Unusually Stable and Long-lived Ligand-induced Conformations of Integrins*

Nina I. Zolotarjova, Gregory F. Hollis, and Richard WynnDagger

From the DuPont Pharmaceuticals Company, Department of Applied Biotechnology, Wilmington, Delaware 19880

Received for publication, October 20, 2000, and in revised form, March 7, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins are a large family of cell surface receptors that are involved in a wide range of biological processes. The integrin alpha IIbbeta 3 (glycoprotein IIb-IIIa) is a major platelet glycoprotein heterodimeric receptor that mediates platelet aggregation and is currently a target for pharmaceutical intervention. Ligand binding to the receptor has been shown to induce conformational changes by physical methods and the exposure of neoepitopes (the ligand-induced binding sites). Here we show that the antagonist XP280 induces a conformation that is stable to treatment with SDS and that the protein retains this conformation for several days even after dissociation of the inhibitor. These ligand-induced conformational changes take place with purified protein and on intact platelets. They are competable with an RGDS peptide and are stable to reduction but not boiling or treatment with EDTA. The retention of an altered conformation in the absence of the ligand implies the possibility of ligand-induced alteration of biological function even in the absence of ligand. Finally, similar behavior is observed with the integrin alpha vbeta 3, suggesting that access to SDS stable conformations may be conserved throughout the integrin superfamily. The unusual stability, long-lived nature, and potential generality of these conformations could have profound implications for integrin biology.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins are a large family of heterodimeric cell surface receptors that mediate cell-matrix, cell-cell, and cell-protein interactions (1). The adhesive interactions have been shown to be important for regulation of microfilament organization and activation of various signaling pathways. Additionally, increasing interest is being focused on "inside-out signaling," whereby events inside the cell are known to modulate the affinity and specificity of the extracellular receptor domains (2). Integrins perform functions central to tissue development, inflammation, tumor cell growth and metastasis, and programmed cell death (3). They have been implicated in the pathology of a wide variety of diseases and therefore make logical targets for therapeutic intervention (4).

Integrins are made up of alpha  and beta  subunits. To date, 16 alpha  and 8 beta  subunits have been identified (5). Pairing of subunits is semispecific, with some subunits being specific for a single partner (alpha IIb pairs only with beta 3) while others pair less strictly (alpha v pairs with beta 1, beta 3, beta 5, beta 6, and beta 8) (3). beta  subunits usually consist of a short intracellular domain, which appears to be important for interaction with the cytoskeleton, a membrane-spanning domain, multiple cysteine-rich repeats, and a conserved domain containing a MIDAS (metal ion-dependent adhesion site) motif. alpha  subunits can be processed post-translationally to a heavy and light chain, which are held together by an extracellular disulfide bond. These subunits also have short intracellular domains and additionally multiple EF-hand-like domains. Both subunits contain significant amounts of carbohydrate and cation binding sites, some of which are important for ligand binding. The large size, transmembrane domains, and complexity due to high amounts of carbohydrate have precluded study by many physical techniques.

