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J. Biol. Chem., Vol. 280, Issue 44, 36873-36882, November 4, 2005

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Two Functional Active Conformations of the Integrin 21, Depending on Activation Condition and Cell Type^*

Gerlinde R. Van de Walle, Karen Vanhoorelbeke1, Zsuzsa Majer, Eszter Illyés, Johan Baert¶, Inge Pareyn, and Hans Deckmyn2

From the Laboratories for Thrombosis Research and ^¶Histology, Interdisciplinary Research Centre, Katholieke Universiteit Leuven, Campus Kortrijk, 8500 Kortrijk, Belgium and the Department of Organic Chemistry, Eötvös Loránd University, 1518 Budapest, Hungary

Received for publication, July 26, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
For several integrins, the existence of multiple conformational states has been studied intensively. For the integrin 21, a major collagen receptor on platelets and other cell types, however, no such experimental data were available thus far. Recently, our group has developed a monoclonal antibody IAC-1 sensitive to the molecular conformation of 21 because it only binds to the activated state of 21 on platelets, induced upon inside-out signaling. By investigating IAC-1 binding in combination with collagen binding after inside-out stimulation and outside manipulation, we demonstrated the existence of three different conformations of 21 on platelets and Chinese hamster ovary cells as follows: (i) a nonactivated, resting state with no collagen nor IAC-1 binding; (ii) an intermediate state, induced by outside manipulation, with collagen but no IAC-1 binding; and (iii) a fully activated state, induced after inside-out stimulation, with both collagen and IAC-1 binding. Moreover, these different conformational states of 21 are dependent on the cell type where 21 is expressed, as IAC-1 binding to peripheral blood mononuclear cells and Jurkat cells could also be induced by outside manipulation, in contrast to platelets and 21-expressing Chinese hamster ovary cells. Finally, we revealed a functional relevance for these different conformational states because the conformation of 21, induced after outside manipulation, resulted in significantly more cell spreading on coated collagen compared with nonactivated or inside-out stimulated cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Integrins belong to a family of heterodimeric () surface receptors that play a major role in cell-cell and cell-matrix interactions (1). Each subunit typically contains a relatively large extracellular domain, a single-pass transmembrane domain, and a short cytoplasmic tail (2). Integrins can become activated and transduce signals across the plasma membrane in both directions; intracellular binding of cellular components to the integrin cytoplasmic domains activates the ligand binding capacity of the extracellular domain (inside-out signaling), whereas ligand binding to the extracellular domain or other stimuli acting at the outside of the integrin (outside manipulation) can also lead to conformational alterations that then again can result in the activation of intracellular pathways (outside-in signaling) (3). Thus, integrin receptors are able to switch from an inactive (low affinity) to an active (high affinity) state, and vice versa, in response to binding events taking place at both the ligand binding area and the cytoplasmic domains. Generally, it is accepted that intersubunit associations at the cytoplasmic and transmembrane domains maintain integrins in the low affinity state and that interruption of this association results in activation of the integrin (4, 5). In particular, for several integrins like IIb3, 21, L2, and M2, it has been demonstrated that a conserved cytoplasmic GFFXX sequence of the subunit modulates inside-out activation by acting as a hinge, keeping the integrin in an inactive conformation (6–8).

Significant progress is currently made in demonstrating that integrins contain a number of flexible joints and connections, leading to a broad spectrum of possible conformational states with distinct effects on the overall ligand binding affinity of the receptor (9). For the I domain lacking integrins V3 and IIb3, three different conformational states have been demonstrated by electron microscopy and crystal structures as follows: (i) a closed conformation (resting state), not able to bind its natural ligand; (ii) an extended conformation with closed headpiece (low affinity state); and (iii) an extended conformation with open headpiece (high affinity state) (10–13). These two extended, active conformations are both favored by outside-in and inside-out signaling and can bind their natural ligand. The integrins 41 and 51 also exhibit multiple affinity states on the cell surface depending on the activation condition, similar to that described for V3 and aIIb3 (14, 15). For the I domain containing integrins L2 and M2, the existence of such conformational states has been demonstrated as well. In the study by Jin et al. (16), molecular dynamic simulations, with pulling forces applied to the 7 helix of the I domain, were used to reproduce intermediate and open conformations of the integrin L subunit, in agreement with the crystal structures described previously (17).

The integrin 21 (also designated VLA-2, GPIaIIa, or CD49b/CD29) is an I domain-containing integrin, expressed on a variety of cell types including platelets, white blood cells, endothelial cells, and fibroblasts (18–20). This integrin acts mainly as a collagen receptor on platelets (21), and on other cell types it can serve both as a collagen and laminin receptor (22). On platelets, 21 plays an important role in primary hemostasis by binding to collagen in the subendothelium exposed after vessel injury (23). On white blood cells, 21 is thought to be involved in tissue injury and inflammation situations (18, 19). More recently, 21 on activated T lymphocytes and Jurkat cells was found to influence Fas-induced apoptosis (24).

For 21, studies on the existence of different conformational states have been hampered, mainly because until recently no antibodies were available that specifically recognize the activated or ligand-bound forms of 21. Our group developed a monoclonal antibody (mAb),³ IAC-1, that recognizes an epitope within the 2 I domain on platelets, hidden in the resting state but accessible when platelets are stimulated with agonists such as thrombin, ADP, or the snake venom convulxin (25). Moreover, because this antibody does not interfere with platelet collagen binding, it defines a new class of antibodies that is distinct from those belonging to the "ligand-induced binding sites" and the "ligand mimetic" groups.

