Affinity Modulation of Platelet Integrin alpha IIbbeta 3 by beta 3-Endonexin, a Selective Binding Partner of the beta 3 Integrin Cytoplasmic Tail

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

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

Abstract
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
References


Abstract

Platelet agonists increase the affinity state of integrin alpha IIbbeta 3, 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. beta 3-Endonexin is a novel 111-amino acid protein that binds selectively to the beta 3 tail. Since beta 3-endonexin is present in platelets, we asked whether it can affect alpha IIbbeta 3 function. When beta 3-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 IIbbeta 3 affinity state in transfected cells by flow cytometry. Cells transfected with GFP and alpha IIbbeta 3 bound little or no PAC1. However, those transfected with GFP/beta 3-endonexin and alpha IIbbeta 3 bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/beta 3-endonexin did not affect levels of surface expression of alpha IIbbeta 3 nor did it modulate the affinity of an alpha IIbbeta 3 mutant that is defective in binding to beta 3-endonexin. Affinity modulation of alpha IIbbeta 3 by GFP/beta 3-endonexin was inhibited by coexpression of either a monomeric beta 3 cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that beta 3-endonexin can modulate the affinity state of alpha IIbbeta 3 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 IIbbeta 3.


Integrins are alpha beta 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 IIbbeta 3. 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 IIbbeta 3 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 IIbbeta 3. 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 IIbbeta 3 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 beta 3 cytoplasmic tail fail to aggregate in response to agonists despite near normal levels of alpha IIbbeta 3 (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 IIbbeta 3 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 beta 3endonexin, which is capable of binding to the cytoplasmic tail of the beta 3 integrin subunit, both in yeast and in vitro (63). However, it fails to bind to other integrin tails, including those of beta 1, beta 2, and alpha IIb. Since beta 3-endonexin is expressed in platelets, the present studies were carried out to determine whether this protein can modulate the ligandbinding function of alpha IIbbeta 3. Using a CHO cell model system to transiently express alpha IIbbeta 3 and beta 3-endonexin, we now report that this protein can increase the affinity state and the adhesive function of alpha IIbbeta 3. Moreover, these effects are structurally specific and subject to metabolic regulation.


Materials and Methods

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 IgMkappa 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 beta 3-endonexin as a protein fused to the carboxy terminus of GFP, beta 3-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 beta 3-endonexin with T4 DNA ligase (Boehringer Mannheim Biochemicals). After transformation of DH5alpha , clones in the correct orientation were selected by PCR using a sense primer in GFP and an antisense primer in beta 3-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, beta 3, a mutant form of beta 3 containing a single amino acid substitution (S752P), and H-Ras (G12V) have been described (33, 49). Tac-beta 3 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 beta 3 and varying amounts of the GFP plasmids to obtain equivalent degrees of GFP expression (e.g., 4 µg of pS65T, 4 µg of pS65T/beta 3-endonexin, 0.02 µg of pEGFP, 0.2 µg of pEGFP/beta 3-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-beta 3, Tac-alpha 5, or H-Ras (G12V) plasmid was cotransfected along with pEGFP/beta 3-endonexin and the plasmids for alpha IIb and beta 3. 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/beta 3-Endonexin Expression in CHO Cells

Expression of GFP/beta 3-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 beta 3-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/beta 3-endonexin to the beta 3 integrin cytoplasmic tail, the 47-amino acid beta 3 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/beta 3-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-beta 3 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/beta 3-endonexin could be specifically coimmunoprecipitated with the beta 3 integrin subunit from CHO cells, immunoprecipitation studies were carried out in the Trition X-100 lysis buffer essentially as described (30). The beta 3 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 beta 3 integrin subunit in the immunoprecipitates, blots were stripped and reprobed with Ab 8035, a rabbit polyclonal antibody specific for beta 3.

Evaluation of alpha IIbbeta 3 Affinity State

48 h after transfection, CHO cells were resuspended at 1-2 × 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-beta 3 antibody (2% anti-LIBS6 ascites) that stabilizes alpha IIbbeta 3 in a high affinity state (30). Others contained an alpha IIbbeta 3-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 × (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 beta 3 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 IIbbeta 3 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 IIbbeta 3 (49) were labeled with a red fluorescent tracer, hydroxyethidine (Polysciences Inc., Junction City, OR). Then 250 µl of these cells (4 × 106/ml) were added to siliconized glass cuvettes containing 250 µl of cells (2 × 106/ml) that had been transfected with GFP/beta 3-endonexin (or GFP) and alpha IIbbeta 3. 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/beta 3-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 beta 3 as described (32). HMEC-1 human endothelial cells, which express alpha Vbeta 3, 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 beta 3.