Glycoprotein (GP)1 IIb-IIIa is an abundant platelet receptor of the integrin family. It has been shown to play a primary role in platelet aggregation and may interact with fibrinogen, von Willebrand factor, fibronectin, and vitronectin via an RGDS sequence. GP IIb-IIIa is maintained on the resting platelet surface predominantly in an inactive or lower affinity conformation. Platelet activation results in a conformation of GP IIb-IIIa that has higher affinity and is competent to bind soluble plasma fibrinogen and other RGDS containing ligands (6). Binding of ligands to GP IIb-IIIa induces conformational changes, which have been studied by proteolysis patterns (7, 8), sucrose gradient ultracentrifugation (9), fibrinogen binding measurements (10), light scattering and electron microscopy (11), and antibody recognition of ligand-induced binding sites (LIBS) (12-16). Here we present evidence that some ligands can induce unusually stable conformations and that these conformations can be long-lived even after removal of the ligand. The technique used here is based on electrophoretic mobility changes. It offers a simple and rapid probe of integrin conformational changes without the need for specific reagents such as conformation-dependent antibodies. Additionally, it is shown that another integrin, alpha vbeta 3, shows similar behavior when treated with antagonists, suggesting that this behavior is conserved throughout the integrin superfamily. The unusual stability and long-lived nature of these conformations may have profound implications for platelet biology and drug development strategies.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- alpha vbeta 3 was purchased from Chemicon International, Inc. Concanavalin A-Sepharose 4B, 6-aminohexanoic acid N-hydroxysuccinimide ester-Sepharose 4B, and subtilisin were purchased from Sigma. RGDS peptide was purchased from Bachem. Electrophoresis reagents were purchased from Novex. GelCode blue stain reagent was from Pierce. SZ21 and SZ22 monoclonal antibodies were purchased from Immunotech. 3-[[[(5S)-3-[4-(Aminoiminomethyl)phenyl]-4,5-dihydro-5-isoxazolyl]acetyl]amino]-N-(butoxycarbonyl)-L-alanine monobenzenesulfonate (XP280) and 3-[(4-[2-(2-amino-1 6-dihydro-6-oxo-4-pyrimidinyl)ethyl]benzoyl]amino]-N-[(2,4,6-trimethylphenyl)sulfonyl]-L-alanine monosodium salt (SS496) were synthesized at the DuPont Pharmaceuticals Co. XP280 and SS496 are tight binding ligands with dissociation constants of 0.2 and 1.1 nM for GP IIb-IIIa and alpha vbeta 3, respectively. XL086 is the D-lysine thiourea fluorescein adduct of cyclic[D-Lys-N2-methyl-L-arginyl-glycyl-L-aspartyl-3-(aminomethyl-benzoic acid)]. It has an affinity for GP IIb-IIIa of 55 nM (17, 18).

Protein Purification-- GP IIb-IIIa was purified from outdated platelets using the protocol of Kouns et al. (8). Briefly, platelets were solubilized with Triton X-100 and the lysates were passed over a concanavalin A-Sepharose column. The eluate was then purified over an RGDS peptide affinity column. The GP IIb-IIIa eluted with RGDS peptide will be referred to as the active protein. The GP IIb-IIIa in the flow-through fraction was further purified by size exclusion chromatography and will be referred to as inactive protein. Unless otherwise stated, active protein was used for all experiments described herein.

Gel Shift Protocol-- Unless otherwise stated, protein and XP280 were incubated at concentrations of 3 and 6 µM, respectively, at 37 °C for 1 h prior to electrophoresis. Given the low dissociation constant for this compound and IIb-IIIa, it is expected that IIb-IIIa will be saturated under these conditions. For SDS gels, an equal volume of 2× SDS buffer (Novex, 0.125 M Tris-HCl, 4% SDS, 20% glycerol, 0.005% bromphenol blue, pH = 6.8) was added to each sample immediately prior to electrophoresis. Unless otherwise noted, samples were not boiled prior to electrophoresis. 4-20% Tris-glycine SDS gels were purchased from Novex. Electrophoresis was carried out at 185 V for 70 min using a running buffer of 250 mM Tris, 192 mM glycine, 0.1% SDS, pH = 8.3. Native gels were poured with 4% acrylamide in 125 mM Tris, 0.1% Triton X-100, pH = 6.8 for the stacking gels and 6.0% acrylamide in 375 mM Tris, 0.1% Triton X-100, pH = 8.8 for the separating gels. 25 mM Tris-HCl, 192 mM glycine, 0.1% Triton X-100, pH = 8.3, was used as the running buffer. Electrophoresis was carried our for 4 h at 100 V. An equal volume of sample buffer (25 mM Tris, 192 mM glycine, 0.1% Triton X-100, pH = 8.3) was added to each sample prior to electrophoresis. All gels were stained with GelCode Blue from Pierce. For experiments where GP IIb-IIIa was reduced prior to analysis, the 2× SDS sample buffer contained 5% beta -mercaptoethanol and the sample was incubated 10 min prior to electrophoresis. For competition experiments with RGDS peptide, a concentration of RGDS of 10 mM was used. Higher concentrations of peptide caused distortion of the electrophoresis.