The objective of this study was to use IAC-1 to provide further insight into the conformational states of 21 after inside-out and outside manipulations and to do this on four different cell types (platelets, 21-expressing CHO cells, PBMC, and Jurkat cells). Moreover, the functional relevance of these different conformational states of 21 was evaluated.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antibodies and Proteins
The anti-2 I domain mAb IAC-1 recognizes the activated state of 21 and was described previously (25). The anti-2 mAb AK7 was from BD Biosciences. The anti-2 mAbs Gi9 (both unlabeled and FITC-labeled) and 15D7 were from Immunotech (Marseille, France) or made in-house, respectively, and both inhibit collagen-induced platelet aggregation. Other monoclonal antibodies used are as follows: 7E3, a mouse anti-human IIb3 that also recognizes the activated state of Mac-1 (a kind gift of Dr. T. Edgington, Scripps Institute, La Jolla, CA) (26); TS2/16, an activating mouse anti-human 1 (a kind gift of Dr. M. Kahn, University of Pennsylvania); MOPC-21-FITC, an unrelated mouse anti-human IgG1 (Sigma); and CD62P-PE, mouse anti-human P-selectin (BD Biosciences). Polyclonal rabbit anti-mouse IgG-FITC was from Dako (Glostrup, Denmark).

Monoclonal antibodies and soluble human collagen type I (type VIII, Sigma) were labeled with FITC according to the manufacturer's instructions (Pierce). The FITC labeling of collagen was performed in 50 mM borate buffer, pH 8.5, after which the excess of fluorescent dye was removed by overnight dialysis against PBS. As a consequence of this treatment, the resulting collagen-FITC consists of reconstituted collagen fibrils.

Activating Agents and Cells
Activating agents used were manganese(II) chloride tetrahydrate (Mn²⁺, Sigma), cobalt chloride (Co²⁺, Sigma), dithiothreitol (DTT, Sigma), and synthetic peptides (see under "Peptide Design" and TABLE ONE). Prostaglandin E₁ (PGE₁, Sigma) was used as an agent to block platelet activation.

Platelets and peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy volunteers who did not use acetylsalicylic acid for the last 10 days. Blood was taken on acid/citrate/dextrose (10:1.5 v/v), and platelets were washed by several centrifugation steps essentially as described previously (27). Platelets were finally resuspended at a concentration of 3 x 10⁵ cells/µl in Hepes/Tyrode (13.7 mM NaHCO₃; 0.2 mM KCl; 0.1 mM MgCl; 0.03 mM Na₂HPO₄; 1.2 mM NaHCO₃; 0.5 mM Hepes; 0.01% (w/v) glucose; pH 7.4) containing 0.3% BSA. For the isolation of PBMC, blood was taken on low molecular weight heparin (250 units/ml final, Clexane, Aventis Pharma, Brussel, Belgium) and separated on Ficoll-Paque (Amersham Biosciences) following the manufacturer's instructions. PBMC were finally resuspended at a concentration of 5 x 10⁵ cells/ml in MEM (Invitrogen).

For the activation experiments, 21-expressing CHO cells and Jurkat cells were harvested, washed with XL buffer (13.7 mM NaCl; 0.5 mM KCl; 0.1 mM MgCl₂; 0.5 mM Hepes; 0.01% (w/v) glucose; pH 7.4), and resuspended at 5 x 10⁵ cells/ml, except where indicated otherwise. Platelets and PBMC were resuspended in their respective buffer or medium as described above. Reaction mixtures of cells (50 µl in a total of 100 µl) and an agonist were incubated for 30 min at room temperature. The agonists Mn²⁺ and Co²⁺ were used at a final concentration of 1 mM DTT at a final concentration of 10 mM. The peptide Ac-KRFYVVMWK-NH₂ and the cell-permeable peptides were used at a final concentration of 50 and 200 µM, respectively. Where indicated, platelets were also incubated with the antagonist PGE₁ at 5 µM final concentration. After washing twice with an excess of their respective buffer, cells were used for further experimentation.

Peptide Design
All peptides (see TABLE ONE) were synthesized by a manual solid phase technique on 4-methylbenzhydrylamine resin using the standard t-butoxycarbonyl/benzyl protocol (Pierce) (28). Some peptides were N-terminally acetylated. Purification was performed by reverse phase-high performance liquid chromatography (RP-HPLC; Jasco Co.), and all peptides were characterized by electrospray ionization-mass spectrometry (Bruker Daltonics Esquire 3000 plus) and amino acid analysis. Analytical data of peptides showed more than 95% purity.

The agonist peptide Ac-KRFYVVMWK-NH₂, based on the binding domain of thrombospondin-1 to the CD47 receptor, was used to stimulate 21 on different cell types via inside-out signaling (29, 30). Cell-permeable chimeric peptides were designed by placing the hydrophobic delivery sequence VTVLALGALAGVGVG to the human 2 cytoplasmic tail sequence KLGFFKR, as described by Wang et al. (31). The control peptide, consisting of the non-sense sequence TKSKYNE, was used as a negative control (31).

