anti-phosphotyrosine Western blot. An anti-
(9D4) and
three anti-
mAbs (P4, P5, and P9)
all stimulate phosphorylation of the characteristic FAK doublet. Two
control antibodies, P3 and P10, do not. Numbers at left indicate position of the indicated molecular mass
standards.
mAbs were tested through a range
of concentrations and, at optimal concentrations, stimulated
phosphorylation to comparable levels. The optimal concentration for
phosphorylation for each antibody generally was below saturation (not
shown). Table 1shows the antibodies tested and some of their
characteristics. In summary, phosphorylation can be stimulated equally
well by blocking, nonblocking, anti-
, or anti-complex
antibodies.
Clusters Mediated by an Activating mAb Do Not Stimulate
Phosphorylation
Different results were obtained with the
activating mAb we have described recently, Ap5(36) . Fig. 4demonstrates that treatment of Clone B with AP5 plus
secondary antisera did not induce phosphorylation of FAK. Tyrosine
phosphorylation could not be detected in any trials with AP5 at
concentrations ranging from saturation to those below detection for
binding (see below for binding data). In some trials, incubation was
carried out for 15 min, three times longer than that normally required
for maximal phosphorylation, with no effect. Moreover, three different
secondary antisera known to cooperate with other mAbs in stimulating
FAK phosphorylation did not do so with AP5 (data not shown). Anti-FAK
Western blots showed that FAK could be recovered equally well from
lysates of AP5-stimulated cells as from those of cells stimulated by
other antibodies (data not shown).
in Clone B was performed with the
anti-
mAb, AP5, which is an activating, anti-LIBS
antibody. AP5 was used at the indicated concentrations. Secondary
antiserum was included in all cases at a concentration of 1/2 the
concentration of AP5 used (several other ratios were tested, with the
same result). Experiments in the right-hand panel were
performed in the presence of a soluble RGD analog, which dramatically
enhances binding of AP5 (see Fig. 5). The positive controls, pos, were lysates of cells stimulated by
clustering by P4 in the absence (left) or presence (right) of the same RGD analog. Numbers at left indicate position of the indicated
molecular mass standards.
on Clone B and exhibits anti-LIBS
behavior. Clone B was incubated with the indicated concentrations of
FITC-conjugated AP5 in the presence or absence of the RGD analog and
subjected to direct immunofluorescence flow cytometry as described
under ``Materials and Methods.'' The receptor-minus parent,
293, was incubated with the highest concentration of AP5 used (200
µg/ml). Relative fluorescence is displayed on the x axis
and the number of cells of a given fluorescence level is displayed on
the y axis. The inset shows a plot of the mean
fluorescence intensity for each curve. Note that in both the plus and
minus RGD graphs, AP5 shows saturable
binding.
on the cell surface. In the
presence of the RGD, AP5 exhibited a saturation binding level similar
to that of the other antibodies in this study (data not shown). Note
that the concentrations used in the phosphorylation experiment in Fig. 4cover the range from saturation binding to no detectable
binding.
by confocal immunofluorescence
microscopy. For these experiments, cells were adhered to
poly-L-lysine-coated coverslips and treated with the primary
and secondary antibodies exactly as for the phosphorylation
experiments. Following a 5-min incubation at 37 °C, the cells were
fixed with paraformaldehyde and exposed to a tertiary, FITC-labeled
anti-goat antibody for visualization of the clusters. Fig. 6depicts 0.8-µm confocal sections beginning near the
center of a cell. Note the absence of any internal staining (Fig. 6, images 1 and 2). This is expected
since the cells were not permeablized. In these sections through the
center of the cell, peripheral staining can be detected and dense areas
of staining can be seen. In the final image, the apical rim of the cell
is in view. The punctate pattern clearly indicates
``capping'' or cluster formation.
on the
surface of Clone B. Confocal immunofluorescence was performed on cells
treated with AP5 plus secondary (in the presence of the RGD).
Conditions for incubation with primary and secondary antibodies were
identical to those used in solution. Clusters were detected with a
FITC-conjugated tertiary antiserum. Four optical sections of 0.8
microns each are shown, representing sections through the middle of the
cell up to the apical rim of the cell. Sections 1 and 2 show peripheral staining with bright and dark areas. Note that no internal staining can be seen, which is
expected since the cells were not permeablized. In section 4 the plane of the membrane is in view. Punctate staining
characteristic of clustering can be seen clearly on the surface of the
cells.
on the cell surface is
extremely diffuse, exhibiting an extremely faint halo of fluorescence
(not shown).
on Clone B for binding of soluble
fibrinogen. Cells were pretreated either with AP5 or a nonactivating,
nonblocking antibody (P4) and then allowed to bind
I-fibrinogen at the indicated concentrations for 30 min
on ice in the presence or absence of the soluble RGD. The
RGD-inhibitable counts bound (specific binding) at each concentration
of fibrinogen is expressed in counts/min (cpm). Values are the average
of duplicate experiments.
alone is sufficient to induce
FAK phosphorylation.
