©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Activation State of the Integrin Affects Outside-in Signals Leading to Cell Spreading and Focal Adhesion Kinase Phosphorylation (*)

(Received for publication, February 27, 1995; and in revised form, May 1, 1995)

Anthony J. Pelletier (1)(§) Thomas Kunicki (2) Zaverio M. Ruggeri (2) Vito Quaranta (1)

From the  (1)Department of Cell Biology and the (2)Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 (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.


INTRODUCTION

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()(8, 9) .

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 (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.

The role of 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).

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 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) .

We used Clone B (5) and a panel of activating or nonactivating mAbs to different epitopes on 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.


MATERIALS AND METHODS

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.


RESULTS

Antibody-mediated Clustering of Stimulates FAK Phosphorylation

Anti-phosphotyrosine Western blots showed that treatment of Clone B cells with a combination of a mAb to (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.


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 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.




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 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.


Figure 3: FAK phosphorylation by antibody-mediated clustering can be induced by several anti- 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.



All 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).


Figure 4: The activating, anti-LIBS antibody, AP5, does not stimulate FAK phosphorylation. Antibody-mediated clustering of 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.




Figure 5: AP5 binds to 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.



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 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.

We next determined that AP5 mediated cluster formation of 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.


Figure 6: AP5 supports clustering of 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.



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 on the cell surface is extremely diffuse, exhibiting an extremely faint halo of fluorescence (not shown).


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 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.



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 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 Does Not Stimulate FAK Phosphorylation

Activating antibodies such as AP5 and LIBS6 induce binding of soluble fibrinogen to (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.

Adhesion to solid-phase fibrinogen, which occurs via basally active , 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.


Figure 9: Binding of soluble fibrinogen to antibody-activated 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).


DISCUSSION

Our results indicate that in our transfected model system, Clone B, clustering of 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.

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 not recognized by activating antibodies must be clustered in order to induce FAK phosphorylation.

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 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.

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 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.

Shattil and co-workers (3) have studied -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) .

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 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.

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 , 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.

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.

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.

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 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.

Other investigators have reported data that suggest that conformation changes in the extracellular domain of 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.

Phosphorylation of FAK appears to be a general effect of 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.

Our data demonstrate clearly that clustering of 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.

Very recently, Miyamoto et al.(35) have reported that for 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.

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 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Training Grant AI07244 and Markey Fellowship 92-10 (to A. J. P.) and National Institutes of Health Grants HL46979, HL48728, and GM46902 (to T. K., Z. M. R., and U. Q., respectively). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: FAK, focal adhesion kinase; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; mAb, monoclonal antibody; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate.

A. J. Pelletier, unpublished observations.

A. J. Pelletier, submitted for publication.


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