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Address correspondence to Xiaoping Du, Department of Pharmacology, College of Medicine, University of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612. Tel.: (312) 355-0237. Fax: (312) 996-1225. E-mail: xdu{at}uic.edu
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Abstract |
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Key Words: integrin; platelet; calpain; signaling; platelet activation
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Introduction |
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Inside-out and outside-in signaling of integrins have been suggested to involve the interaction between the cytoplasmic domains of integrins and intracellular signaling molecules. Interactions between the membrane-proximal regions of the integrin IIb and ß3 cytoplasmic domains are important in maintaining a resting conformation of the integrin, and inside-out signaling is associated with disruption of this interaction (Hughes et al., 1996; Vinogradova et al., 2002). The cytoplasmic domain of
IIbß3 interacts with several intracellular proteins, including cytoskeletal proteins such as talin (Calderwood et al., 1999; Knezevic et al., 1996) and myosin (Phillips et al., 2001), calcium-binding protein CIB (Naik et al., 1997), phosphotyrosine-binding proteins SHC and GRB2 (Law et al., 1996), protein kinases such as integrin-linked protein kinase (Hannigan et al., 1996), and ß3 endonexin (Shattil et al., 1995). Binding of talin (Calderwood et al., 1999, 2002) and ß3 endonexin (Kashiwagi et al., 1997) to ß3 has been implicated in promoting integrin activation. Binding of phosphotyrosine-binding proteins and focal adhesion kinase has been suggested to play roles in outside-in signaling (Law et al., 1996). Also, the ß3 cytoplasmic domain can be chemically modified during platelet activation. For example, T753 (Lerea et al., 1999), Y747, and Y759 (Law et al., 1996) are phosphorylated by protein kinases, and the COOH-terminal region of ß3 is cleaved by calpain at the COOH-terminal side of Y741, T747, F754, and Y759 (Du et al., 1995). Phosphorylation at both Y747 and Y759 is critical to outside-in signaling of the integrin (Law et al., 1999). The roles of calpain cleavage of the ß3 cytoplasmic domain in bidirectional signaling of the integrin, however, are not clear.
The requirement for specific sequences in the ß3 cytoplasmic domain in outside-in integrin signaling has been indicated by mutagenesis studies in transfected CHO cell models (Chen et al., 1994; Hughes et al., 1995; Ylanne et al., 1995; Patil et al., 1999) and transgenic mouse models (Law et al., 1999). Deletions or mutations that disrupted the two NXXY motifs in the cytoplasmic domain of the integrin abolished outside-in signal-dependent integrin functions such as stable cell adhesion and cell spreading (Ylanne et al., 1993, 1995; Chen et al., 1994; Patil et al., 1999). In a transgenic mouse model, mutations replacing both Y747 and Y759 in the NXXY motifs with phenylalanine to disrupt tyrosine phosphorylation inhibited outside-in signaling but had no significant effect on inside-out signaling (Law et al., 1999). The requirement for the cytoplasmic domain of ß3 in inside-out signaling has been indicated by defective integrin activation in variant Glanzmann's thrombasthenia patients whose ß3 cytoplasmic domain either has a point mutation replacing S752 with proline (Chen et al., 1992, 1994) or has a truncation mutation at R724 (Wang et al., 1997). However, mapping of functional sites important to inside-out signaling has been hampered by the lack of a suitable inside-out signaling model in cultured cells deficient in endogenous wild-type ß3, as recombinant integrin IIbß3 expressed in CHO cells is not activated by platelet agonists such as thrombin and ADP (O'Toole et al., 1990). Although constitutively active integrin mutants have been useful in characterizing the affinity regulation by integrin cytoplasmic domains (O'Toole et al., 1991, 1994; Hughes et al., 1995), these mutants obviously bypass the normal onoff switch mechanism of inside-out signaling. We and others have recently shown that integrin can be activated in a reconstituted CHO cell model via the GPIb-IX pathway (Gu et al., 1999; Zaffran et al., 2000; Li et al., 2001). In this model, and as shown here, binding of vWF to GPIb-IX induces inside-out signaling and results in activation of the fibrinogen-binding function of
IIbß3, which replicates the GPIb-IXinduced inside-out signaling mechanism in platelets. To understand the structural requirement of the ß3 cytoplasmic domain in integrin inside-out signaling and the role of calpain cleavage in regulating integrin signaling, we have expressed different truncation mutants of ß3 in this reconstituted integrin activation model. We show that removal of the NITY sequence abolished inside-out signaling, whereas truncation of RGT sequence COOH terminal to the NITY motif reduced outside-in signaling without affecting inside-out signaling. Furthermore, a point mutation changing Y759 to alanine abolished inside-out signaling and reduced outside-in signaling. Thus, the NITY sequence is essential for both inside-out and outside-in signaling of
IIbß3, and the RGT sequence is important for outside-in signaling. Furthermore, localized calpain cleavage of ß3 during platelet activation mainly occurs at a site COOH terminal to Y759, suggesting that calpain cleavage may selectively regulate outside-in signaling.
