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© The Rockefeller University Press, 0021-9525/1997//1433 $5.00
The Journal of Cell Biology, Volume 137, Number 6, , 1997 1433-1443

Article

Affinity Modulation of Platelet Integrin {alpha}IIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail



Hirokazu Kashiwagi*, Martin A. Schwartz*, Martin Eigenthaler*, K.A. Davis§, Mark H. Ginsberg*, and Sanford J. Shattil*,{ddagger}

* Department of Vascular Biology, {ddagger} Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037; and § Becton-Dickinson Immunocytometry Systems, San Jose, California 95131

Platelet agonists increase the affinity state of integrin {alpha}IIbβ3, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β3-Endonexin is a novel 111–amino acid protein that binds selectively to the β3 tail. Since β3-endonexin is present in platelets, we asked whether it can affect {alpha}IIbβ3 function. When β3-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor {alpha}IIbβ3 affinity state in transfected cells by flow cytometry. Cells transfected with GFP and {alpha}IIbβ3 bound little or no PAC1. However, those transfected with GFP/β3-endonexin and {alpha}IIbβ3 bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β3-endonexin did not affect levels of surface expression of {alpha}IIbβ3 nor did it modulate the affinity of an {alpha}IIbβ3 mutant that is defective in binding to β3-endonexin. Affinity modulation of {alpha}IIbβ3 by GFP/β3-endonexin was inhibited by coexpression of either a monomeric β3 cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β3-endonexin can modulate the affinity state of {alpha}IIbβ3 in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of {alpha}IIbβ3.

Integrins are {alpha}β heterodimers and each subunit contains a relatively large extracellular domain, a membrane-spanning domain, and a 20–70–amino acid cytoplasmic tail. They function in cell adhesion and signaling by interacting with extracellular matrix proteins or cellular counter-receptors on the one hand, and with intracellular proteins on the other (8, 34, 59). The adhesive function of many integrins is subject to rapid regulation by two processes collectively referred to as "inside-out" signaling: (a) a structural change intrinsic to the heterodimer, and (b) clustering of heterodimers within the plane of the plasma membrane. The former modulates the affinity of the ligand–receptor interaction and thus is often referred to as "affinity modulation." The latter increases the valency and, therefore, the avidity of the interaction. These two types of regulation are not mutually exclusive, and their relative contributions probably vary with the integrin and the cell type (12, 20, 62, 71).

A good example of the pathophysiological significance of rapid integrin regulation involves platelet {alpha}IIbβ3. Circulating platelets ordinarily do not interact with each other or with the blood vessel wall. However, when the vessel is damaged by trauma or disease, platelets become activated and {alpha}IIbβ3 is converted within seconds into a functional receptor for several Arg-Gly-Asp–containing ligands, including fibrinogen and von Willebrand factor. Since ligand binding is required for platelet aggregation, inside-out signaling is a prerequisite for primary hemostasis and for formation of occlusive platelet thrombi in vascular diseases (9, 27). Affinity modulation is thought to be responsible for the initial, reversible phase of fibrinogen binding to platelets, while integrin clustering may be involved in stabilizing the interaction (14, 52).

Studies with intact and permeabilized platelets indicate that specific intracellular mediators promote rapid increases or decreases in ligand binding to {alpha}IIbβ3. Excitatory platelet agonists, such as thrombin, increase ligand binding by a process that involves heterotrimeric G proteins and protein and lipid kinases (38, 61, 69, 74). On the other hand, substances such as prostacyclin and nitric oxide, which stimulate protein kinase A and protein kinase G, respectively, inhibit or reverse ligand binding (22, 28). In addition to intracellular mediators, the cytoplasmic tails of {alpha}IIbβ3 appear to participate in the regulation of fibrinogen binding. Platelets from patients with variant forms of Glanzmann thrombasthenia due to a deletion or mutation in the β3 cytoplasmic tail fail to aggregate in response to agonists despite near normal levels of {alpha}IIbβ3 (6; Wang, R., D.R. Ambruso, and P.J. Newman. 1994. Blood. 84:244a). However, it is not clear how intracellular signals affect the cytoplasmic tails of {alpha}IIbβ3 or how changes at the level of these tails regulate ligand binding. One hypothesis is that specific intracellular proteins bind to the tails and promote a structural change that is propagated across the plasma membrane to the extracellular face of the receptor. Accordingly, recent efforts have focused on identifying proteins that interact with integrin cytoplasmic tails (11).

Using a yeast two-hybrid screening strategy, we recently discovered a novel 111–amino acid polypeptide called β3endonexin, which is capable of binding to the cytoplasmic tail of the β3 integrin subunit, both in yeast and in vitro (63). However, it fails to bind to other integrin tails, including those of β1, β2, and {alpha}IIb. Since β3-endonexin is expressed in platelets, the present studies were carried out to determine whether this protein can modulate the ligandbinding function of {alpha}IIbβ3. Using a CHO cell model system to transiently express {alpha}IIbβ3 and β3-endonexin, we now report that this protein can increase the affinity state and the adhesive function of {alpha}IIbβ3. Moreover, these effects are structurally specific and subject to metabolic regulation.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Mammalian expression vectors for green fluorescent protein (GFP)1 (pS65T-C1 and pEGFP-C1) were obtained from Clontech (Palo Alto, CA). Monoclonal antibodies PAC1, A2A9, D57, anti-LIBS1, and anti-LIBS6 were obtained from ascites and purified as described (30). PAC1 was conjugated to phycoerythrin (PE) by first derivatizing it with N-succinimidyl S-acetylthioacetate (Pierce Chemical Co., Rockford, IL). SH groups were deprotected with hydroxylamine, and the antibody was then coupled to PE that had been derivatized with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Pierce Chemical Co.). PE:PAC1 conjugates (1:1 mol/mol) were isolated by sizing on a Superose 6 column (Pharmacia Fine Chemicals, Piscataway, NJ). In one experiment, PAC1 IgM{kappa} was first reduced to the 185-kD monomer at pH 8.6 with 20 mM cysteine before conjugating to PE (46).