For platelet gel-shift experiments, 0.25 units of fresh concentrated platelets were obtained from Interstate Blood Bank and washed three times with 200 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH = 7.2, by repeated pelleting at 5000 rpm. The final pellet was suspended in 0.75 ml of 20 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.5 mM AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride), 100 µM E-64, 20 µM leupeptin, pH = 7.5. One hundred µl of this sample was mixed with 1 µl of compound or water and incubated at 37 °C for 1 h. One hundred µl of 2× SDS buffer were added, and the samples were loaded onto 4-20% Tris-glycine gradient gels and run as above. Proteins were transferred onto polyvinylidene difluoride membranes for 2 h at 50 V with ice pack cooling unit using 10 mM CAPS-NaOH, pH = 11.0, 10% methanol as a transfer buffer. Polyvinylidene difluoride membranes were blocked in 5% milk with shaking for 2 h. Incubation with primary antibody was carried out overnight at 4 °C. The primary antibodies used were a mixture of the commercial antibodies SZ21 and SZ22, which target the IIIa and IIb chains, respectively. Detection was carried out with horseradish peroxidase-conjugated secondary antibodies.

Protease Digestion Experiments-- Protein was preincubated with or without ligands according to the gel-shift protocol described above. Subtilisin was added at various weight ratios, and the incubation was continued at 37 °C for 1 h. After addition of one volume of 2× SDS buffer, samples were boiled for 5 min before loading onto a 4-20% SDS-PAGE gel. Electrophoresis was carried out as described above.

Time Course for Reversibility of Complex Formation-- For time courses using purified protein, protein was incubated with ligand as above and then bound to concanavalin A-Sepharose resin. The resin was continually washed with buffer A (0.1% Triton X-100 (v/v), 20 mM Tris-HCl, 1 mM MgCl2, 1 mM CaCl2, 150 mM NaCl, pH = 7.4) for 1 h. The protein was then eluted with 100 mM methyl-alpha -D-mannopyranoside. Eluted samples were incubated at 37 °C either in the elution buffer or with dilution of 2× SDS buffer and then analyzed by SDS-PAGE as described above. Stained gels were scanned and band volumes integrated using the IPLab gel software system. Control experiments with varying loads of protein showed that these experiments were carried out in the linear range of detection. For time courses on platelets, platelets were incubated with ligand as described above. Ligand was removed by repeated centrifugation and resuspension of the platelets. Control experiments using tritiated ligand showed that greater than 99% of the drug was removed. Analysis was carried out as described above, and the blots were scanned as described for gels above. Analysis of different sample loads indicated that detection was within the linear range of the system.

Gel Shift of alpha vbeta 3-- Protein in 0.1% Triton X-100 (v/v), 20 mM Tris-HCl, 2 mM MgCl2, 0.1 mM CaCl2, 150 mM NaCl, pH = 7.5, was mixed with SS496 at 3 and 6 µM concentrations, respectively, and incubated at 37 °C for 1 h. The dissociation constant for SS496 and alpha vbeta 3 is 1.1 nM. Under these experimental conditions, the protein is expected to be saturated. An equivalent volume of 2× SDS buffer was added and electrophoresis was carried out as described above for GP IIb-IIIa. Samples were not boiled prior to electrophoresis. For examination of platelets, experiments were conducted as described above for GP IIb-IIIa. Western analysis of alpha v was carried out using monoclonal antibody 1960 (Chemicon) at 1:1000 dilution. Detection by ECL was performed using reagents from Amersham Pharmacia Biotech under manufacturer's protocols.