Results

Expression of beta 3-Endonexin in CHO Cells

CHO cells provide a useful model system for characterizing the adhesive and signaling functions of ectopically expressed alpha IIbbeta 3 (43, 49, 50). Therefore, beta 3-endonexin was transiently coexpressed with alpha IIbbeta 3 in these cells to study its effects on the ligand-binding function of this integrin. beta 3-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/beta 3-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/beta 3-endonexin expression, and the plasmids were used interchangeably in the following experiments. GFP/beta 3-endonexin was also detectable with polyclonal antibodies raised against either recombinant human beta 3-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/beta 3-endonexin can be expressed in CHO cells.


Fig. 1. Expression of GFP and GFP/beta 3-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/beta 3-endonexin; (lower arrow) position of GFP. The band in the "mock" lane stained with anti-beta 3-endonexin carboxy-terminal antibody migrated more slowly than GFP/beta 3-endonexin and was nonspecific.
[View Larger Version of this Image (39K GIF file)]

Previous studies have shown that beta 3-endonexin binds in vitro to the beta 3 integrin subunit from detergent-solubilized platelets and CHO cells (13, 63). To determine if beta 3-endonexin retains its ability to bind to the beta 3 integrin subunit after its fusion to GFP, lysates from CHO cells expressing GFP/beta 3-endonexin were passed over an affinity matrix containing the bacterially expressed beta 3 cytoplasmic tail. GFP/beta 3-endonexin, but not GFP, was specifically retained by and eluted from this affinity matrix (Fig. 2 A). Moreover, GFP/beta 3-endonexin and the beta 3 integrin subunit could be specifically coprecipitated from CHO cell lysates (Fig. 2 B). Finally, CHO cells containing GFP/beta 3-endonexin were strongly fluorescent in the FL1 channel of a flow cytometer (see below). Thus, fusion of beta 3-endonexin to the carboxy terminus of GFP abrogates neither the integrin-binding function of beta 3-endonexin nor the fluorescent properties of GFP.


Fig. 2. Interactions of GFP/beta 3-endonexin with the beta 3 integrin cytoplasmic tail. (A) CHO cells were transfected with 4 µg of pS65T-GFP/beta 3-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-beta 3 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/beta 3endonexin; (lower arrow) position of GFP. Previous studies of this kind have already documented that beta 3-endonexin interacts with the beta 3 but not the beta 1 cytoplasmic tail (63). (B) CHO cells expressing both alpha IIbbeta 3 and GFP/beta 3-endonexin (+ GFP/beta 3-EN) or alpha IIbbeta 3 alone (- GFP/beta 3-EN) were lysed in Triton X-100 lysis buffer, and clarified lysates were immunoprecipitated either with a monoclonal anti-beta 3 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-beta 3 antibody. The first two lanes represent 30 µg of lysate. Lys, lysate; FT, flow-through; W1, first wash; WL, last wash; E, eluate.
[View Larger Version of this Image (67K GIF file)]

beta 3-Endonexin Increases the Affinity State of Integrin alpha IIbbeta 3

48 h after cotransfection of CHO cells with expression plasmids encoding alpha IIbbeta 3 alpha nd GFP/beta 3-endonexin, the affinity state of alpha IIbbeta 3 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 IIbbeta 3, as assessed by staining with an antibody specific for the alpha IIbbeta 3 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 IIbbeta 3 (either Ro 43-5054 or Integrilin). Maximal PAC1 binding was determined in the presence of an activating anti-beta 3 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/beta 3- endonexin and alpha IIbbeta 3 exhibited a relatively high activation index of 44 (Fig. 3 A). In contrast, PAC1 binding to cells transfected with GFP and alpha IIbbeta 3 exhibited a lower activation index of 18 (Fig. 3 D), a value similar to that observed previously for alpha IIbbeta 3 transfectants in the absence of GFP (31, 49). Thus, expression of beta 3-endonexin appears to activate alpha IIbbeta 3 and increase its affinity for a cognate ligand.


Fig. 3. Effect of GFP/beta 3endonexin on PAC1 binding to alpha IIbbeta 3 in CHO cells. CHO cells were transfected with alpha IIbbeta 3 and either GFP/beta 3-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-beta 3 antibody, anti-LIBS6. Note that there was more PAC1 binding to GFP/beta 3- 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%).
[View Larger Version of this Image (63K GIF file)]

This impression was confirmed by the series of experiments summarized in Fig. 4. Compared with cells expressing GFP, those expressing GFP/beta 3-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/beta 3-endonexin cells (44 ± 5) began to approach that for cells expressing a constitutively active form of alpha IIbbeta 3 (alpha IIbalpha 6Abeta 3; 61 ± 6; n = 3) (49). Expression of GFP/beta 3-endonexin or the other GFP proteins did not affect levels of surface expression of alpha IIbbeta 3, as determined by the binding of antibody D57. All together, these results indicate that expression of beta 3-endonexin can increase the affinity state of alpha IIbbeta 3.