DNA Constructs
To express β3-endonexin as a protein fused to the carboxy terminus of GFP, β3-endonexin cDNA was excised from a yeast expression vector with XbaI and BamHI (63), and the recessed 3' termini were filled in using Klenow (Boehringer Mannheim Biochemicals, Indianapolis, IN). The GFP vectors, pS65T-C1 and pEGFP-C1, were cut with XhoI, blunt-ended with Klenow, and ligated to β3-endonexin with T4 DNA ligase (Boehringer Mannheim Biochemicals). After transformation of DH5{alpha}, clones in the correct orientation were selected by PCR using a sense primer in GFP and an antisense primer in β3-endonexin.

Plasmid DNA encoding the cytoskeletal protein, VASP, was a gift from Ulrich Walter and Thomas Jarchau (25) (Medizinische Universitatsklinik, Wurzburg, Germany). The coding sequence of VASP was amplified with Pfu polymerase (Stratagene, La Jolla, CA) using a sense primer containing an XhoI site and an antisense primer with an HindIII site. The digested PCR fragment was subcloned into XhoI- and HindIII-cut pEGFP-C1 so that VASP would be expressed in-frame at the carboxy terminus of GFP. Plasmid DNA encoding FRNK, an autonomously expressed carboxy-terminal segment of pp125FAK, was a gift from Michael Schaller (University of North Carolina, Chapel Hill, NC) (57). FRNK was amplified with Pfu polymerase with a sense primer containing a BglII site and an antisense primer containing an EcoRI site. The digested PCR fragment was subcloned into BglII- and EcoRI-cut pEGFP-C1 so that FRNK would be expressed in-frame at the carboxy terminus of GFP.

pCDM8 expression vectors encoding wild-type {alpha}IIb, β3, a mutant form of β3 containing a single amino acid substitution (S752P), and H-Ras (G12V) have been described (33, 49). Tac-β3 and Tac-{alpha}5 chimeras were in the vector, CMV-IL2R (7). All expression plasmids were amplified in Escherichia coli and purified (Plasmid Maxi Kit; Qiagen, Inc., Chatsworth, CA). Before use in transfection experiments, each plasmid was sequenced in the Scripps Research Institute DNA Core Facility to confirm the authenticity of the coding sequences.

Transient Protein Expression in CHO Cells
cDNAs were transfected into CHO-K1 cells with Lipofectamine (GIBCO BRL, Gaithersburg, MD). A total of 5 µg of plasmid DNA and 20 µl of Lipofectamine solution was incubated for 10 min in 200 µl of DME and then diluted with 3.8 ml of DME. Unless otherwise indicated, the amount of DNA per transfection included 0.5 µg each of {alpha}IIb and β3 and varying amounts of the GFP plasmids to obtain equivalent degrees of GFP expression (e.g., 4 µg of pS65T, 4 µg of pS65T/β3-endonexin, 0.02 µg of pEGFP, 0.2 µg of pEGFP/β3-endonexin, or 0.05 µg of pEGFP/VASP). When necessary, an empty vector (pcDNA3; Invitrogen, San Diego, CA) was included to equalize the amount of DNA transfected. In some experiments, 2 µg of the Tac-β3, Tac-{alpha}5, or H-Ras (G12V) plasmid was cotransfected along with pEGFP/β3-endonexin and the plasmids for {alpha}IIb and β3. DNA/ Lipofectamine mixtures were added to CHO cells at 30–50% confluence in a 100-mm tissue-culture plate. 6 h later, the medium was changed to DME containing 10% FBS, 1% nonessential amino acids, 2 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. 48 h after transfection, the cells were evaluated biochemically and by flow cytometry.

Evaluation of GFP/β3-Endonexin Expression in CHO Cells
Expression of GFP/β3-endonexin fusion proteins was confirmed by immunoblotting. 48 h after transfection, the cells were lysed for 30 min at 4°C in a lysis buffer containing 1% Triton X-100, 0.9% NaCl, 1 mM CaCl2, 50 mM Tris, pH 7.2, and protease inhibitors (100 U/ml aprotinin, 0.5 mM leupeptin, 4 mM Pefabloc) (63). After clarification of the lysate in a microfuge, protein concentration was determined with a bicinchoninic acid reagent (BCA; Pierce Chemical Co.). 30 µg of each sample was then electrophoresed in 14% SDS-polyacrylamide gels under reducing conditions. After transfer to nitrocellulose, immunoblotting was performed with a rabbit polyclonal antibody reactive with GFP (Clontech) or rabbit antibodies reactive with β3-endonexin (63). After the addition of affinity-purified, HRP-conjugated goat anti–rabbit IgG, the blots were developed for 0.1–1 min using the enhanced chemiluminescence reaction (ECL; Amersham Corp., Arlington Heights, IL).

To study the binding of GFP/β3-endonexin to the β3 integrin cytoplasmic tail, the 47–amino acid β3 tail was expressed in bacteria with a (His)6 tag at its amino terminus (pET His Tag System; Novagen, Inc., Madison, WI), and then immobilized on a nickel-agarose matrix. Transiently transfected CHO cells expressing equivalent amounts of GFP/β3-endonexin or GFP were lysed in 0.4 ml of the Triton X-100 lysis buffer. After clarification, 0.35 ml was diluted with an equal volume of lysis buffer containing no Triton, and each sample was batch-incubated with 0.1 ml packed volume of the His-β3 tail affinity matrix for 12 h at 4°C while shaking. After washing the matrices five times with 10 vol of lysis buffer, bound proteins were eluted into 0.7 ml of lysis buffer by addition of 1 M imidazole. Samples were electrophoresed on 12% SDS-polyacrylamide gels under reducing conditions and transferred to nitrocellulose. Immunoblotting was performed with the polyclonal anti-GFP antibody.