Flow Cytometry
Cells were incubated in the presence or absence of an activating agent for 30 min as described above. After washing, cells were incubated with either 10 µg/ml IAC-1-FITC or 40 µg/ml collagen-FITC (final concentration) for 30 min at room temperature. Afterward, cells were washed in their respective buffer or medium and finally resuspended in 400 µlof PBS, supplemented with 7-aminoactinomycin (Molecular Probes, Eugene, OR) diluted 1:400. When an indirect staining was used, cells were incubated with 10 µg/ml of primary antibody for 30 min at room temperature, followed by incubation with a polyclonal rabbit-anti-mouse IgG-FITC (diluted 1:100) for 30 min at room temperature. Cells were immediately analyzed by flow cytometry (Beckman Coulter, Miami, FL), and the mean fluorescence intensity (MFI) was determined (10,000 cells for each condition).

Cell Adhesion Assays
Cell Adhesion to Collagen Under Static Conditions—Wells of a microtiter plate (Greiner Bio-One GmbH, Frickenhauser, Germany) were coated with human collagen type I (25 µg/ml in PBS) overnight at 4 °C. Wells were blocked with 3% milk powder in PBS for 2 h at room temperature. 21-Expressing CHO cells or Jurkat cells were preincubated for 30 min in the presence or the absence of an activating agent. After washing with XL buffer, a dilution series of activated and nonactivated cells was added to the wells and incubated for 0, 5, 10, 20, 30, 60, and 120 min at room temperature. Cell adhesion was determined by measuring endogenous phosphatase activity colorimetrically at 405 nm, after lysis of the cells with Triton X-100, using p-nitrophenyl phosphate (Merck) as a substrate (32). Wells were washed six times after coating and blocking and three times elsewhere. All assays were performed in duplicate and repeated at least three times.

Cell Attachment and Spreading Assay—Cell attachment and spreading was studied as described previously with modifications (33). Coverslips (18 x 18 mm, Menzel-Glaser, Braunschweig, Germany) were coated with human collagen type I (25 µg/ml in PBS) overnight at 4 °C. Coverslips were blocked with 1% BSA and 0.1% glucose in Hepes/Tyrode buffer for 30 min at room temperature. Here the 21-expressing CHO cells and Jurkat cells were used at a concentration of 6 x 10⁵ cells/ml in Iscove's medium and were preincubated for 30 min in the presence or the absence of an activating agent. Coverslips were washed twice with Hepes/Tyrode buffer before the addition of 300 µl of cell suspension and incubated for either 10 or 60 min at room temperature. Coverslips were thoroughly washed to remove unbound cells and then fixed with 2% paraformaldehyde in PBS for 30 min at room temperature. After washing, coverslips were stained with Mayer's hematoxylin (2 min), followed by Fuchsin Ponceau (5 min), and mounted with DPX Mountant (Canemenco and Marivac Inc., Quebec, Canada). Adhesion to BSA-coated coverslips and adhesion of CHO-dhfr⁺cells to collagen-coated coverslips, used as negative controls, never exceeded 0.2–1.0% of the cell input.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Inside-out activation of 21 on platelets and 21-expressing CHO cells using the KLGFFKR peptide. Washed human platelets or 21-expressing CHO cells were incubated with the activating peptide H-VTVLALGALAGVGVGKLGFFKR-NH2 (200 µM, black line or gray bar) or the control peptide H-VTVLALGALAGVGVGTKSYNE-NH2 (200 µM, gray shaded or white bar) for 30 min at room temperature. Next, cells were incubated with either collagen-FITC (40 µg/ml) or IAC-1-FITC (10 µg/ml) for 30 min at room temperature. A representative histogram is shown for each cell type (A). Data are presented as the mean ± S.E. of the MFI of triplicate assays.*, p < 0.05 (B).

Analysis was performed with an inverted Nikon Eclipse TE200 microscope connected to an Image Analyzer (Lucia, Laboratory Imaging Ltd., Analis, Namur, Belgium). Quantitative results were obtained by determining the number of adherent cells by light microscopy, and fluorescence microscopy was used to visualize actin and 21, using standard emission and excitation filters.