Fig. 4. GFP/beta 3-endonexin causes activation of alpha IIbbeta 3 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 IIbbeta 3 and either GFP (open bar), GFP/beta 3-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/beta 3- endonexin and three experiments with GFP/VASP. (Right) CHO cells were cotransfected with the signaling-deficient integrin mutant, alpha IIbbeta 3 (S752P), and either GFP (open bar) or GFP/beta 3- endonexin (black bar). Data represent the means ± SEM of three experiments.
[View Larger Version of this Image (24K GIF file)]

Platelets containing alpha IIbbeta 3 with a specific point mutation in the beta 3 cytoplasmic tail at position 752 (Sright-arrow P) fail to bind fibrinogen or aggregate (6). Furthermore, the binding of beta 3-endonexin to this mutant beta 3 integrin subunit is markedly reduced (63). When alpha IIbbeta 3 (S752P) was coexpressed with GFP/beta 3-endonexin in CHO cells, no increase in PAC1 binding was observed (Fig. 4). This suggests that beta 3endonexin modulates the affinity state of alpha IIbbeta 3 in a structurally specific manner.

Functional Consequences of Integrin Affinity Modulation by beta 3-Endonexin

To determine whether the changes in PAC1 binding induced by GFP/beta 3-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 beta 3 subunit (19). Specific fibrinogen binding was defined as that inhibitable by a function-blocking anti-alpha IIbbeta 3 antibody, A2A9. CHO cells expressing wild-type alpha IIbbeta 3 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 IIbbeta 3. However, those expressing GFP/beta 3-endonexin and alpha IIbbeta 3 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/beta 3-endonexin can lead to an increase in fibrinogen binding to alpha IIbbeta 3.


Fig. 5. GFP/beta 3-endonexin induces fibrinogen binding to alpha IIbbeta 3. 48 h after transfection of CHO cells with alpha IIbbeta 3 and either GFP (open bar) or GFP/beta 3-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-beta 3 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.
[View Larger Version of this Image (19K GIF file)]

When fibrinogen binds to activated alpha IIbbeta 3 on the surface of platelets or CHO cells under stirring conditions, the cells aggregate (4, 16). To determine whether GFP/beta 3-endonexin can trigger this aggregation response, CHO cells expressing GFP/beta 3-endonexin and alpha IIbbeta 3 were mixed with cells containing alpha IIbbeta 3 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 IIbbeta 3 on the GFP/beta 3- endonexin cells, this cell-bound fibrinogen should then be able to recruit the red fluorescent cells into mixed aggregates, even though the alpha IIbbeta 3 on the red fluorescent cells is initially in a low affinity state (Fig. 6 A) (16).


Fig. 6. GFP/beta 3-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 IIbbeta 3 and GFP (left) or with alpha IIbbeta 3 and GFP/beta 3-endonexin (center and right) were mixed with CHO cells that had been stably transfected with alpha IIbbeta 3, 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.
[View Larger Version of this Image (51K GIF file)]

In the experiment shown in Fig. 6 B, it can be seen that GFP/beta 3-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/beta 3endonexin 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 IIbbeta 3. These results indicate that affinity modulation of alpha IIbbeta 3 by beta 3- endonexin can cause fibrinogen-dependent cell aggregation.

Factors That Influence Integrin Activation by beta 3-Endonexin

Additional experiments were conducted to clarify the mechanism of action of GFP/beta 3-endonexin. Although PAC1 is a multimeric IgM antibody, GFP/beta 3-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/beta 3-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/beta 3endonexin is primarily a modulator of alpha IIbbeta 3 affinity rather than avidity.

Next, GFP/beta 3-endonexin was studied in CHO cells expressing both alpha IIbbeta 3 and a beta 3 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 IIbbeta 3 for beta 3-endonexin. If so, it should prevent beta 3-endonexin from binding to and modulating the function of alpha IIbbeta 3. Indeed, expression of the Tac/ beta 3 chimera prevented GFP/beta 3-endonexin from activating alpha IIbbeta 3 (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 beta 3-endonexin modulates integrin affinity through an interaction with the beta 3 cytoplasmic tail.


Fig. 7. Factors influencing integrin activation by GFP/beta 3-endonexin. CHO cells were transfected with alpha IIbbeta 3 alpha nd either GFP or GFP/beta 3-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/beta 3 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.
[View Larger Version of this Image (17K GIF file)]

Affinity modulation of alpha IIbbeta 3 by platelet agonists requires metabolic energy (68). In CHO cells, PAC1 binding induced by GFP/beta 3-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 beta 3-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/beta 3-endonexin on PAC1 binding (Fig. 7). Taken together with the energy depletion experiments, this indicates that the function of GFP/beta 3-endonexin is subject to metabolic regulation.

Subcellular Localization of beta 3-Endonexin

In order for beta 3-endonexin to directly influence the function of the beta 3 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 Vbeta 3, were microinjected with DNA encoding GFP/beta 3-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/ beta 3-endonexin (Fig. 8). An identical pattern of GFP/beta 3- endonexin localization was observed in CHO cells that had been allowed to spread on fibrinogen through alpha IIbbeta 3 (not shown). These results are consistent with a generalized cytoplasmic distribution of GFP/beta 3-endonexin and with a nuclear localization that may be promoted by a consensus nuclear localization signal in beta 3-endonexin (see Discussion).