To evaluate whether GFP/β3-endonexin could be specifically coimmunoprecipitated with the β3 integrin subunit from CHO cells, immunoprecipitation studies were carried out in the Trition X-100 lysis buffer essentially as described (30). The β3 integrin subunit was immunoprecipitated from 2-mg aliquots of cell lysate using 10 µg/ml of the murine mAb, SSA6, and protein A–Sepharose (1). Control immunoprecipitations were carried out with affinity-purified mouse IgG (Zymed Laboratories, Inc., South San Francisco, CA) and with an isotype-matched mAb against von Willebrand factor, RG 7 (a gift from Zaverio Ruggeri; Scripps Research Institute, La Jolla, CA). Samples were electrophoresed on 10% SDS-polyacrylamide gels under reducing conditions and subsequent Western blots were probed with the polyclonal anti-GFP antibody. To demonstrate equivalent recovery of the β3 integrin subunit in the immunoprecipitates, blots were stripped and reprobed with Ab 8035, a rabbit polyclonal antibody specific for β3.

Evaluation of {alpha}IIbβ3 Affinity State
48 h after transfection, CHO cells were resuspended at 1–2 x 106 cells per ml in Tyrode's buffer containing 2 mM CaCl2 and MgCl2 (49). Cells were then incubated in the dark in 50-µl aliquots with 20 µg/ml PE-PAC1 for 30 min at room temperature. Some samples also contained an anti-β3 antibody (2% anti-LIBS6 ascites) that stabilizes {alpha}IIbβ3 in a high affinity state (30). Others contained an {alpha}IIbβ3-selective inhibitor of ligand binding (either 2 µM Ro 43-5054 or 10 µM Integrilin) (2, 56). Samples were then diluted with 0.5 ml Tyrode's buffer containing 10 µg/ml propidium iodide (Sigma Chemical Co., St. Louis, MO) and analyzed on a FACS®can or FACS®Calibur flow cytometer (Becton Dickinson, San Jose, CA). In one set of experiments, the cells were preincubated for 30 min at room temperature with 4 mg/ml of 2-deoxy-d-glucose (Sigma Chemical Co.) and 0.2% sodium azide before incubation with PE-PAC1.

After electronic compensation of the FL1, FL2, and FL3 fluorescence channels, PE-PAC1 binding (FL2) was analyzed on the gated subset of live cells (propidium iodide–negative, FL3 channel) that were positive for GFP expression (FL1 channel). PAC1 binding was expressed as an "activation index" calculated from median fluorescence intensity measurements (49). The activation index is defined as 100 x (Fx-Fi)/(Fm-Fi), where Fx is PAC1 fluorescence in the absence and Fi is PAC1 fluorescence in the presence of Ro 43-5054 or Integrilin. Fm is PAC1 fluorescence in the presence of anti-LIBS6.

Fibrinogen Binding Assay
Fibrinogen binding to GFP-positive cells was determined by flow cytometry using biotinylated anti-LIBS1, which recognizes a fibrinogen-sensitive epitope on the β3 subunit (19). Cells were prepared as for the PAC1 binding studies and incubated for 30 min in the dark at room temperature with fibrinogen (250 µg/ml; Enzyme Research Laboratories, South Bend, IN), biotin-LIBS1 (20 µg/ml), and phycoerythrin-streptavidin (4% final dilution; Molecular Probes, Inc., Eugene, OR). To calculate the activation index, some aliquots were incubated with anti-LIBS6 to induce maximal fibrinogen binding, while others were incubated with the function-blocking anti-{alpha}IIbβ3 {alpha}ntibody, A2A9, to determine nonspecific fibrinogen binding. Cells were then diluted with Tyrode's buffer containing 10 µg/ml propidium iodide, and analyzed by flow cytometry.

CHO Cell Aggregation Assay
Fibrinogen-dependent aggregation of CHO cells was quantitated by flow cytometry as described (16), with minor modifications. First, CHO cells stably expressing {alpha}IIbβ3 (49) were labeled with a red fluorescent tracer, hydroxyethidine (Polysciences Inc., Junction City, OR). Then 250 µl of these cells (4 x 106/ml) were added to siliconized glass cuvettes containing 250 µl of cells (2 x 106/ml) that had been transfected with GFP/β3-endonexin (or GFP) and {alpha}IIbβ3. After addition of 300 µg/ml fibrinogen, the cells were stirred with a magnetic stir bar at 1,000 rpm for 20 min at room temperature. In some cases, the incubations with fibrinogen were also carried out in the presence of 20 µg/ml A2A9 or 10 µM Integrilin to inhibit fibrinogen binding. Incubations were stopped by addition of 0.25% formaldehyde, and the samples were kept on ice for 30 min before flow cytometric detection of mixed red–green cellular aggregates.

Subcellular Localization of GFP/β3-Endonexin
48 h after transfection, CHO cells were cultured on fibrinogen-coated coverslips for 2 h at 37°C, and then processed and analyzed by fluorescence microscopy for expression of GFP, {alpha}IIb, and β3 as described (32). HMEC-1 human endothelial cells, which express {alpha}Vβ3, were similarly cultured on fibrinogen-coated coverslips, and then microinjected with plasmid DNA (0.5 µg/µl) encoding various GFP proteins (45). After 4 h at 37°C, the cells were processed and analyzed by fluorescence microscopy for expression of GFP, {alpha}V, and β3.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Expression of β3-Endonexin in CHO Cells
CHO cells provide a useful model system for characterizing the adhesive and signaling functions of ectopically expressed {alpha}IIbβ3 (43, 49, 50). Therefore, β3-endonexin was transiently coexpressed with {alpha}IIbβ3 in these cells to study its effects on the ligand-binding function of this integrin. β3-Endonexin cDNA was fused in-frame to the 3' end of two different versions of GFP in a mammalian expression plasmid. One form (S65T) is red-shifted and the other (EGFP) is both red-shifted and codon-optimized for mammalian expression. 48 h after transfection, expression of recombinant proteins was assessed by Western blotting of cell lysates. GFP/β3-endonexin was detectable using an anti-GFP antibody, and the codon-optimized plasmid provided higher levels of protein expression for a given amount of DNA transfected (Fig. 1). Subsequently, therefore, the amount of each plasmid used was adjusted to obtain roughly equivalent amounts of GFP/β3-endonexin expression, and the plasmids were used interchangeably in the following experiments. GFP/β3-endonexin was also detectable with polyclonal antibodies raised against either recombinant human β3-endonexin or a synthetic peptide consisting of the carboxy-terminal 17 residues of the protein (Fig. 1). No hamster protein cross-reactive with these antibodies was detected in CHO cells. These results indicate that full-length GFP/β3-endonexin can be expressed in CHO cells.