The number of adherent cells per coverslip was calculated as follows: (number of adherent cells at time point x/number of cells added per coverslip) x 100. Adherent cells are defined as cells that are immobilized to the substrate, independently of cell morphology. Cell morphology was used to distinguish between attached and spread cells. Attached cells are defined as cells with a rounded morphology, whereas spread cells had a clear elongated morphology with clearly visible cytoplasm around the nucleus (34).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Effect of PGE1 on anti-P-selectin, collagen, and IAC-1 binding by platelets stimulated with the KLGFFKR peptide. Washed human platelets were incubated with the activating peptide H-VTVLALGALAGVGVGKLGFFKR-NH2 (200 µM) in the absence (black line or white bar) or presence (gray shaded or gray bar) of 5 µM PGE1 for 30 min at room temperature. Next, cells were incubated with either anti-P-selectin-PE (10 µg/ml), collagen-FITC (40 µg/ml), or IAC-1-FITC (10 µg/ml) for 30 min at room temperature. A representative histogram is shown for each cell type (A). Data are presented as the mean ± S.E. of the MFI of triplicate assays (B).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Inside-out Signaling and Outside Manipulation Result in Different Conformational States of 21—We demonstrated previously that inside-out activation of human platelets by convulxin, for example, resulted in exposure of both the collagen-binding sites in 21 and a neo-epitope of the receptor, recognized by the mAb IAC-1 (25). Here we extended this observation by manipulating the inside of the cell by using a cell-permeable peptide containing the KLGFFKR sequence of the human 2 cytoplasmic tail sequence (31). Addition of this peptide induced activation of 21 on the platelet membrane as demonstrated by a significant increase in collagen binding (p < 0.05) (Fig. 1). This binding was specific because of the following: (i) addition of the control non-sense peptide had no effect on collagen binding (Fig. 1), and (ii) addition of the inhibitory anti 2-I domain mAb 15D7 could reverse the collagen binding (data not shown). The inside manipulation by the cell-permeable peptide also resulted in a significant increase in IAC-1 binding (p < 0.05) (Fig. 1). Addition of this peptide did not result in an up-regulation of 21, because no increase in MFI was observed when staining with the control mAbs Gi9 and AK-7, nor was a significant binding of the unrelated mouse anti-human IgG1 MOPC-21 observed (data not shown). Besides, addition of this peptide resulted in an overall activation of platelets as determined by P-selectin exposure (Fig. 2). However, addition of PGE1 before addition of the peptide still induced collagen and IAC-1 binding without the exposure of P-selectin (Fig. 2), demonstrating that addition of the peptide per se induces conformational changes of 21, independent of general cell activation.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Outside activation of 21 on platelets and 21-expressing CHO cells. Washed human platelets (A) or 21-expressing CHO cells (B) were incubated without an agonist or with Mn2+ (1 mM), DTT (10 mM) or TS2/16 (50 µg/ml) for 30 min at room temperature. Next, the cells were incubated with either collagen-FITC (40 µg/ml, gray bars) or IAC-1-FITC (10 µg/ml, white bars). Data are presented as the mean ± S.E. of the MFI of triplicate assays.

Next, we investigated the binding of collagen and IAC-1 to platelets and 21-expressing CHO cells after outside manipulation with divalent cations (Mn²⁺ and Co²⁺), the reducing agent DTT or TS2/16, an activating anti-1 antibody, and found that all resulted in a significant increase in collagen binding, indicating that 21 was activated (p < 0.05, Fig. 3). Most interestingly, this outside manipulation was not accompanied by an increase in IAC-1 binding (Fig. 3). The different outside manipulations did not result in an up-regulation of 21 on the cells, as determined by Gi9 or AK-7 binding, nor was P-selectin exposed (data not shown). Hence, because these results demonstrate that the neo-epitope of IAC-1 is not exposed after outside manipulation in contrast to inside-out signaling, we presumed that at least two different active conformations of 21 exist.

The Different Conformational States of 21 Regulate Adhesion and Spreading to Collagen—To evaluate whether these two different active conformations have a functional role in cell adhesion, the adhesion of 21-expressing CHO cells to coated collagen type I was studied under static conditions.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Static adhesion, attachment, and spreading of 21-expressing CHO cells on collagen after stimulation. Unstimulated 21-expressing CHO cells ( or white bar)or 21-expressing CHO cells stimulated with Mn2+ (1 mM, • or striped bar)or the peptide H-VTVLALGALAGVGVGKLGFFKR-NH2 (200µM, or gray bar) were allowed to adhere to reconstituted human collagen type I-coated wells (A) or coverslips (B) for different periods up to 120 min. A, adherent cells were permeabilized and stained, and the optical density (OD) was determined. Specific binding was measured by subtracting nonspecific adhesion to noncoated wells. Data are presented as the mean ± S.E. of triplicate assays, performed in duplicate. B, coverslips were fixed with 2% paraformaldehyde and stained with hematoxilin/fuchsin, and the number of spread cells was determined and expressed relatively to the number of adherent cells (= 100%). Data are presented as the mean ± S.E. of triplicate assays. A representative picture of 21-expressing CHO cells, at 60 min and stimulated with Mn2+, is shown. Arrows indicate attached cells and arrowheads indicate spread cells. ns, nonspecific. Bar, 15 µM.

Next, the effect of outside and inside-out stimulation on cell spreading was evaluated as described before (33). Therefore, 21-expressing CHO cells, stimulated or not, were allowed to adhere to collagen-coated coverslips for 10 or 60 min, after which the cells were fixed and stained. Both outside and inside-out manipulation gave similar adhesion results as observed above (data not shown). Moreover, the adhesion to collagen was 21-specific because (i) CHO-dhfr⁺cells did not adhere to collagen, and (ii) preincubation with the inhibitory mAb 15D7 almost completely abolished adhesion (data not shown). When the adherent cells were analyzed in more detail, an interesting observation was made. At 10 min, almost all adherent cells were simply attached to collagen, independent of activation, with 94 ± 1.3% of attached nonactivated cells and 98 ± 2.1% attached cells after outside and 96 ± 1.9% after inside-out stimulation, respectively. At 60 min, however, a significant proportion of the adherent 21-expressing CHO cells stimulated with Mn²⁺ (outside) obtained a spread morphology in contrast to nonactivated and inside-out stimulated 21-expressing CHO cells (p < 0.05; Fig. 4B). These results indicate that the different active conformational states, induced after inside-out and outside manipulation, have a functional relevance in adhesion and spreading. Finally, we could demonstrate by fluorescent images that spread 21-expressing CHO cells, obtained after outside manipulation, did not stained positive for IAC-1 in contrast to attached 21-expressing CHO cells, obtained after inside-out manipulation (Fig. 5). Staining of 21-expressing CHO cells with the unrelated mouse anti-human IgG MOPC-21 did not result in a fluorescence signal (data not shown), and staining with the anti-a2 I domain mAb AK7 always resulted in a positive FITC signal, independent of stimulation (Fig. 5).