Fig. 8. Expression of GFP, GFP/beta 3-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.
[View Larger Version of this Image (45K GIF file)]

When CHO cells containing alpha Vbeta 3 or alpha IIbbeta 3 are allowed to spread on fibrinogen, the beta 3 cytoplasmic tail is necessary and sufficient for localization of the beta 3 integrins to focal adhesions (40, 72). Immunostaining of HMEC-1 cells revealed that alpha V and beta 3 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/beta 3-endonexin to these focal adhesions, excluding the possibility that beta 3-endonexin might associate tightly with the beta 3 cytoplasmic tail during cytoskeletal assembly. However, some weak staining of beta 3-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/beta 3-endonexin is not strongly or consistently concentrated in focal adhesions.


Fig. 9. Subcellular localization of GFP/beta 3-endonexin and alpha Vbeta 3 in HMEC-1 cells. Cells were allowed to spread for 2 h on fibrinogen and then were microinjected with GFP/beta 3-endonexin. 4 h later, the cells were fixed, stained with rhodamine-labeled antibodies to alpha V or beta 3, 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/beta 3-endonexin and alpha Vbeta 3 in focal adhesions. Bar, 5 µm.
[View Larger Version of this Image (100K GIF file)]


Discussion

These studies demonstrate that: (a) in CHO cells, expression of beta 3-endonexin as a fusion protein with GFP is associated with an increase in the affinity state of integrin alpha IIbbeta 3. This affinity change enables the cells to undergo fibrinogen-dependent aggregation. (b) Affinity modulation of alpha IIbbeta 3 by GFP/beta 3-endonexin is structurally specific in that other GFP proteins (GFP; GFP/VASP) do not promote this response. Furthermore, GFP/beta 3-endonexin does not affect the function of alpha IIbbeta 3 (S752P), a mutant integrin that is defective in binding to beta 3-endonexin and in integrin signaling. (c) Affinity modulation by beta 3-endonexin may be the consequence of its direct interaction with alpha IIbbeta 3 since it is prevented by coexpression of a Tac-beta 3 cytoplasmic tail chimera. (d) The effect of beta 3-endonexin on alpha IIbbeta 3 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/beta 3-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 beta 3 integrin. Taken together, these results indicate that beta 3-endonexin may play a significant role in cell adhesion and signaling through integrin alpha IIbbeta 3.

We interpret the effect of GFP/beta 3-endonexin on PAC1 and fibrinogen binding to CHO cells to represent an example of inside-out signaling in which beta 3-endonexin increases the affinity of individual alpha IIbbeta 3 heterodimers for specific ligands. An alternative interpretation that beta 3-endonexin triggers oligomerization of alpha IIbbeta 3 complexes and therefore increases receptor avidity cannot be excluded, but it seems less likely for several reasons. First, changes within alpha IIbbeta 3 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 IIbbeta 3 is not absolutely dependent on the valency of the ligand or, presumably, the receptor. Second, the effect of GFP/beta 3-endonexin on alpha IIbbeta 3 was detected using either native PAC1 or a monomeric fragment of the antibody. Third, agonist-induced clustering of beta 2 integrins in leukocytes and possibly alpha IIbbeta 3 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/beta 3-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 beta 3-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 beta 3- 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 beta 3-endonexin with the cytoplasmic tail of the beta 3 integrin subunit. A mutational analysis of the beta 3 tail has shown that membrane-distal residues near the carboxy terminus of the tail (N756ITY) are required for the interaction with beta 3-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 beta 3 tail chimera, but not an alpha 5 tail chimera, prevented affinity modulation by beta 3-endonexin, possibly because the former chimera but not the latter could compete with alpha IIbbeta 3 for binding to beta 3endonexin. Finally, when other recombinant GFP proteins such as GFP and GFP/VASP were expressed in CHO cells, they failed to increase alpha IIbbeta 3 affinity.