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Results |
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Differential cleavage at different sites of the ß3 cytoplasmic domain by calpain in platelets
We have shown previously that calpain may cleave the cytoplasmic domain of ß3 at sites COOH terminal to T741, Y747, F754, and Y759, which generates ß3 fragments identical to the above-described truncation mutants of ß3 (741, 747, 754, and 759). As we showed above that truncations at these different sites result in different functional effects on integrin signaling, our results also indicate that cleavage of the ß3 cytoplasmic domain at these different sites has the potential to differentially regulate outside-in and inside-out signaling of integrin IIbß3. To determine if calpain differentially cleaves the ß3 integrin at different sites during platelet activation, washed platelets were treated with or without thrombin (0.1 U/ml) and then immunoblotted for calpain cleavage by the cleavage-specific antibodies (Du et al., 1995). We found that anti-759 antibody, which only recognizes ß3 molecules with cleavage at Y759, reacted with
0.8% of the integrin
IIbß3 molecules in washed "resting" platelets (Fig. 7). This reaction was unlikely to result from cross-reaction of this antibody with the intact ß3 subunit, because we showed that the antibody did not react with the intact ß3 subunit but reacted with the
759 mutant expressed in CHO cells (Fig. 2). Thus a very small percentage of the ß3 molecules in resting platelets has been cleaved at the Y759 site. Stimulation of platelets with thrombin caused a time-dependent and significant increase in the cleavage at Y759. In contrast to the 759 site, calpain cleavage at T754 or Y747 (Fig. 7) occurred to a much lesser degree and only after a much longer exposure to thrombin. Calpain cleavage at the 741 site was not detectable in thrombin-stimulated platelets (unpublished data) but was detected in platelets treated with calcium ionophore A23187 (Du et al., 1995). Thus, in thrombin-activated platelets, calpain preferentially cleaves ß3 at Y759. As we showed above that cleavage at Y759 selectively reduced integrin outside-in signaling without affecting inside-out signaling, this result indicates that calpain cleavage has the potential to selectively regulate outside-in signaling during platelet activation.
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Discussion |
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We conclude that the NITY sequence of the ß3 cytoplasmic domain is critical to the inside-out signaling function of IIbß3. This conclusion is supported by the finding that deletion of the RGT sequence COOH terminal to the NITY motif did not affect vWF-induced integrin activation, but deletion of the TNITY sequence completely abolished integrin activation. The importance of the NITY sequence in inside-out signaling is further supported by the data that the Y759A point mutation inhibited vWF-induced integrin activation. Also, we show that inhibition of vWF-induced fibrinogen binding is not caused by loss of the fibrinogen-binding function per se, as RGDS-induced fibrinogen binding to either 1b9/754 or 1b9/Y759A mutant cells was not different from the wild-type integrin. Furthermore, our data indicate that the NITY sequence and the COOH-terminal RGT sequence are both important for outside-in signaling of
IIbß3 and integrin-dependent stable cell adhesion and spreading. This is consistent with previous work showing that this region of the ß3 cytoplasmic domain is important in cell spreading and focal adhesion formation (Ylanne et al., 1993, 1995). Thus, the NITY motif is important for both inside-out and outside-in signaling of the integrin
IIbß3.