View larger version (39K): [in this window] [in a new window]   FIGURE 5. Double immunofluorescence staining of actin and 21 on 21-expressing CHO cells. Inside-out or outside stimulated 21-expressing CHO cells were allowed to adhere to reconstituted human collagen type I coated cover glasses for 60 min. After fixation and permeabilization, cells were incubated with phalloidin-Texas Red to stain actin and IAC-1-FITC to stain activated 21. Incubation of stimulated cells with AK7, followed by rabbit-anti-mouse IgG-FITC, was used as a positive control to stain 21 Representative images are shown of attached cells (inside-out) and spread cells (outside). Bar, 10 µm.

We started by inducing inside-out signaling on these cell types with the synthetic peptide Ac-KRFYVVMWK-NH₂, known to activate indirectly 21 on platelets and smooth muscle cells and V3 on white blood cells by binding to the thrombospondin-1 receptor CD47 (29, 30, 35). As expected, incubation with Ac-KRFYVVMWK-NH₂ resulted in a significant increase of both collagen and IAC-1 binding on platelets from 1.8 ± 0.8 to 29.7 ± 1.9% and from 1.5 ± 0.9 to 24.5 ± 1.5%, respectively (p < 0.05, Fig. 6A). Moreover, addition of Ac-KRFYVVMWK-NH₂ to PBMC was also able to induce both collagen and IAC-1 binding (Fig. 6), demonstrating that CD47 on white blood cells cannot only modulate 3 integrins but also the 1 integrin 21. This peptide, however, was not able to activate 21 on Jurkat cells because no increase in collagen nor IAC-1 binding was observed (Fig. 6). This is consistent with previous reports where it has been suggested that the function of CD47, although highly expressed on Jurkat cells (36), appears to be integrin-independent (37, 38).

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Inside-out activation of 21 on platelets, PBMC, and Jurkat cells using the Ac-KRFYVVMWK-NH2 peptide. Washed human platelets, human PBMC, or Jurkat cells were incubated without (gray shaded or white bar) or with the activating peptide Ac-KRFYVVMWK-NH2 (50 µM, black line or gray bar) for 30 min at room temperature. Next, cells were incubated with either collagen-FITC (40 µg/ml) or IAC-1-FITC (10 µg/ml) for 30 min at room temperature. A representative histogram is shown for each cell type (A). Data are presented as the mean ± S.E. of the MFI of triplicate assays.*, p < 0.05 (B).

Just as for platelets and 21-expressing CHO cells, we stimulated PBMC and Jurkat cells via outside manipulation. Here we only used divalent cations for activation because TS2/16 had no effect on these cell types, and addition of DTT resulted in a marked down-regulation of 21, as determined by AK-7 binding (data not shown). The latter is consistent with previous reports describing that DTT can affect integrin expression on lymphocytes (39). Activation of 21 on PBMC and Jurkat cells by the divalent cations Mn²⁺ and Co²⁺ resulted in a significant increase in collagen binding, indicating that 21 was activated (p < 0.05; Fig. 8). Most surprisingly, and in striking contrast to platelets and 21-expressing CHO cells, this outside activation of 21 was accompanied by a significant increase in IAC-1 binding (p < 0.05; Fig. 8). Next, in line with 21-expressing CHO cells, a time course of adhesion to coated collagen type I was determined for the Jurkat cells. Also, a significantly increased cell adhesion after stimulation was observed, with no difference between outside and inside-out manipulation (Fig. 9A). Evaluation of the cell morphology of the adherent Jurkat cells revealed at every time point and after every stimulation merely attached cells (Fig. 9B). Taken together, these results demonstrated that the different conformational states of 21 are cell type-dependent and confirmed their functional relevance in adhesion and spreading.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
During the last years, much progress has been made in unraveling the complexity of the broad spectrum of possible conformational states in integrins (9, 40, 41). Collective observations from electron microscopy, crystal structures, mutational introduction of disulfides, and other functional studies suggest that integrins may assume at least three different activation states, represented by different quaternary conformations as follows: (i) a closed conformation (resting state), not able to bind its natural ligand; (ii) an extended conformation with closed headpiece (low affinity state); and (iii) an extended conformation with open headpiece (high affinity state) (10, 15–17, 42). These two extended, active conformations are both favored by outside-in and inside-out signaling and can bind their natural ligand.

For the integrin 21, a major collagen receptor on platelets, no such experimental data were reported thus far, mainly because until recently, no antibodies were available that specifically recognize activated or ligand-bound forms. We have developed mAb IAC-1, sensitive to the molecular conformation of 21 because it only binds to the activated form of 21 on platelets, induced via inside-out signaling (25). The aim of the this study was to use this mAb IAC-1 to evaluate the effects of different inside-out and outside manipulations on the conformational state of 21.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Inside-out activation of 21 on PBMC and Jurkat cells using the KLGFFKR peptide. Human PBMC or Jurkat cells were incubated with the activating peptide H-VTVLALGALAGVGVGKLGFFKR-NH2 (200 µM, black line or gray bar) or the control peptide H-VTVLALGALAGVGVGTKSYNE-NH2 (200 µM, gray shaded or white bar) for 30 min at room temperature. Next, cells were incubated with either collagen-FITC (40 µg/ml) or IAC-1-FITC (10 µg/ml) for 30 min at room temperature. A representative histogram is shown for each cell type (A). Data are presented as the mean ± S.E. of the MFI of triplicate assays.*, p < 0.05 (B).