Additional studies will be required to determine how cellular energy depletion or coexpression of activated H-Ras inhibits affinity modulation by beta 3-endonexin. Nonetheless, these results imply that this function of beta 3endonexin 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 IIbbeta 3. In contrast, compounds that activate protein kinase A or G inhibit fibrinogen binding (22, 28). Perhaps beta 3-endonexin is a direct substrate of specific kinases or phosphatases or is a target of downstream effectors of these enzymes. For example, beta 3-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 beta 1 and beta 3 integrins, it seems unlikely that the pathways triggered by these GTPases converge directly on beta 3-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 beta 3-endonexin with the beta 3 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 beta 3 subunit relative to the alpha IIb subunit. This is plausible given the biophysical evidence for interactions between the cytoplasmic tails of alpha IIb and beta 3 (24) and for agonist- induced structural changes in the extracellular domains of alpha IIbbeta 3 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 (beta  tails) (51), talin (beta ) (29), and filamin (beta 2) (60), and potential signaling molecules, such as calreticulin (alpha  tails) (10), pp125FAK (beta ) (58), integrin-linked kinase (beta ) (26), and cytohesin-1 (beta 2) (37). Of note, the expression of calreticulin and cytohesin-1 appears to stimulate or stabilize a high affinity state of integrins alpha 2beta 1 and alpha Lbeta 2, respectively (10, 37). There is no sequence similarity between either of these proteins and beta 3-endonexin. Thus, a structurally diverse group of cytoplasmic tail-binding proteins may function to regulate integrins. Some like cytohesin-1 and beta 3-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 beta 3- 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 beta 3-endonexin (K62RKK) (35, 63). Interestingly, several proteins implicated in cell adhesion and adhesive signaling, including ZO-1, beta -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 beta 3-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 beta 3-endonexin to beta 3-rich focal adhesions suggests that it may interact most strongly with the beta 3 cytoplasmic tail while cells are in suspension or are in the early phases of adhesion. Thus, it is attractive to speculate that beta 3-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 beta 3-endonexin, but they leave several questions unanswered. Does beta 3-endonexin influence outside-in signaling events, such as protein tyrosine phosphorylation (8)? Is beta 3endonexin subject to posttranslational modifications in vivo, and does this affect its subcellular localization or function? Does beta 3-endonexin modulate the adhesive function of alpha Vbeta 3, which like alpha IIbbeta 3 appears to be subject to rapid regulation in some cell types (67, 73)? Does beta 3-endonexin regulate alpha IIbbeta 3 in platelets?


Footnotes

Received for publication 23 December 1996 and in revised form 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).

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.