The NITY motif contains a tyrosine residue that becomes phosphorylated during platelet aggregation (Law et al., 1996). The phosphorylated NXXY motifs have been shown to interact with several intracellular molecules, including myosin and phosphotyrosine-binding proteins such as GRB2 and SHC (Cowan et al., 2000), which are implicated in integrin outside-in signaling. It has been shown that mutation of both tyrosine residues to phenolalanines selectively abolished outside-in signaling in transgenic mouse platelets (Law et al., 1999), suggesting that phosphorylation of one or both of these tyrosine residues is important for outside-in signaling but not for integrin activation (inside-out signaling). Thus, although we conclude that the NITY sequence is essential for inside-out signaling, phosphorylation at Y759 is unlikely to be involved in this process. In this regard, a functional difference between Y759F and Y759A mutations has been shown previously (Schaffner-Reckinger et al., 1998). Y759A, but not Y759F, inhibited integrin-dependent cell adhesion. Furthermore, tyrosine phosphorylation occurs only after platelet aggregation, suggesting that inside-out signaling does not involve tyrosine phosphorylation in the NXXY motifs (Law et al., 1996, 1999).
It is interesting to note that although the NITY sequence is important for both inside-out and outside-in signaling, there is a significant difference in the structural requirement between inside-out signaling (integrin activation) and outside-in signaling. Cleavage of the COOH-terminal three residues did not affect inside-out signaling but significantly inhibited outside-in signalingdependent integrin function, such as cell spreading and stable cell adhesion. On the other hand, disruption of the NITY sequence by Y759A mutation abolished vWF-induced integrin activation (as indicated by soluble fibrinogen binding) and partially (although significantly) inhibited cell spreading and stable cell adhesion on fibrinogen. In addition, previous work suggests that outside-in signaling, but not inside-out signaling, requires phosphorylation at tyrosine residues (Law et al., 1999). The difference in the structural requirements between inside-out and outside-in signals suggests that inside-out and outside-in signals may be mediated by different molecules (or mechanisms) that interact with the COOH-terminal region of ß3 during platelet adhesion and aggregation. This also suggests that inside-out and outside-in signals can be differentially regulated.
The family of the calcium-dependent intracellular proteases, calpain, plays important roles in cytoskeletal reorganization, cell migration, platelet aggregation, and clot retraction (Fox et al., 1983; Huttenlocher et al., 1997; Croce et al., 1999; Bialkowska et al., 2000; Azam et al., 2001). We have shown previously that the ß3 cytoplasmic domain is cleaved by either calpain I or calpain II at sites flanking two NXXY motifs in human platelets (Du et al., 1995; Pfaff et al., 1999). Calpain cleavage of the ß3 cytoplasmic domain also occurs during endothelial cell apoptosis (Meredith et al., 1998). The physiological roles of calpain cleavage of ß3 have been unclear. Calpain I knockout in mouse inhibited platelet aggregation but did not affect ß3 cleavage. This suggests that cleavage of the ß3 subunit is not involved in promoting platelet aggregation (Azam et al., 2001). Here we show that one of the functional consequences of calpain cleavage of ß3 is to negatively regulate the signaling functions of integrin IIbß3. Furthermore, we show that cleavage by calpain at different sites of ß3 may result in different regulatory effects. Cleavage at Y759 has no significant effect on inside-out signaling but significantly reduces the integrin functions associated with outside-in signaling. In contrast, cleavage at F754 or further NH2-terminal sites abolishes both inside-out and outside-in signaling. Nevertheless, cleavage at these sites occurs much later during platelet activation (Figs. 7 and 8), suggesting that such cleavages are not important for the early phase of integrin activation. On the other hand, we found that calpain cleavage of integrin ß3 subunit in intact platelets mainly occurs at the most COOH-terminal Y759 site during platelet activation and adhesion (Figs. 7 and 8). Furthermore, cleavage of ß3 does not appear to occur to the integrin molecules at the leading edge of spreading platelets but occurs to more centrally localized integrin molecules. Thus, it is likely that cleavage at Y759 serves to selectively down-regulate outside-in signaling in the integrincytoskeletal signaling complexes that have been formed during the earlier stage of platelet spreading, thereby facilitating the dynamic reorganization of the integrincytoskeleton signaling complex during platelet spreading.