For L2, the mAb MEM148 has been described that recognizes an epitope not expressed in the resting integrin state but exposed upon Mg²⁺/EGTA treatment (49). Just recently, the epitope of this mAb has been mapped to a critical proline residue located on the inner face of the 2 hybrid domain (50). In this study, L2-expressing MOLT-4 cells could bind MEM148 after Mg²⁺/EGTA treatment and were able to bind the ligand ICAM-1 as expected, but most interestingly, treatment with the activator phorbol ester 12-myristate 13-acetate (PMA) was not able to induce detectable expression of the MEM148 epitope on these cells. This was rather unexpected because PMA is known to promote L2 ligand binding (51). So it appears that the mAb MEM148 recognizes an epitope that become accessible after outside manipulation (by the divalent cation magnesium) but not after inside-out stimulation (by PMA). The specificity of this mAb for different conformational changes in the integrin is in line with our present data; however, we observed no binding of IAC-1 after outside manipulation but only after inside-out stimulation. Still, we could demonstrate also that our mAb IAC-1 can distinguish between conformational states of 21 induced after different agonistic stimulations.

View larger version (24K): [in this window] [in a new window]   FIGURE 8. Outside activation of 21 on PBMC and Jurkat cells. Human PBMC or Jurkat cells were incubated without an agonist (gray shaded) or with Mn2+ (1 mM, black line) or Co2+ (1 mM, dotted gray line) for 30 min at room temperature. Next, cells were incubated with either collagen-FITC (40 µg/ml, gray bars) or IAC-1-FITC (10 µg/ml, white bars) for 30 min at room temperature. A representative histogram is shown for each cell type (A). Data are presented as the mean ± S.E. of the MFI of triplicate assays (B).

View larger version (27K): [in this window] [in a new window]   FIGURE 9. Static adhesion, attachment, and spreading of Jurkat cells on collagen after stimulation. Unstimulated Jurkat cells ( or white bar) or Jurkat cells stimulated with Mn2+ (1 mM, • or striped bar) or the peptide H-VTVLALGALAGVGVGKLGFFKR-NH2 (200 µM, or gray bar) were allowed to adhere to reconstituted human collagen type I-coated wells (A) or coverslips (B) for different periods up to 120 min. A, adherent cells were fixed and stained, and the optical density (OD) was determined. Specific binding was measured by subtracting nonspecific (ns) adhesion to noncoated wells. Data are presented as the mean ± S.E. of triplicate assays, performed in duplicate. B, coverslips were fixed with 2% paraformaldehyde and stained with hematoxilin/fuchsin, and the number of spread cells was determined and expressed relatively to the number of adherent cells (= 100%). Data are presented as the mean ± S.E. of triplicate assays. A representative picture of Jurkat cells at 60 min and stimulated with Mn2+ is shown. Bar, 15 µM.

Apart from studying the effect of outside manipulation on platelets and 21-expressing CHO cells, we also performed experiments with PBMC and Jurkat cells. Also, inside-out signaling of 21 always resulted in both collagen and IAC-1 binding. Most unexpectedly, outside manipulation with divalent cations not only resulted in collagen binding but was accompanied by an increase in IAC-1 binding, which is in contrast to what was seen upon outside manipulation of platelets or 21-expressing CHO cells. Although this discrepancy in IAC-1 binding after outside manipulation between platelets and 21-expressing CHO cells on the one hand and PBMC and Jurkat cells on the other hand remains to be clarified, it is known that integrins can display different ligand binding properties and functions, depending on the cell type on which the integrin is expressed (55). Similar to our findings with IAC-1, a very recent study reported that the exposure of the neo-epitope of MEM148 (already mentioned above) could vary after stimulation, depending on the cell type where the integrin is expressed (50). This study demonstrated that MEM148 could bind to L2-expressing MOLT-4 cells after Mg²⁺/EGTA stimulation but not after PMA treatment (50), which is in contrast to experiments performed previously, where L2-expressing myeloid cells did bind MEM148 after PMA stimulation (56).

In conclusion, the data presented here show that at least two different functionally active conformational states of 21 exist depending on the way the integrin is stimulated and on the cell type where 21 is expressed, and furthermore that these different conformations correlate with the different activities of the integrin.

	FOOTNOTES

 
^* This work was supported by Grant GOA/2004/09 from the Katholieke Universiteit Leuven, the Hungarian Research Fund/OTKA Grant T037719, and a Bilateral Collaboration Grant BIL/04/36. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen.

² To whom correspondence should be addressed: Laboratory for Thrombosis Research, Interdisciplinary Research Centre, Katholieke Universiteit Leuven, Campus Kortrijk, E. Sabbelaan 53, 8500 Kortrijk, Belgium. Tel.: 32-56246422; Fax: 32-56246997; E-mail: Hans.deckmyn{at}kulak.ac.be.