References

1. Abrams, C., J. Deng, B. Steiner, and S.J. Shattil. 1994. Determinants of specificity of a baculovirus-expressed antibody Fab fragment that binds selectively to the activated form of integrin alpha IIbbeta 3. J. Biol. Chem. 269: 18781-18788 [Abstract/Free Full Text].
2. Alig, L., A. Edenhofer, P. Hadváry, M. Hürzeler, D. Knopp, M. Müller, B. Steiner, A. Trzeciak, and T. Weller. 1992. Low molecular weight, nonpeptide fibrinogen receptor antagonists. J. Med. Chem. 35: 4393-4407
3. Behrens, J., J.P. Vonkries, M. Kuhl, L. Bruhn, D. Wedlich, R. Grosschedl, and W. Birchmeier. 1996. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature (Lond.). 382: 638-642
4. Bennett, J.S., and G. Vilaire. 1979. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J. Clin. Invest. 64: 1393-1401
5. Boudreau, N., C. Myers, and M.J. Bissell. 1995. From laminin to lamin-regulation of tissue-specific gene expression by the ECM. Trends Cell Biol. 5: 1-4 .
6. Chen, Y.-P., I. Djaffar, D. Pidard, B. Steiner, A.-M. Cieutat, J.P. Caen, and J.-P. Rosa. 1992. Ser-752 right-arrow Pro mutation in the cytoplasmic domain of integrin beta 3 subunit and defective activation of platelet integrin alpha IIbbeta 3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia. Proc. Natl. Acad. Sci. USA. 89: 10169-10173 [Abstract].
7. Chen, Y.-P., T.E. O'Toole, T. Shipley, J. Forsyth, S.E. LaFlamme, K.M. Yamada, S.J. Shattil, and M.H. Ginsberg. 1994. "Inside-out" signal transduction inhibited by isolated integrin cytoplasmic domains. J. Biol. Chem. 269: 18307-18310 [Abstract/Free Full Text].
8. Clark, E.A., and J.S. Brugge. 1995. Integrins and signal transduction pathways. The road taken. Science (Wash. DC). 268: 233-239
9. Coller, B.S.. 1995. Blockade of platelet GPIIb/IIIa receptors as an antithrombotic strategy. Circulation. 92: 2373-2380 [Free Full Text].
10. Coppolino, M., C. Leung-Hagesteijn, S. Dedhar, and J. Wilkins. 1995. Inducible interaction of integrin alpha 2beta 1 with calreticulin: dependence on the activation state of the integrin. J. Biol. Chem. 270: 23132-23138 [Abstract/Free Full Text].
11. Dedhar, S., and G.E. Hannigan. 1996. Integrin cytoplasmic interactions and bidirectional transmembrane signalling. Curr. Opin. Cell Biol. 8: 657-669
12. Diamond, M.S., and T.A. Springer. 1994. The dynamic regulation of integrin adhesiveness. Curr. Biol. 4: 506-517
13. Eigenthaler, M., L. Hofferer, S.J. Shattil, and M.H. Ginsberg. 1997. A conserved sequence motif in the integrin beta 3 cytoplasmic domain is required for its specific interaction with beta 3-endonexin. J. Biol. Chem. 272: 7693-7698 [Abstract/Free Full Text].
14. Fox, J., S.J. Shattil, R. Kinlough-Rathbone, M. Richardson, M.A. Packham, and D.A. Sanan. 1996. The platelet cytoskeleton stabilizes the interaction between alpha IIbbeta 3 and its ligand and induces selective movements of ligandoccupied integrin. J. Biol. Chem. 271: 7004-7011 [Abstract/Free Full Text].
15. Gadella, T.W.J., Jr, and T.M. Jovin. 1995. Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. J. Cell Biol. 129: 1543-1558 [Abstract].
16. Gawaz, M.P., J.C. Loftus, M.L. Bajt, M.M. Frojmovic, E.F. Plow, and M.H. Ginsberg. 1991. Ligand bridging mediates integrin alpha IIbbeta 3 (platelet GPIIB-IIIA)-dependent homotypic and heterotypic cell-cell interactions. J. Clin. Invest. 88: 1128-1134
17. Geiger, B., S. Yehuda-Levenberg, and A. D. Bershadsky. 1995. Molecular interactions in the submembrane plaque of cell-cell and cell-matrix adhesions. Acta Anat. 154: 46-62
18. Gilmore, A.P., and K. Burridge. 1996. Molecular mechanisms for focal adhesion assembly through regulation of protein-protein interactions. Structure (Lond.). 4: 647-651
19. Ginsberg, M.H., A.L. Frelinger, S.C.-T. Lam, J. Forsyth, R. McMillan, E.F. Plow, and S.J. Shattil. 1990. Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies. Blood. 76: 2017-2023 [Abstract].
20. Ginsberg, M.H., X. Du, and E.F. Plow. 1992. Inside-out integrin signalling. Curr. Opin. Cell Biol. 4: 766-771
21. Gottardi, C.J., M. Arpin, A.S. Fanning, and D. Louvard. 1996. The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts. Proc. Natl. Acad. Sci. USA. 93: 10779-10784 [Abstract/Free Full Text].
22. Graber, S.E., and J. Hawiger. 1982. Evidence that changes in platelet cyclic AMP levels regulate the fibrinogen receptor on human platelets. J. Biol. Chem. 257: 14606-14609 [Abstract/Free Full Text].
23. Guinebault, C., B. Payrastre, C. Sultan, G. Mauco, M. Breton, S. LevyToledano, M. Plantavid, and H. Chap. 1993. Tyrosine kinases and phosphoinositide metabolism in thrombin-stimulated human platelets. Biochem. J. 292: 851-856
24. Haas, T.A., and E.F. Plow. 1996. The cytoplasmic domain of alpha IIbbeta 3. A ternary complex of the integrin alpha  and beta  subunits and a divalent cation. J. Biol. Chem. 271: 6017-6026 [Abstract/Free Full Text].