Figure 1
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  		Figure 1 Expression of GFP and GFP/β3-endonexin in CHO cells. CHO cells were either mock transfected or transfected with 4 µg of the indicated plasmids as described in Materials and Methods. 48 h later, the cells were lysed in a buffer containing Triton X-100, and 30 µg of each sample was probed on Western blots with the indicated polyclonal antibodies. (Upper arrow) Position of GFP/β3-endonexin; (lower arrow) position of GFP. The band in the "mock" lane stained with anti–β3-endonexin carboxy-terminal antibody migrated more slowly than GFP/β3-endonexin and was nonspecific.

 
Previous studies have shown that β3-endonexin binds in vitro to the β3 integrin subunit from detergent-solubilized platelets and CHO cells (13, 63). To determine if β3-endonexin retains its ability to bind to the β3 integrin subunit after its fusion to GFP, lysates from CHO cells expressing GFP/β3-endonexin were passed over an affinity matrix containing the bacterially expressed β3 cytoplasmic tail. GFP/β3-endonexin, but not GFP, was specifically retained by and eluted from this affinity matrix (Fig. 2 A). Moreover, GFP/β3-endonexin and the β3 integrin subunit could be specifically coprecipitated from CHO cell lysates (Fig. 2 B). Finally, CHO cells containing GFP/β3-endonexin were strongly fluorescent in the FL1 channel of a flow cytometer (see below). Thus, fusion of β3-endonexin to the carboxy terminus of GFP abrogates neither the integrin-binding function of β3-endonexin nor the fluorescent properties of GFP.


Figure 2
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  		Figure 2 Interactions of GFP/β3-endonexin with the β3 integrin cytoplasmic tail. (A) CHO cells were transfected with 4 µg of pS65T-GFP/β3-endonexin or pS65T-GFP. 48 h later, the cells were lysed in a Triton X-100–containing buffer, and equal volumes of the lysates were batch-incubated with a His-β3 cytoplasmic tail affinity matrix. After extensive washing, proteins were eluted with 1 M imidazole, and equal volumes of the indicated fractions were transferred to nitrocellulose and probed with a polyclonal antibody to GFP. (Upper arrow) Position of GFP/β3endonexin; (lower arrow) position of GFP. Previous studies of this kind have already documented that β3-endonexin interacts with the β3 but not the β1 cytoplasmic tail (63). (B) CHO cells expressing both {alpha}IIbβ3 and GFP/β3-endonexin (+ GFP/β3-EN) or {alpha}IIbβ3 alone (– GFP/β3-EN) were lysed in Triton X-100 lysis buffer, and clarified lysates were immunoprecipitated either with a monoclonal anti-β3 antibody (SSA6), an isotype control antibody (RG 7), or mouse IgG (mIgG). Western blotting was performed initially with a polyclonal antibody to GFP and blots were then reprobed with a polyclonal anti-β3 antibody. The first two lanes represent 30 µg of lysate. Lys, lysate; FT, flow-through; W1, first wash; WL, last wash; E, eluate.

 
β3-Endonexin Increases the Affinity State of Integrin {alpha}IIbβ3
48 h after cotransfection of CHO cells with expression plasmids encoding {alpha}IIbβ3 {alpha}nd GFP/β3-endonexin, the affinity state of {alpha}IIbβ3 was determined by flow cytometry using a PE conjugate of the fibrinogen-mimetic mAb, PAC1. Since transfection efficiencies varied from 15–45%, data acquisition included only live cells positive for GFP fluorescence. About 75% of these cells were also positive for {alpha}IIbβ3, as assessed by staining with an antibody specific for the {alpha}IIbβ3 complex (D57). To standardize the results of PAC1 binding from experiment to experiment, binding was expressed as an activation index calculated from median fluorescence values (49). To obtain this index, nonspecific PAC1 binding was determined in the presence of a selective inhibitor of ligand binding to {alpha}IIbβ3 (either Ro 43-5054 or Integrilin). Maximal PAC1 binding was determined in the presence of an activating anti-β3 antibody (anti-LIBS6) (49). After subtraction of nonspecific binding, this maximal binding was assigned an activation index of 100. Consequently, the activation index for PAC1 binding can range from 0 to 100.

Fig. 3 shows the results of a representative experiment. PAC1 binding to CHO cells transfected with GFP/β3- endonexin and {alpha}IIbβ3 exhibited a relatively high activation index of 44 (Fig. 3 A). In contrast, PAC1 binding to cells transfected with GFP and {alpha}IIbβ3 exhibited a lower activation index of 18 (Fig. 3 D), a value similar to that observed previously for {alpha}IIbβ3 transfectants in the absence of GFP (31, 49). Thus, expression of β3-endonexin appears to activate {alpha}IIbβ3 and increase its affinity for a cognate ligand.


Figure 3
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  		Figure 3 Effect of GFP/β3endonexin on PAC1 binding to {alpha}IIbβ3 in CHO cells. CHO cells were transfected with {alpha}IIbβ3 and either GFP/β3-endonexin (contour plots A, B, and C) or GFP (plots D, E, and F). 48 h later, binding of PEPAC1 (x-axis) to green fluorescent–positive cells (y-axis) was analyzed by flow cytometry. Each contour plot represents 10,000 cells. Plots B and E represent nonspecific PAC1 binding determined in the presence of Ro 43-5054. Plots C and F represent maximal PAC1 binding in the presence of an activating anti-β3 antibody, anti-LIBS6. Note that there was more PAC1 binding to GFP/β3- endonexin cells (plot A) than to GFP cells (plot D), a conclusion supported by the calculated activation indices (plot A, activation index = 44%; plot D, activation index = 18%).