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Materials and methods |
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Construction of truncation mutants of ß3 subunit
Truncation mutagenesis was performed using PCR to introduce stop codons into integrin ß3 cDNA at sites corresponding to the carboxy side of amino acid residues 741, 747, 754, or 759, respectively. The forward primer has the sequence of AGAGCTTAAGGACAC at an AflII site of ß3 cDNA. The reverse primers contain an XhoI digestion site, a stop codon, and the 18-nucleotide ß3 sequences at the intended COOH terminus of each mutant. The PCR products were digested with restriction enzymes AspI and XhoI and ligated into a ß3 cDNA construct in a modified cDM8 vector containing only the 3'-end XhoI site that was digested with the same restriction enzymes. All mutant constructs were verified by DNA sequencing.
Expression of mutant ß3 cDNA constructs in CHO cells
CHO cells expressing GPIb-IX (1b9) were maintained in DEAE medium supplemented with 10% FBS, glutamine, and nonessential amino acid. Transfection was performed using LipofectAMINE 2000 (Life Technologies). Each mutant ß3 cDNA was cotransfected together with wild-type IIb and pcDNA3.1/Hyg plasmid at a ratio of 5:5:1. Stable cell lines expressing mutant ß3 were selected in 0.2 mg/ml of hygromycin (Invitrogen). Expression of integrin
IIbß3 was monitored by flow cytometry using D57. Expression of GPIb-IX was detected using SZ2. Cells expressing both GPIb-IX and integrin
IIbß3 were selected by cell sorting. All cell lines were sorted using the expression levels of 123 cells as a gate until similar levels of expression were achieved. To verify correct expression of calpain cleavagemimicking mutants, cells were also solubilized and electrophoresed on 7% polyacrylamide gels and then immunoblotted with various cleavage-specific antibodies followed by detection using the ECL kit from Amersham.
Fibrinogen binding to transfected CHO cells activated by vWF
Activation of IIbß3 induced by ristocetin and vWF was examined by flow cytometric analysis of Oregon green 488conjugated fibrinogen binding to
IIbß3, as previously described (Gu et al., 1999; Li et al., 2001). In brief, transfected CHO cells were resuspended to 5 x 105/ml in modified Tyrode's solution (2.5 mM Hepes, 150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 5.5 mM D-glucose, 1 mM CaCl2, 1 mM MgCl2, 0.1% BSA, pH 7.4), incubated with Oregon green 488conjugated fibrinogen (30 µg/ml) and ristocetin (1 mg/ml) in the presence or absence of purified human vWF (25 µg/ml) at 22°C for 30 min, and analyzed by flow cytometry. Nonspecific binding of fibrinogen was estimated by measuring fibrinogen binding in the presence of an integrin inhibitor, RGDS (1 mM).
Fibrinogen binding induced by RGDS peptide
RGDS pretreatmentinduced fibrinogen binding was assayed according to a previously described method (Du et al., 1991). Cells in modified Tyrode's solution were incubated with 1 mM RGDS peptide at 22°C for 10 min. ParaformaldehydePBS was then added (to a final concentration of 1%), and the mixture was incubated at 22°C for 1 h. After adding 250 mM NH4Cl, the fixed cells were washed, resuspended in modified Tyrode's solution, and incubated with Oregon green 488conjugated fibrinogen (30 µg/ml) for 30 min before flow cytometric analyses.