25. Haffner, C., T. Jarchau, M. Reinhard, J. Hoppe, S.M. Lohmann, and U. Walter. 1995. Molecular cloning, structural analysis and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP. EMBO (Eur. Mol. Biol. Organ.) J. 14: 19-27 [Abstract].
26. Hannigan, G.E., C. Leung-Hagesteijn, L. Fitz-Gibbon, M.G. Coppolino, G. Radeva, J. Filmus, J.C. Bell, and S. Dedhar. 1996. Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase. Nature (Lond.). 379: 91-96
27. Hawiger, J.. 1995. Mechanisms involved in platelet-vessel wall interaction. Thromb. Haemostasis. 74: 369-372
28. Horstrup, K., B. Jablonka, P. Honig-Liedl, M. Just, K. Kochsiek, and U. Walter. 1994. Phosphorylation of focal adhesion vasodilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrinogen receptor inhibition. Eur. J. Biochem. 225: 21-27 [Abstract].
29. Horwitz, A., K. Duggan, C. Buck, M.C. Beckerle, and K. Burridge. 1986. Interaction of plasma membrane fibronectin receptor with talin: a transmembrane linkage. Nature (Lond.). 320: 531-533
30. Huang, M.-M., L. Lipfert, M. Cunningham, J.S. Brugge, M.H. Ginsberg, and S.J. Shattil. 1993. Adhesive ligand binding to integrin alpha IIbbeta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK. J. Cell Biol. 122: 473-483 [Abstract].
31. Hughes, P.E., T.E. O'Toole, J. Ylanne, S.J. Shattil, and M.H. Ginsberg. 1995. The conserved membrane-proximal region of an integrin cytoplasmic domain specifies ligand-binding affinity. J. Biol. Chem. 270: 12411-12417 [Abstract/Free Full Text].
32. Hughes, P.E., F. Diaz-Gonzalez, L. Leong, C.Y. Wu, J.A. McDonald, S.J. Shattil, and M.H. Ginsberg. 1996. Breaking the integrin hinge: a defined structural constraint regulates integrin signaling. J. Biol. Chem. 271: 6571-6574 [Abstract/Free Full Text].
33. Hughes, P.E., M.W. Renshaw, M. Pfaff, J. Forsyth, V.M. Keivens, M.A. Schwartz, and M.H. Ginsberg. 1996. Suppression of integrin activation: a novel function of a Ras/Raf-initiated MAP-kinase pathway. Cell. 88: 521-530 .
34. Hynes, R.O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 69: 11-25
35. Jans, D.A.. 1995. The regulation of protein transport to the nucleus by phosphorylation. Biochem. J. 311: 705-716
36. Kieffer, J.D., G. Plopper, D.E. Ingber, J.H. Hartwig, and T.S. Kupper. 1995. Direct binding of F-actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro. Biochem. Biophys. Res. Commun. 217: 466-474
37. Kolanus, W., W. Nagel, B. Schiller, L. Zeitlmann, S. Godar, H. Stockinger, and B. Seed. 1996. Alpha-L-Beta-2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell. 86: 233-242
38. Kovacsovics, T.J., C. Bachelot, A. Toker, C.J. Vlahos, B. Duckworth, L.C. Cantley, and J.H. Hartwig. 1995. Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation. J. Biol. Chem. 270: 11358-11366 [Abstract/Free Full Text].
39. Deleted in proof.
40. LaFlamme, S.E., L.A. Thomas, S.S. Yamada, and K.M. Yamada. 1994. Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly. J. Cell Biol. 126: 1287-1298 [Abstract].
41. Law, S.F., J. Estojak, B.L. Wang, T. Mysliwiec, G. Kruh, and E.A. Golemis. 1996. Human enhancer of filamentation 1, a novel p130cas-like docking protein, associates with focal adhesion kinase and induces pseudohyphal growth in Saccharomyces cerevisiae. Mol. Cell. Biol. 16: 3327-3337 [Abstract].
42. Lee, J.O., L.A. Bankston, M.A. Arnaout, and R.C. Liddington. 1995. Two conformations of the integrin A-domain (I-domain): a pathway for activation? Structure (Lond.). 3: 1333-1340
43. Leong, L., P.E. Hughes, M.A. Schwartz, M.H. Ginsberg, and S.J. Shattil. 1995. Integrin signaling: roles for the cytoplasmic tails of alpha IIbbeta 3 in the tyrosine phosphorylation of pp125FAK. J. Cell Sci. 108: 3817-3825 [Abstract/Free Full Text].
44. Lewis, J.M., R. Baskaran, S. Taagepera, M.A. Schwartz, and J.Y.J. Wang. 1996. Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic-nuclear transport. Proc. Natl. Acad. Sci. USA. 93: 15174-15179 [Abstract/Free Full Text].
45. Meredith, J., Y. Tahada, M. Fornaro, L. Languino, and M.A. Schwartz. 1995. Inhibition of cell cycle progression by the alternatively spliced integrin beta(1C). Science (Wash. DC). 269: 1570-1572
46. Miller, F., and H. Metzger. 1965. Characterization of a human macroglobulin. II. Distribution of the disulfide bonds. J. Biol. Chem. 240: 4740-4745 [Free Full Text].
47. Nachmias, V.T., and R. Golla. 1991. Vinculin in relation to stress fibers in spread platelets. Cell Motil. Cytoskeleton. 20: 190-202
48. O'Toole, T.E., J.C. Loftus, X. Du, A.A. Glass, Z.M. Ruggeri, S.J. Shattil, E.F. Plow, and M.H. Ginsberg. 1990. Affinity modulation of the alpha IIbbeta 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor. Cell Regul. 1: 883-893
49. O'Toole, T.E., Y. Katagiri, R.J. Faull, K. Peter, R. Tamura, V. Quaranta, J.C. Loftus, S.J. Shattil, and M.H. Ginsberg. 1994. Integrin cytoplasmic domains mediate inside-out signaling. J. Cell Biol. 124: 1047-1059 [Abstract].
50. O'Toole, T.E., J. Ylanne, and B.M. Culley. 1995. Regulation of integrin affinity states through an NPXY motif in the beta  subunit cytoplasmic domain. J. Biol. Chem. 270: 8553-8558 [Abstract/Free Full Text].