 
This impression was confirmed by the series of experiments summarized in Fig. 4. Compared with cells expressing GFP, those expressing GFP/β3-endonexin consistently showed an increase in PAC1 binding, and the difference was statistically significant (P < 0.03). In contrast, PAC1 binding to cells expressing an unrelated GFP fusion protein, GFP/VASP, was not increased despite similar levels of recombinant protein expression. VASP was chosen because it is present in platelets and localizes to integrin-rich focal adhesions (25). Although not shown, the PAC1 activation index for GFP/β3-endonexin cells (44 ± 5) began to approach that for cells expressing a constitutively active form of {alpha}IIbβ3 ({alpha}IIb{alpha}6Aβ3; 61 ± 6; n = 3) (49). Expression of GFP/β3-endonexin or the other GFP proteins did not affect levels of surface expression of {alpha}IIbβ3, as determined by the binding of antibody D57. All together, these results indicate that expression of β3-endonexin can increase the affinity state of {alpha}IIbβ3.


Figure 4
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  		Figure 4 GFP/β3-endonexin causes activation of {alpha}IIbβ3 in a structurally specific manner. PAC1 binding to transfected CHO cells was analyzed by flow cytometry as described in Materials and Methods and in the legend to Fig. 3. (Left) CHO cells were cotransfected with wild-type {alpha}IIbβ3 and either GFP (open bar), GFP/β3-endonexin (black bar), or GFP/VASP (shaded bar). PAC1 binding was expressed as an activation index, and the data represent means ± SEM for 15 experiments with GFP and GFP/β3- endonexin and three experiments with GFP/VASP. (Right) CHO cells were cotransfected with the signaling-deficient integrin mutant, {alpha}IIbβ3 (S752P), and either GFP (open bar) or GFP/β3- endonexin (black bar). Data represent the means ± SEM of three experiments.

 
Platelets containing {alpha}IIbβ3 with a specific point mutation in the β3 cytoplasmic tail at position 752 (S-> P) fail to bind fibrinogen or aggregate (6). Furthermore, the binding of β3-endonexin to this mutant β3 integrin subunit is markedly reduced (63). When {alpha}IIbβ3 (S752P) was coexpressed with GFP/β3-endonexin in CHO cells, no increase in PAC1 binding was observed (Fig. 4). This suggests that β3endonexin modulates the affinity state of {alpha}IIbβ3 in a structurally specific manner.

Functional Consequences of Integrin Affinity Modulation by β3-Endonexin
To determine whether the changes in PAC1 binding induced by GFP/β3-endonexin translate into increased binding of a physiological ligand, the binding of fibrinogen to CHO cells was studied by flow cytometry. Bound fibrinogen was detected with a biotinylated mAb (anti-LIBS1) specific for a fibrinogen-sensitive epitope on the β3 subunit (19). Specific fibrinogen binding was defined as that inhibitable by a function-blocking anti-{alpha}IIbβ3 antibody, A2A9. CHO cells expressing wild-type {alpha}IIbβ3 bind little or no fibrinogen at a saturating concentration of ligand (250 µg/ml) (48). The same was true for cells expressing GFP and {alpha}IIbβ3. However, those expressing GFP/β3-endonexin and {alpha}IIbβ3 bound increased amounts of fibrinogen (Fig. 5). Similar results were obtained when fibrinogen binding was measured directly with biotinylated fibrinogen (not shown). Thus, expression of GFP/β3-endonexin can lead to an increase in fibrinogen binding to {alpha}IIbβ3.


Figure 5
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  		Figure 5 GFP/β3-endonexin induces fibrinogen binding to {alpha}IIbβ3. 48 h after transfection of CHO cells with {alpha}IIbβ3 and either GFP (open bar) or GFP/β3-endonexin (black bar), the cells were resuspended in Ca2+- and Mg2+-containing Tyrode's buffer and incubated for 30 min at room temperature in the presence of 250 µg/ml fibrinogen. Then fibrinogen binding to the transfected cells was assessed by flow cytometry using phycoerythrin-streptavidin and a biotinylated anti-β3 antibody (anti-LIBS 1) sensitive to the presence of bound fibrinogen. Specific binding was defined as that blocked by antibody A2A9 (20 µg/ml), and it was expressed as an activation index. Data represent the means ± SEM of two experiments, each performed in triplicate.

 
When fibrinogen binds to activated {alpha}IIbβ3 on the surface of platelets or CHO cells under stirring conditions, the cells aggregate (4, 16). To determine whether GFP/β3-endonexin can trigger this aggregation response, CHO cells expressing GFP/β3-endonexin and {alpha}IIbβ3 were mixed with cells containing {alpha}IIbβ3 and a red fluorescent tracer, hydroxyethidine. After stirring for 20 min in the presence of 300 µg/ml fibrinogen, the formation of mixed, red–green cellular aggregates was monitored by flow cytometry. The rationale for this experimental design is that if fibrinogen first becomes bound to activated {alpha}IIbβ3 on the GFP/β3- endonexin cells, this cell-bound fibrinogen should then be able to recruit the red fluorescent cells into mixed aggregates, even though the {alpha}IIbβ3 on the red fluorescent cells is initially in a low affinity state (Fig. 6 A) (16).


Figure 6
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  		Figure 6 GFP/β3-endonexin causes fibrinogen-dependent aggregation of CHO cells. A illustrates the rationale for this aggregation protocol, which is discussed in the text. In B, as detailed in Materials and Methods, CHO cells that had been transfected with {alpha}IIbβ3 and GFP (left) or with {alpha}IIbβ3 and GFP/β3-endonexin (center and right) were mixed with CHO cells that had been stably transfected with {alpha}IIbβ3, and then stained with the red fluorescent dye, hydroxyethidine. After stirring for 20 min in the presence of 300 µg/ml fibrinogen, the cells were fixed with formaldehyde, and 10,000 propidium iodide–negative and GFP-positive cells (y-axis) were analyzed by flow cytometry. (B, right) The incubation with fibrinogen was carried out in the presence of 20 µg/ml antibody A2A9 to inhibit fibrinogen binding. Mixed red–green cellular aggregates appear to the right of the vertical line on the FL2 axis.