Other Activating Antibodies Also Fail to Stimulate FAK
Phosphorylation
In order to determine whether lack of FAK
phosphorylation is a general characteristic of activating antibodies,
we tested the activating anti-LIBS antibody, LIBS6(25) , and
mAb P41. Through a wide range of concentrations, these activating
antibodies also failed to stimulate FAK phosphorylation (Table 1). Flow cytometry was used to establish that these
antibodies bound to on the surface
of Clone B (data not shown).
Binding of Soluble Fibrinogen-activated
Activating antibodies such as AP5 and LIBS6
induce binding of soluble fibrinogen to
Does Not Stimulate FAK
Phosphorylation
(18, 25, 36) .
We examined directly the ability of AP5 to activate
on the surface of Clone B in a
soluble fibrinogen binding assay. Cells were pretreated with 100
µg/ml AP5 or a nonactivating, nonblocking antibody, P4, for 15 min
on ice. They then were incubated with various concentrations of soluble
I-fibrinogen in the presence or absence of 40 µM G4120. The results are shown in Fig. 8, presented as the
raw counts (left panel) and specific binding (right
panel). Specific or RGDdependent binding is defined as that which
could be inhibited by G4120 (see ``Materials and Methods'').
Values represent average of duplicate experiments. Cells pretreated
with P4 (or with no antibody; not shown) exhibited virtually no
specific binding of fibrinogen. This indicates that, by this criterion,
on Clone B is in the basal state.
In contrast, dose-dependent binding of
I-fibrinogen is
evident in cells treated with AP5. We have repeated this experiment
several times using FITC-labeled fibrinogen and flow cytometry as an
assay with consistent results (data not shown). Using this approach, we
were able to determine that all cells in the population were stimulated
to bind fibrinogen.
, induces rapid
tyrosine phosphorylation of FAK ( (5) and see Fig. 11).
We therefore examined whether soluble fibrinogen bound to
antibody-activated
on the surface
of Clone B would stimulate phosphorylation of FAK (Fig. 9).
Clone B was preincubated with 100 µg/ml AP5 or LIBS6 for 15 min on
ice (or mock-treated with buffer alone). Fibrinogen at 50, 100, or 200
µg/ml was added to the cells, which then were incubated for 10 min
at 37 °C. Even though fibrinogen binds under these conditions (Fig. 8), no phosphorylation of FAK was induced, in striking
contrast to what is seen when Clone B adheres to solid-phase
fibrinogen.
does not
stimulate FAK phosphorylation in Clone B. Cells were pretreated for 15
min on ice with either no antibody, AP5 (100 µg/ml), or LIBS 6 (100
µg/ml) and incubated with the indicated concentrations of
fibrinogen for 10 min. The positive control (pos) is lysate
from P4-stimulated cells. Numbers at left indicate
position of the indicated molecular mass
standards.
AP5 Supports Adhesion, but Not Spreading, of Clone
B
We examined the adhesion of clone B to activating and
nonactivating antibodies. Various antibodies or fibrinogen were coated
onto 24-well tissue culture plates. Clone B cells were allowed to
adhere to these substrates for 30 min at 37 °C in serum-free
medium. Fig. 10compares the morphology of cells plated either
on fibrinogen (12A), P9 (12B), or AP5 (12C). Note the cells adhere to
all three substrates. However, although P9 and fibrinogen both support
spreading of the majority of cells, AP5 does not. The experiment was
performed either with or without 40 µM G4120 (to promote
adhesion to AP5). G4120 is included in 12B and 12C and not in 12A.
G4120 had no effect on adhesion or spreading of cells plated on P9 and
completely abolished adhesion to fibrinogen (not shown). We also have
tested 9D4, CP8, and LIBS6 and found that 9D4 and CP8 both support
spreading, whereas LIBS6 does not (data not shown). We examined FAK
phosphorylation in cells adhered under these conditions and found that
phosphorylation was not detected in cells adhered to the activating
mAbs, which do not support spreading, but was detected in cells adhered
to and spread on the nonactivating mAbs or fibrinogen (Fig. 11).
is not
sufficient to stimulate FAK phosphorylation. We found that clustering
of the resting form of the receptor, whether achieved by antibodies or
by adhesion to solid-phase fibrinogen, results in rapid FAK
phosphorylation. In contrast, clustering of the active from of the
receptor, whether achieved with activating antibodies or by binding of
soluble fibrinogen to the activated receptor, did not stimulate FAK
phosphorylation. Other characteristics of the antibodies, such as
subunit or complex specificity, affinity, or ligand-blocking ability do
not correlate with the ability to stimulate FAK phosphorylation.
not recognized by activating
antibodies must be clustered in order to induce FAK phosphorylation.
in Clone B. Although we have not established a causal
relationship between the ability of cells to spread and the ability to
phosphorylate FAK, the two clearly are correlated.