Cell adhesion under static conditions
Microtiter wells were coated with 10 µg/ml human vWF or fibrinogen in 0.1 M NaHCO3, pH 8.3, at 4°C overnight and then blocked with 5% BSAPBS at 22°C for 2 h. Cell suspension (106/ml in modified Tyrode's buffer with 1% BSA) was added to ligand-coated microtiter wells and incubated for 60 min at 37°C in a CO2 incubator. After three washes, cell spreading was examined under an inverted microscope (20x objective lens). In quantitative assays, 50 µl of 0.3% p-nitrophenyl phosphate in 1% Triton X-100, 50 mM sodium acetate, pH 5.0, was added to microtiter wells and incubated at 37°C for 1 h. The reaction was stopped by adding 50 µl of 1 M NaOH. Results were determined by reading the OD at a 405-nm wavelength.
Cell adhesion under flow
Purified human vWF (100 µg/ml with 0.1 mM NaHCO3, pH 8.3) was added into a glass capillary tube (0.59 mm ID, 75 mm in length; Harvard Apparatus Inc.) and then incubated overnight in a humid environment at 4°C (Englund et al., 2001). The capillary tubes were rinsed with PBS and then blocked with 5% BSA in PBS. CHO cells expressing human platelet receptors (5 x 106/ml in modified Tyrode's buffer containing 5% BSA) were perfused with a syringe pump (Harvard Apparatus Inc.) through the capillary tube at various shear rates for 2 min followed by perfusion for 10 min with cell-free buffer at the same shear rates. Shear rate was calculated as described by Slack and Turitto (1994). Cell interaction with immobilized vWF was observed in real time under an inverted microscope and recorded on videotapes. The number of stable adherent cells on immobilized vWF was counted on images obtained in 10 randomly selected fields in the vWF-coated tubes. The statistical difference between 123 cells and each one of the mutant cell lines was determined using the t test.
Calpain cleavage of integrin ß3 subunit in human platelets
Preparation of washed human platelets has been described previously (Li et al., 2003). Platelet aggregation was induced in a Chronolog lumi-aggregometer by adding 0.1 U/ml thrombin with constant stirring at 1,000 rpm for various lengths of time. The reaction was stopped by adding an equal volume of 2x SDS-PAGE sample buffer containing 1 mM EDTA, 1 mM PMSF, and 0.1 mM E64. Samples were then analyzed by SDS-PAGE on a 7% gel and immunoblotted with cleavage-specific antibodies (Du et al., 1995). The densities of reactive bands were scanned and then quantitated using NIH Image. Lysates prepared from 123, 1b9/747, 1b9/754, and 1b9/579 CHO cell lines were used as internal calibration standards for each of the cleavage-specific antibodies. The ratio of cleaved ß3 molecules was calculated as the ratio between the optical density of the reaction of an antibody with platelet lysates and the reaction of the same antibody with the corresponding standard CHO cell lysates multiplied by the ratio between the ß3 levels in the standard CHO cell lysates and the ß3 level in platelet lysates (as determined by immunoblotting with Mab15 directed against the extracellular domain of ß3).
Immunofluorescence analysis of calpain-cleaved ß3 in spreading platelets
Platelets in modified Tyrode's buffer (1 x 108/ml) were allowed to adhere to the Lab-Tek chamber slides (Nunc) precoated with 20 µg/ml of fibrinogen at 37°C for 2 h as previously described (Bodnar et al., 1999). The chamber slides were rinsed three times. Adherent platelets were fixed with 1% paraformaldehyde and permeabilized with 0.1 M Tris, 10 mM EGTA, 0.15 M NaCl, 5 mM MgCl2, 1 mM PMSF, 0.1 mM E64, 0.1% Triton X-100, 1% BSA, pH 7.5. The samples were incubated first with Mab15 and one of the cleavage-specific antibodies and then with Alexa Fluor 488conjugated goat antimouse IgG and Alexa Fluor 546conjugated goat antirabbit IgG. The slides were then scanned under a Carl Zeiss MicroImaging, Inc. LSM510 confocal microscope (63x objective lens).
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Acknowledgments |
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Submitted: 18 March 2003
Revised: 6 June 2003
Accepted: 10 June 2003
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References |
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