 
In the experiment shown in Fig. 6 B, it can be seen that GFP/β3-endonexin promoted the formation of mixed aggregates (center), an effect that could be inhibited by the function-blocking antibody, A2A9 (right), or the cyclic peptide, Integrilin (not shown). In three such experiments, an average of 7.0 ± 1.6% of the cells expressing GFP/β3endonexin were engaged in red–green aggregates, compared with 3.5 ± 1.9% of cells expressing GFP. While this effect may seem small, it was statistically significant (P < 0.01). Moreover, it should be emphasized that the extent of mixed aggregation was limited by the required use of red fluorescent cells expressing low affinity {alpha}IIbβ3. These results indicate that affinity modulation of {alpha}IIbβ3 by β3- endonexin can cause fibrinogen-dependent cell aggregation.

Factors That Influence Integrin Activation by β3-Endonexin
Additional experiments were conducted to clarify the mechanism of action of GFP/β3-endonexin. Although PAC1 is a multimeric IgM antibody, GFP/β3-endonexin was also found to increase the binding of a monomeric form of PAC1 obtained by enzyme digestion. In addition, PAC1 binding because of GFP/β3-endonexin was not affected by preincubation of the cells with 10 µM cytochalasin D, an inhibitor of actin polymerization (data not shown). Since actin polymerization promotes integrin clustering (12, 71), which would be expected to influence preferentially the binding of multivalent ligands, these results suggest that GFP/β3endonexin is primarily a modulator of {alpha}IIbβ3 affinity rather than avidity.

Next, GFP/β3-endonexin was studied in CHO cells expressing both {alpha}IIbβ3 and a β3 cytoplasmic tail chimera containing the extracellular and transmembrane domains of the Tac subunit of the IL-2 receptor. We reasoned that the chimera, which does not dimerize with {alpha}IIb (7, 40), would compete intracellularly with {alpha}IIbβ3 for β3-endonexin. If so, it should prevent β3-endonexin from binding to and modulating the function of {alpha}IIbβ3. Indeed, expression of the Tac/ β3 chimera prevented GFP/β3-endonexin from activating {alpha}IIbβ3 (Fig. 7). In contrast, a Tac chimera containing the structurally unrelated {alpha}5 cytoplasmic tail exhibited no such effect. This is consistent with the idea that β3-endonexin modulates integrin affinity through an interaction with the β3 cytoplasmic tail.


Figure 7
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  		Figure 7 Factors influencing integrin activation by GFP/β3-endonexin. CHO cells were transfected with {alpha}IIbβ3 {alpha}nd either GFP or GFP/β3-endonexin. As detailed in Materials and Methods, some transfectants were subjected to additional treatments before determination of PAC1 binding. These included (a) cotransfection with a Tac/β3 tail chimera; (b) cotransfection with a Tac/{alpha}5 tail chimera; (c) cotransfection with a constitutively active form of H-Ras (G12V); or (d) energy depletion by preincubation for 30 min with 0.2% sodium azide and 4 mg/ml 2-deoxy-d-glucose. Data are the means ± SEM of three experiments.

 
Affinity modulation of {alpha}IIbβ3 by platelet agonists requires metabolic energy (68). In CHO cells, PAC1 binding induced by GFP/β3-endonexin was not observed if the cells were pretreated with sodium azide and 2-deoxy-dglucose to inhibit oxidative metabolism (Fig. 7). In this respect, the effect of β3-endonexin in the CHO cell system is similar to that of excitatory agonists in the platelet system.

In platelets, heterotrimeric GTP-binding proteins have been implicated in affinity modulation. On the other hand, the role of the small GTPase, H-Ras, which is also present in these cells, has not been examined. Recently, Hughes and co-workers found that a constitutively active form of H-Ras (G12V) acts as a general suppressor of integrin adhesive function in CHO cells (33). Similarly, we found that the expression of H-Ras (G12V) inhibited the effects of GFP/β3-endonexin on PAC1 binding (Fig. 7). Taken together with the energy depletion experiments, this indicates that the function of GFP/β3-endonexin is subject to metabolic regulation.

Subcellular Localization of β3-Endonexin
In order for β3-endonexin to directly influence the function of the β3 integrin cytoplasmic tail, these proteins must be located together in the cell. To address this question, HMEC-1 human endothelial cells, which attach and spread on immobilized fibrinogen through {alpha}Vβ3, were microinjected with DNA encoding GFP/β3-endonexin or GFP. 4 h later, specific green fluorescence could be observed diffusely in the cytoplasm and the nucleus. The degree of nuclear fluorescence was much greater in the case of GFP/ β3-endonexin (Fig. 8). An identical pattern of GFP/β3- endonexin localization was observed in CHO cells that had been allowed to spread on fibrinogen through {alpha}IIbβ3 (not shown). These results are consistent with a generalized cytoplasmic distribution of GFP/β3-endonexin and with a nuclear localization that may be promoted by a consensus nuclear localization signal in β3-endonexin (see Discussion).


Figure 8
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  		Figure 8 Expression of GFP, GFP/β3-endonexin, and GFP/VASP in HMEC-1 cells. Cells were allowed to spread for 2 h on fibrinogencoated coverslips, and then were microinjected with the indicated expression plasmids. 4 h later, green fluorescence was visualized in a fluorescence microscope using an FITC filter set. Uninjected cells were not visible under these conditions. Bar, 10 µm.

 
When CHO cells containing {alpha}Vβ3 or {alpha}IIbβ3 are allowed to spread on fibrinogen, the β3 cytoplasmic tail is necessary and sufficient for localization of the β3 integrins to focal adhesions (40, 72). Immunostaining of HMEC-1 cells revealed that {alpha}V and β3 were localized both in a diffuse pattern consistent with a generalized plasma membrane distribution and in discrete foci characteristic of focal adhesions (Fig. 9). There was no strong or consistent localization of GFP/β3-endonexin to these focal adhesions, excluding the possibility that β3-endonexin might associate tightly with the β3 cytoplasmic tail during cytoskeletal assembly. However, some weak staining of β3-endonexin in focal adhesions was observed, suggesting that a weaker or more transient association may occur (Fig. 9, arrowheads). No localization of GFP to focal adhesions was detected. As a positive control, GFP was fused to FRNK, an autonomously expressed segment of pp125FAK that contains a focal adhesion targeting sequence (57). After microinjection, GFP/FRNK significantly localized to focal adhesions, demonstrating that a GFP fusion protein can target to these structures under the experimental conditions used here (Fig. 8). Thus, GFP/β3-endonexin is not strongly or consistently concentrated in focal adhesions.