51. Otey, C.A., G.B. Vasquez, K. Burridge, and B.W. Erickson. 1993. Mapping of the alpha -actinin binding site within the beta 1 integrin cytoplasmic domain. J. Biol. Chem. 268: 21193-21197 [Abstract/Free Full Text].
52. Peerschke, E.I.. 1995. Regulation of platelet aggregation by post-fibrinogen binding events. Thromb. Haemostasis. 73: 862-867
53. Peerschke, E.I.B.. 1995. Bound fibrinogen distribution on stimulated platelets: examination by confocal scanning laser microscopy. Am. J. Pathol. 147: 678-687 [Abstract].
54. Peerschke, E.I.B., and J.A. Wainer. 1985. Examination of irreversible platelet-fibrinogen interactions. Am. J. Physiol. 248: C466-C472 [Abstract].
55. Qu, A.D., and D.J. Leahy. 1996. The role of divalent cation in the structure of the I-domain from the CD11a/CD18 integrin. Structure (Lond.). 4: 931-942
56. Scarborough, R.M., M.A. Naughton, W. Teng, J.W. Rose, D.R. Phillips, L. Nannizzi, A. Arfsten, A.M. Campbell, and I.F. Charo. 1993. Design of potent and specific integrin antagonists. Peptide antagonists with high specificity for glycoprotein IIb-IIIa. J. Biol. Chem. 268: 1066-1073 [Abstract/Free Full Text].
57. Schaller, M.D., C.A. Borgman, and J.T. Parsons. 1993. Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK. Mol. Cell. Biol. 13: 785-791 [Abstract].
58. Schaller, M.D., C.A. Otey, J.D. Hildebrand, and J.T. Parsons. 1995. Focal adhesion kinase and paxillin bind to peptides mimicking beta  integrin cytoplasmic domains. J. Cell Biol. 130: 1181-1187 [Abstract].
59. Schwartz, M.A., M.D. Schaller, and M.H. Ginsberg. 1995. Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Biol. 11: 549-599
60. Sharma, C.P., R.M. Ezzell, and M.A. Arnaout. 1995. Direct interaction of filamin (ABP-280) with the beta 2-integrin subunit CD18. J. Immunol. 154: 3461-3470 [Abstract/Free Full Text].
61. Shattil, S.J., M. Cunningham, T. Wiedmer, J. Zhao, P.J. Sims, and L.F. Brass. 1992. Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9. J. Biol. Chem. 267: 18424-18431 [Abstract/Free Full Text].
62. Shattil, S.J., M.H. Ginsberg, and J.S. Brugge. 1994. Adhesive signaling in platelets. Curr. Opin. Cell Biol. 6: 695-704
63. Shattil, S.J., T. O'Toole, M. Eigenthaler, V. Thon, M. Williams, B.M. Babior, and M.H. Ginsberg. 1995. beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin beta 3 subunit. J. Cell Biol. 131: 807-816 [Abstract].
64. Shock, D.D., K. He, J.D. Wencel-Drake, and L.V. Parise. 1997. Ras activation in platelets following stimulation of the thrombin receptor, thromboxane A2 receptor or protein kinase C.  Biochem. J. 321: 525-530
65. Sims, P.J., M.H. Ginsberg, E.F. Plow, and S.J. Shattil. 1991. Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex. J. Biol. Chem. 266: 7345-7352 [Abstract/Free Full Text].
66. Stock, J.. 1996. Signaling across membranes: a one and a two and a . . .  Science (Wash. DC). 274: 370-371 [Abstract/Free Full Text].
67. Stupack, D.G., C. Shen, and J.A. Wilkins. 1992. Induction of alpha vbeta 3 integrinmediated attachment to extracellular matrix in beta 1 integrin (CD29)-negative B cell lines. Exp. Cell Res. 203: 443-448
68. Van Willigen, G., and J.-W.N. Akkerman. 1991. Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIb-IIIa complex of human platelets. Biochem. J. 273: 115-120
69. Van Willigen, G., I. Hers, G. Gorter, and J.-W.N. Akkerman. 1996. Exposure of ligand-binding sites on platelet integrin alpha IIb/beta 3 by phosphorylation of the beta 3 subunit. Biochem. J. 314: 769-779
70. Deleted in proof.
71. Weber, C., J. Kitayama, and T.A. Springer. 1996. Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils. Proc. Natl. Acad. Sci. USA. 93: 10939-10944 [Abstract/Free Full Text].
72. Ylanne, J., J. Huuskonen, T.E. O'Toole, M.H. Ginsberg, I. Virtanen, and C.G. Gahmberg. 1995. Mutation of the cytoplasmic domain of the integrin beta 3 subunit: differential effects on cell spreading, recruitment to adhesion plaques, endocytosis and phagocytosis. J. Biol. Chem. 270: 9550-9557 [Abstract/Free Full Text].
73. Zanetti, A., G. Conforti, S. Hess, I. Martìn-Padura, E. Ghibaudi, K.T. Preissner, and E. Dejana. 1994. Clustering of vitronectin and RGD peptides on microspheres leads to engagement of integrins on the luminal aspect of endothelial cell membrane. Blood. 84: 1116-1123 [Abstract/Free Full Text].
74. Zhang, J., S.J. Shattil, M. Cunningham, J.R. Falck, K.K. Reddy, and S.E. Rittenhouse. 1996. Phosphoinositide 3-kinase (gamma ) and p85/phosphoinositide 3-kinase in platelets: relative activation by thrombin receptor or beta -phorbol myristate acetate and roles in promoting the ligand-binding function of alpha IIbbeta 3 integrin. J. Biol. Chem. 271: 6265-6272 [Abstract/Free Full Text].
75. Zhang, Z., K. Vuori, H.-G. Wang, J.C. Reed, and E. Ruoshlati. 1996. Integrin activation by R-ras. Cell. 85: 61-69

Copyright © 1997 by The Rockefeller University Press.