³ The abbreviations used are: mAb, monoclonal antibody; PBS, phosphate-buffered saline; Mn²⁺, manganese(II) chloride tetrahydrate; Co²⁺, cobalt chloride; DTT, dithiothreitol, PGE₁, prostaglandin E₁; CHO, Chinese hamster ovary; PBMC, peripheral blood mononuclear cells; RP-HPLC, reverse phase high performance liquid chromatography; MFI, mean fluorescence intensity; PMA, phorbol 12-myristate 13-acetate; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; MEM, minimum Eagle's medium.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. N. Kieffer (Laboratoire de Biologie et Physiologie Intégréé, Université du Luxembourg, Luxembourg) for the 21-expressing CHO cells, Dr. T. Edgington (Scripps Institute, La Jolla, CA) for the antibody 7E3, and Dr. M. Kahn (University of Pennsylvania, PA) for the antibody TS2/16. We also acknowledge C. Bouckaert and B. Strynck for histological assistance and P. Vandenberghe for help with the cell cultures.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Ruoslahti, E., and Vaheri, A. (1997) Curr. Opin. Cell Biol. 9, 605-607[CrossRef][Medline] [Order article via Infotrieve]
2. Humphries, M. J. (2000) Biochem. Soc. Trans. 28, 311-339[Medline] [Order article via Infotrieve]
3. Hynes, R. O. (2002) Cell 110, 673-687[CrossRef][Medline] [Order article via Infotrieve]
4. Hughes, P. E., Diaz-Gonzalez, F., Leong, L., Wu, C., McDonald, J. A., Shattil, S. J., and Ginsberg, M. H. (1996) J. Biol. Chem. 271, 6571-6574[Abstract/Free Full Text]
5. Luo, B. H., Springer, T. A., and Takagi, J. (2004) PLoS. Biol. 2, 776-786
6. O'Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K., Tamura, R., Quaranta, V., Loftus, J. C., Shattil, S. J., and Ginsberg, M. H. (1994) J. Cell Biol. 124, 1047-1059[Abstract/Free Full Text]
7. Pardi, R., Bossi, C., Inverardi, L., Rovida, E., and Bender, J. R. (1995) J. Immunol. 155, 1252-1263[Abstract]
8. Kanse, S. M., Matz, R. L., Preissner, K. T., and Peter, K. (2004) Arterioscler. Thromb. Vasc. Biol. 24, 2251-2256[Abstract/Free Full Text]
9. Mould, A. P., and Humphries, M. J. (2004) Curr. Opin. Cell Biol. 16, 544-551[CrossRef][Medline] [Order article via Infotrieve]
10. Takagi, J., Petre, B. M., Walz, T., and Springer, T. A. (2002) Cell 110, 599-611[CrossRef][Medline] [Order article via Infotrieve]
11. Xiong, J. P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S. L., and Arnaout, M. A. (2002) Science 296, 151-155[Abstract/Free Full Text]
12. Adair, B. D., and Yeager, M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 14059-14064[Abstract/Free Full Text]
13. Xiao, T., Takagi, J., Coller, B. S., Wang, J. H., and Springer, T. A. (2004) Nature 432, 59-67[CrossRef][Medline] [Order article via Infotrieve]
14. Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S., and Sklar, L. A. (2001) J. Biol. Chem. 276, 48670-48678[Abstract/Free Full Text]
15. Takagi, J., Strokovich, K., Springer, T. A., and Walz, T. (2003) EMBO J. 22, 4607-4615[CrossRef][Medline] [Order article via Infotrieve]
16. Jin, M., Andricioaei, L., and Springer, T. A. (2004) Structure (Camb.) 12, 2137-2147[Medline] [Order article via Infotrieve]
17. Shimaoka, M., Xiao, T., Liu, J. H., Yang, Y. T., Dong, Y. C., Jun, C. D., McCormack, A., Zhang, R. G., Joachimiak, A., Takagi, J., Wang, J. H., and Springer, T. A. (2003) Cell 112, 99-111[CrossRef][Medline] [Order article via Infotrieve]
18. Hemler, M. E. (1990) Annu. Rev. Immunol. 8, 365-400[CrossRef][Medline] [Order article via Infotrieve]
19. Goldman, R., Harvey, J., and Hogg, N. (1992) Eur. J. Immunol. 22, 1109-1114[Medline] [Order article via Infotrieve]
20. Gullberg, D. E., and Lundgren-Akerlund, E. (2002) Prog. Histochem. Cytochem. 37, 3-54[CrossRef][Medline] [Order article via Infotrieve]
21. Santoro, S. A. (1986) Cell 46, 913-920[CrossRef][Medline] [Order article via Infotrieve]
22. Elices, M. J., and Hemler, M. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9906-9910[Abstract/Free Full Text]
23. Santoro, S. A., and Zutter, M. M. (1995) Thromb. Haemostasis 74, 813-821[Medline] [Order article via Infotrieve]
24. Gendron, S., Couture, J., and Aoudjit, F. (2003) J. Biol. Chem. 278, 48633-48643[Abstract/Free Full Text]
25. Schoolmeester, A., Vanhoorelbeke, K., Katsutani, S., Depraetere, H., Feys, H. B., Heemskerk, J. M., Hoylaerts, M. F., and Deckmyn, H. (2004) Blood 104, 390-396[Abstract/Free Full Text]