125,000
peptide.
Kanner et al.(27) have reported
an analog of FAK, called FAKb, which is of slightly lower molecular
mass than FAK. The analog we observe here is about 5 kDa larger than
FAK. In anti-FAK blots from Clone B, a band that migrates slightly
faster than FAK can be detected and may correspond to FAKb. The higher
molecular weight band reported here clearly is not FAKb. The cause and
significance of this variant currently are not known.
-mediated signaling in platelets
extensively and found that when platelets adhere to solid-phase
fibrinogen, FAK is one of the prominent phosphotyrosine containing
proteins. However, when
on
platelets was activated by LIBS6 and allowed to bind soluble
fibrinogen, they found that FAK phosphorylation did not occur unless
the platelets were allowed to aggregate(18) . Based on these
and other data, they have concluded that ligation of
is not sufficient to stimulate FAK
phosphorylation and that costimulation of other signaling pathways are
required(15) .
in the active state does not
stimulate FAK phosphorylation in Clone B when clustered or when bound
by fibrinogen, whereas the same receptor in the basal or so-called
resting state does. We do not yet know the mechanism accounting for
this difference. However, in order for a costimulus to be involved in
this difference, that costimulus itself must be induced by the resting,
but not the active receptor. Although cell-type differences might
exist, we think activation state-dependent signaling could play a role
in platelets as well.
, either by
antibodies or by soluble fibrinogen. In contrast, binding of soluble
fibrinogen to activated
in
platelets induces phosphorylation of several proteins other than FAK.
One possible reason for this difference is that these bands represent
proteins not found in Clone B. Consistent with this idea, one of these
bands has been identified by Shattil and co-workers as
pp75
(15) , a tyrosine kinase found only
in hematopoetic cells.
Adhesion Is Both a Regulated and a Regulating Event
Our
concept of ``activation states'' of integrins derives
primarily from the paradigms of platelet and leukocyte integrins, which
mediate the transition of cells from suspension to
adhesion(12, 29, 30, 31) . However,
in a more general sense, the binding of all integrins must be
modulated, for example, in order for a cell to migrate. It is logical
therefore that many, if not most, integrins may exist in different
activation states as the cell makes, breaks, and re-makes contacts, as
in the process of migration. If so, at any given time, a cell might
express subpopulations of a single integrin in different activation
states, which may in turn have discrete roles in mediating cellular
responses. on resting platelets (36) or on Clone B in the absence
of a ligand analog (Fig. 3). Moreover, there are examples of
transient modifications to integrins, such as phosphorylation of
(32) or ADP-ribosylation
of
(33) , that occur to only a subset
(5-40%) of the total population. The functional significance of
these modifications is not known. An intriguing possibility is that
they result in functionally distinct subpopulations with specific
ligand binding and/or signaling properties. A related possibility is
that ligands that bind preferentially to a particular subset of an
integrin on the cell surface might, therefore, initiate different
signals.
can be propagated over a long
range (17) and are regulated by changes to the intracellular
domain (reviewed in (12) ). It seems reasonable to suggest that
alterations to the conformation of the extracellular domain also can
influence the structure and function of the intracellular domain.
Therefore, it might even be possible for some ligands to alter the
conformation and therefore the signaling properties of an integrin.
LIBS/activation domains may be targets for binding of ligand domains or
accessory proteins whose activity is mimicked by the activating
antibodies.
and
integrins. In fact, LaFlamme
and co-workers (34) have reported that the
cytoplasmic domain alone can stimulate phosphorylation of FAK.
Since FAK phosphorylation seems to be a common result of all integrins
that form focal adhesions, the diversity of effects elicited by
different matrices cannot be the result of this common biochemical
change. If integrins are involved at all in regulating a cell's
response to different matrices, other biochemical signals that are more
receptor-specific must be involved. The possibility that
and
integrins could trigger other signals apart
from those involved in the pathway that leads to focal adhesions is an
attractive one.
is not sufficient to stimulate
phosphorylation of FAK. The data suggest further that one important
determinant of whether or not FAK is phosphorylated is the activation
state of
. We propose that other
signaling cascades not involving focal adhesions and FAK
phosphorylation may be stimulated by
in the active conformation. Results reported for platelets
suggests that this indeed may be the case.
integrins, both receptor conformation and clustering are
important in determining with what intracellular proteins the integrin
associates. This is likely to have an effect on the signals induced.
Thus, the role of integrin conformation in modulating outside-in
signals may be a general one.
receptors. We have found recently that
can be induced into a higher avidity
state by AP5.
(
)This form may have different
signaling properties. Thus, activation state-linked signaling may be a
general mechanism by which integrins mediate the diverse cellular
responses induced by integrin-matrix interactions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.