Figure 9
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  		Figure 9 Subcellular localization of GFP/β3-endonexin and {alpha}Vβ3 in HMEC-1 cells. Cells were allowed to spread for 2 h on fibrinogen and then were microinjected with GFP/β3-endonexin. 4 h later, the cells were fixed, stained with rhodamine-labeled antibodies to {alpha}V or β3, and then examined by microscopy for GFP fluorescence (top) and rhodamine fluorescence (bottom). Two different cells are shown, one in the lefthand panels, the other in the righthand panels. Arrowheads denote the occasional coalignment of GFP/β3-endonexin and {alpha}Vβ3 in focal adhesions. Bar, 5 µm.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
These studies demonstrate that: (a) in CHO cells, expression of β3-endonexin as a fusion protein with GFP is associated with an increase in the affinity state of integrin {alpha}IIbβ3. This affinity change enables the cells to undergo fibrinogen-dependent aggregation. (b) Affinity modulation of {alpha}IIbβ3 by GFP/β3-endonexin is structurally specific in that other GFP proteins (GFP; GFP/VASP) do not promote this response. Furthermore, GFP/β3-endonexin does not affect the function of {alpha}IIbβ3 (S752P), a mutant integrin that is defective in binding to β3-endonexin and in integrin signaling. (c) Affinity modulation by β3-endonexin may be the consequence of its direct interaction with {alpha}IIbβ3 since it is prevented by coexpression of a Tac-β3 cytoplasmic tail chimera. (d) The effect of β3-endonexin on {alpha}IIbβ3 may be subject to metabolic regulation since it is not observed if cellular energy is depleted or if the cells are cotransfected with an activated form of H-Ras. (e) GFP/β3-endonexin is found in both the nuclear and cytoplasmic compartments after CHO cells or HMEC-1 cells have spread on a fibrinogen matrix via a β3 integrin. Taken together, these results indicate that β3-endonexin may play a significant role in cell adhesion and signaling through integrin {alpha}IIbβ3.

We interpret the effect of GFP/β3-endonexin on PAC1 and fibrinogen binding to CHO cells to represent an example of inside-out signaling in which β3-endonexin increases the affinity of individual {alpha}IIbβ3 heterodimers for specific ligands. An alternative interpretation that β3-endonexin triggers oligomerization of {alpha}IIbβ3 complexes and therefore increases receptor avidity cannot be excluded, but it seems less likely for several reasons. First, changes within {alpha}IIbβ3 that enable the binding of RGD-containing macromolecular ligands have been detected with both a monovalent Fab fragment of PAC1 as well as with the native, multivalent antibody (1). This indicates that regulated ligand binding to {alpha}IIbβ3 is not absolutely dependent on the valency of the ligand or, presumably, the receptor. Second, the effect of GFP/β3-endonexin on {alpha}IIbβ3 was detected using either native PAC1 or a monomeric fragment of the antibody. Third, agonist-induced clustering of β2 integrins in leukocytes and possibly {alpha}IIbβ3 in platelets is facilitated by polymerization of F-actin (12, 14, 71). However, cytochalasin D, an inhibitor of actin polymerization, had no effect on PAC1 binding induced by GFP/β3-endonexin. While it is not possible to quantitate precisely the relative contributions of affinity and avidity regulation, based on the above considerations, we speculate that β3-endonexin can regulate reversible fibrinogen binding through affinity modulation. Other factors, including actin polymerization and cytoskeletal reorganization, may enhance cell adhesion by promoting receptor clustering and irreversible ligand binding. Consistent with this idea, cytochalasin D has been reported to inhibit primarily the later, irreversible phase of fibrinogen and PAC1 binding to platelets and CHO cells (14, 53, 54).

The present results were obtained by expressing β3- endonexin ectopically in CHO cells. Therefore, it is possible that the function of the endogenous protein in platelets or other cells differs quantitatively or qualitatively from that described here. Despite this caveat, a number of observations indicate that affinity modulation may result directly from the interaction of β3-endonexin with the cytoplasmic tail of the β3 integrin subunit. A mutational analysis of the β3 tail has shown that membrane-distal residues near the carboxy terminus of the tail (N756ITY) are required for the interaction with β3-endonexin (13). Mutation or deletion of these same residues also disrupts insideout integrin signaling in platelets and CHO cells (50; Wang, R., D.R. Ambruso, and P.J. Newman. 1994. Blood. 84:244a). Moreover, coexpression of a β3 tail chimera, but not an {alpha}5 tail chimera, prevented affinity modulation by β3-endonexin, possibly because the former chimera but not the latter could compete with {alpha}IIbβ3 for binding to β3endonexin. Finally, when other recombinant GFP proteins such as GFP and GFP/VASP were expressed in CHO cells, they failed to increase {alpha}IIbβ3 affinity.

Additional studies will be required to determine how cellular energy depletion or coexpression of activated H-Ras inhibits affinity modulation by β3-endonexin. Nonetheless, these results imply that this function of β3endonexin is subject to metabolic regulation. In this context, studies with platelets have suggested that serine-threonine kinases (61), tyrosine kinases (23), and PI 3-kinase (38, 74; Kovacsovics, T.J., J.H. Hartwig, L.C. Cantley, and A. Toker. 1995. Blood. 86:454a) are involved in promoting fibrinogen binding to {alpha}IIbβ3. In contrast, compounds that activate protein kinase A or G inhibit fibrinogen binding (22, 28). Perhaps β3-endonexin is a direct substrate of specific kinases or phosphatases or is a target of downstream effectors of these enzymes. For example, β3-endonexin contains several serine and threonine residues in favorable contexts for phosphorylation by protein kinase C and A (63).