26. Altieri, D. C., and Edgington, T. S. (1988) J. Immunol. 141, 2656-2660[Abstract]
27. Jandrot-Perrus, M., Lagrue, A. H., Okuma, M., and Bon, C. (1997) J. Biol. Chem. 272, 27035-27041[Abstract/Free Full Text]
28. Stewart, J. M. (1976) J. Macromol. Sci.-Chem. 10, 259-288
29. Chung, J., Wang, X. Q., Lindberg, F. P., and Frazier, W. A. (1999) Blood 94, 642-648[Abstract/Free Full Text]
30. Wang, X. Q., and Frazier, W. A. (1998) Mol. Biol. Cell 9, 865-874[Abstract/Free Full Text]
31. Wang, Z., Leisner, T. M., and Parise, L. V. (2003) Blood 102, 1307-1315[Abstract/Free Full Text]
32. Depraetere, H., Wille, C., Gansemans, Y., Stanssens, P., Lauwereys, M., Baruch, D., De Reys, S., and Deckmyn, H. (1997) Thromb. Haemostasis 77, 981-985[Medline] [Order article via Infotrieve]
33. Mekrache, M., Legendre, P., Kieffer, N., and Baruch, D. (2002) Br. J. Haematol. 119, 1024-1032[CrossRef][Medline] [Order article via Infotrieve]
34. Perrault, C., Mekrache, M., Schoevaert, D., Kieffer, N., Melchior, C., Warszawski, J., and Baruch, D. (1998) Cell Adhes. Commun. 6, 335-348[Medline] [Order article via Infotrieve]
35. Gao, A. G., Lindberg, F. P., Dimitry, J. M., Brown, E. J., and Frazier, W. A. (1996) J. Cell Biol. 135, 533-544[Abstract/Free Full Text]
36. Waclavicek, M., Majdic, O., Stulnig, T., Berger, M., Baumruker, T., Knapp, W., and Pickl, W. F. (1997) J. Immunol. 159, 5345-5354[Abstract]
37. Reinhold, M. I., Lindberg, F. P., Kersh, G. J., Allen, P. M., and Brown, E. J. (1997) J. Exp. Med. 185, 1-11[Abstract/Free Full Text]
38. Ticchioni, M., Raimondi, V., Dejoux, O., Bernard, G., and Bernard, A. (1998) FASEB J. 12, 577 (abstr.)
39. Woods, M. L., Cabanas, C., and Shimizu, Y. (2000) Eur. J. Immunol. 30, 38-49[CrossRef][Medline] [Order article via Infotrieve]
40. Takagi, J., and Springer, T. A. (2002) Immunol. Rev. 186, 141-163[CrossRef][Medline] [Order article via Infotrieve]
41. Springer, T. A. (2002) Curr. Opin. Struct. Biol. 12, 802-813[CrossRef][Medline] [Order article via Infotrieve]
42. Xiong, J. P., Stehle, T., Goodman, S. L., and Arnaout, M. A. (2003) Blood 102, 1155-1159[Abstract/Free Full Text]
43. Kawaguchi, S., and Hemler, M. E. (1993) J. Biol. Chem. 268, 16279-16285[Abstract/Free Full Text]
44. Kamata, T., Puzon, W., and Takada, Y. (1994) J. Biol. Chem. 269, 9659-9663[Abstract/Free Full Text]
45. Kamata, T., and Takada, Y. (1994) J. Biol. Chem. 269, 26006-26010[Abstract/Free Full Text]
46. Mould, A. P., Garratt, A. N., Puzon-McLaughlin, W., Takada, Y., and Humphries, M. J. (1998) Biochem. J. 331, 821-828[Medline] [Order article via Infotrieve]
47. Mekrache, M., Kieffer, N., and Baruch, D. (2002) Br. J. Haematol. 116, 636-644[CrossRef][Medline] [Order article via Infotrieve]
48. Ni, H. Y., Li, A. L., Simonsen, N., and Wilkins, J. A. (1998) J. Biol. Chem. 273, 7981-7987[Abstract/Free Full Text]
49. Tan, S. M., Robinson, M. K., Drbal, K., van Kooyk, Y., Shaw, J. M., and Law, S. K. A. (2001) J. Biol. Chem. 276, 36370-36376[Abstract/Free Full Text]
50. Tang, R. H., Tng, E., Law, S. K., and Tan, S. M. (2005) J. Biol. Chem. 280, 29208-29216[Abstract/Free Full Text]
51. Tng, E., Tan, S. M., Ranganathan, S., Cheng, M., and Law, S. K. A. (2004) J. Biol. Chem. 279, 54334-54339[Abstract/Free Full Text]
52. Henderson, R. B., Lim, L. H. K., Tessier, P. A., Gavins, F. N. E., Mathies, M., Perretti, M., and Hogg, N. (2001) J. Exp. Med. 194, 219-226[Abstract/Free Full Text]
53. Salas, A., Shimaoka, M., Kogan, A. N., Harwood, C., von Andrian, U. H., and Springer, T. A. (2004) Immunity 20, 393-406[CrossRef][Medline] [Order article via Infotrieve]
54. Lecut, C., Schoolmeester, A., Kuijpers, M. J., Broers, J. L., Van Zandvoort, M. A., Vanhoorelbeke, K., Deckmyn, H., Jandrot-Perrus, M., and Heemskerk, J. W. (2004) Arterioscler. Thromb. Vasc. Biol. 24, 1727-1733[Abstract/Free Full Text]
55. Hemler, M. E., Elices, M. J., Chan, B. M. C., Zetter, B., Matsuura, N., and Takada, Y. (1990) Cell Differ. Dev. 32, 229-238[CrossRef][Medline] [Order article via Infotrieve]
56. Drbal, K., Angelisova, P., Hilgert, I., Cerny, J., Novak, P., and Horejsi, V. (2001) Blood 98, 1561-1566[Abstract/Free Full Text]

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