Suppression of integrin activation in CHO cells by activated H-Ras involves a Raf-1–initiated MAP kinase pathway and is transcription independent (33). In contrast, an activated variant of R-Ras was recently implicated in the promotion of integrin-mediated cell adhesion (75). Since the reported opposite actions of activated R-Ras and H-Ras affect both β1 and β3 integrins, it seems unlikely that the pathways triggered by these GTPases converge directly on β3-endonexin. Although platelets contain both H-Ras and R-Ras, and platelet stimulation by thrombin activates H-Ras, the functions of these GTPases in this terminally differentiated cell are unknown (64).

Based on the present results, we propose that the interaction of β3-endonexin with the β3 cytoplasmic tail triggers a structural change in the integrin to reconfigure the extracellular face of the receptor so that it can engage fibrinogen. While the nature of this propagated change is unknown, one possibility is that there is a reorientation of the β3 subunit relative to the {alpha}IIb subunit. This is plausible given the biophysical evidence for interactions between the cytoplasmic tails of {alpha}IIb and β3 (24) and for agonist- induced structural changes in the extracellular domains of {alpha}IIbβ3 in platelets (65). In a similar manner, relatively subtle changes within preexisting dimers may play a role in ligand-triggered, "outside-in" signaling across other plasma membrane receptors, including the bacterial aspartate receptor (66) and the mammalian EGF receptor (15).

A complete understanding of the proximate events in inside-out signaling will require identification of all relevant integrin-binding proteins and a more refined knowledge of integrin structure. Progress is beginning to be made in both of these areas (11, 42, 55). Several proteins have been described that bind directly to integrin cytoplasmic tails, at least in vitro. These include structural proteins of the cytoskeleton, such as F-actin (specific for the {alpha}2 tail) (36), {alpha}-actinin (β tails) (51), talin (β) (29), and filamin (β2) (60), and potential signaling molecules, such as calreticulin ({alpha} tails) (10), pp125FAK (β) (58), integrin-linked kinase (β) (26), and cytohesin-1 (β2) (37). Of note, the expression of calreticulin and cytohesin-1 appears to stimulate or stabilize a high affinity state of integrins {alpha}2β1 and {alpha}Lβ2, respectively (10, 37). There is no sequence similarity between either of these proteins and β3-endonexin. Thus, a structurally diverse group of cytoplasmic tail-binding proteins may function to regulate integrins. Some like cytohesin-1 and β3-endonexin may be restricted in their action because of their binding specificities, while others like calreticulin, which recognizes a conserved motif in all integrin {alpha} tails, may be less specific.

While not relevant to platelets, the localization of β3- endonexin to the cytoplasm and nucleus of HMEC-1 and CHO cells suggests that this protein may have more than one function. The nuclear localization may be explained, in part, by the presence of a consensus nuclear localization signal in β3-endonexin (K62RKK) (35, 63). Interestingly, several proteins implicated in cell adhesion and adhesive signaling, including ZO-1, β-catenin, zyxin, c-Abl, and HEF1, either exhibit cytoplasmic and nuclear localization or shuttle between the cytoplasm and the nucleus depending on the adhesive state of the cell (Nix, D.A., and M.C. Beckerle. 1995. Mol. Biol. Cell. 6:366a; 3, 21, 41, 44). The identification of other proteins that can bind to β3-endonexin should help to explain its pattern of subcellular localization.

Focal adhesions are dynamic structures containing integrins, cytoskeletal elements, and signaling molecules that form on the basal surfaces of many types of cells in culture and in platelets during spreading on fibrinogen (47). These macromolecular assemblies may function to optimize traction during cell motility and to promote information flow from the extracellular matrix to the nucleus (5, 17, 18). The lack of consistent and strong localization of β3-endonexin to β3-rich focal adhesions suggests that it may interact most strongly with the β3 cytoplasmic tail while cells are in suspension or are in the early phases of adhesion. Thus, it is attractive to speculate that β3-endonexin may participate in integrin activation but may dissociate at later times to permit cytoskeletal interactions with the integrin tails.

These studies provide the first clues about the functions of β3-endonexin, but they leave several questions unanswered. Does β3-endonexin influence outside-in signaling events, such as protein tyrosine phosphorylation (8)? Is β3endonexin subject to posttranslational modifications in vivo, and does this affect its subcellular localization or function? Does β3-endonexin modulate the adhesive function of {alpha}Vβ3, which like {alpha}IIbβ3 appears to be subject to rapid regulation in some cell types (67, 73)? Does β3-endonexin regulate {alpha}IIbβ3 in platelets?


   Acknowledgments
 
We thank David Phillips for providing Integrilin, Michael Schaller for FRNK cDNA, Beat Steiner for Ro 43-5054, Zaverio Ruggeri for antibody RG 7, and Ulrich Walter and Thomas Jarchau for VASP cDNA.

These studies were supported by research grants from the National Institutes of Health (HL 56595, HL 48728, AR 27214) and from Cor Therapeutics, Inc. H. Kashiwagi is the recipient of a Banyu Fellowship in Lipid Metabolism and Atherosclerosis sponsored by Banyu Pharmaceutical Co., Ltd. and the Merck Company Foundation. M. Eigenthaler is the recipient of a fellowship from the American Heart Association (CA Chapter). This is manuscript 10536-VB from the Scripps Research Institute.

Submitted: 23 December 1996

Revised: 24 March 1997

1. Abbreviations used in this paper: GFP, green fluorescent protein; PE, phycoerythrin.

Please address all correspondence to Sanford J. Shattil, Department of Vascular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, VB-5, La Jolla, CA 92037. Tel.: (619) 784-7148. Fax: (619) 784-7422. e-mail: shattil{at}scripps.edu

This work was presented in part at the Annual Meeting of the American Society of Hematology on December 8, 1996, in Orlando, FL and published in abstract form (1996. Blood. 88:140a).


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