Protein sequencing was carried out on an HP G1000A protein sequencer. Bands of interest from Coomassie-stained gels were cut out, and the protein was eluted into 0.1% SDS solutions overnight. The eluted protein was sequenced according to manufacturer's protocols.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antagonists Induce Formation of an Unusually Stable GP IIb-IIIa Complex-- Fig. 1A shows SDS-PAGE analysis of purified GP IIb-IIIa with and without treatment with the small molecule antagonist XP280 or RGDS peptide. Lane 1 shows untreated protein with the IIb and IIIa subunits migrating at their expected molecular weights. Treatment with RGDS peptide prior to electrophoresis does not change this pattern (lane 2). However, treatment with the antagonist XP280 causes a drastic change in the mobility and banding pattern of the protein (lane 3). The new single band migrates with a molecular mass of slightly less than 220 kDa and close to but less than the combined weights of the IIb and IIIa chains. The fact that the combined weights are slightly less than expected sum of GP IIb and GP IIIa suggests that some amount of native structure is maintained during electrophoresis. This would cause an increase in mobility due to the more compact protein structure. In fact, Western analysis confirms the presence of both IIb and IIIa chains in this band, and protein sequencing indicates the presence of IIb heavy and lights chains and the IIIa chain in roughly equimolar amounts (data not shown). Thus, the binding of XP280 but not RGDS peptide induces a conformation that causes the heterodimerization of the IIb and IIIa chains to be stable to treatment with SDS. This is a highly unusual observation, as protein-protein interactions are rarely maintained in SDS solutions. We will refer to this form of the protein as GP IIb-IIIa* throughout.


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Fig. 1.   Gel shift of GP IIb-IIIa by ligands. Panel A, SDS-PAGE gel shift of GP IIb-IIIa. Lane 1, no compound; lane 2, RGDS peptide; lane 3, XP280; lane 4, RGDS peptide-XP280 mixture; lane 5, no compound + reductant; lane 6, XP280 + reductant. GP IIb-IIIa*, IIb, and IIIa chains are indicated. The IIb light chain is indicated by LC. Molecular size markers are indicated at left. Panel B, native PAGE gel shift of GP IIb-IIIa. Lane 1, no compound; lane 2, RGDS peptide; lane 3, XP280.

A similar pattern is obtained when the protein is reduced prior to electrophoresis (lanes 5 and 6 for untreated and XP280-treated, respectively). In this case, a high molecular mass band corresponding to GP IIb-IIIa* is observed in addition to a band corresponding to the light chain of IIb at ~20 kDa. It cannot be concluded that all disulfide bonds are reduced under the conditions used here. However, the appearance of the light chain separate from GP IIb-IIIa* shows that it is not a critical part of the subunit interface that holds together the heterodimer in SDS solutions. Boiling of the sample prior to electrophoresis or incubation with EDTA after incubation with compound results in disruption of GP IIb-IIIa* formation (data not shown). The stability of GP IIb-IIIa* to reduction but not boiling or EDTA treatment suggests that the complex is held together through non-covalent intersubunit interactions. In particular, if disulfide exchange were taking place to form an intersubunit disulfide, boiling or EDTA would not be expected to reverse formation of GP IIb-IIIa*. Control experiments have shown that boiling or treatment with reducing reagents does not alter the ability of XP280 to induce formation of GP IIb-IIIa*. Treatment of a different integrin, alpha vbeta 3, with XP280 does not result in a change in the banding pattern (Fig. 6, lane 3). Thus, the behavior observed here is not due to nonspecific chemical cross-linking. Experiments with inactive (non-RGDS affinity-purified) protein give similar results (data not shown). Subequimolar amounts of XP280 resulted in fractional formation of GP IIb-IIIa*. The above results suggest that 1) binding of XP280 to GP IIb-IIIa induces a conformation that confers stability to SDS treatment, 2) the interactions that hold together the IIb and IIIa chains are noncovalent in nature, and 3) this effect is specific to GP IIb-IIIa for XP280.

We pursued an alternative format to test the stability of GP IIb-IIIa heterodimerization. It is well known that the native structure of integrins is dependent upon the presence of calcium and magnesium ions. Native PAGE was run in the absence of these ions to test for ligand-conferred stability toward ion removal. Fig. 1B shows a native PAGE analysis of GP IIb-IIIa with and without treatment of antagonist. Untreated or RGDS peptide-treated protein migrates as a diffuse band (lanes 1 and 2, respectively). Western analysis confirms the presence of both chains in this region. As expected, removal of the necessary ions during electrophoresis results in a dissociation of the heterodimer. Incubation of GP IIb-IIIa with XP280 results in a change in mobility relative to untreated or RGDS-treated protein (lane 3). Western analysis using IIb- and IIIa-specific monoclonal antibodies shows the presence of both subunits in the gel-shifted band (data not shown). The change in mobility and banding pattern during native electrophoresis again indicates conformational and stability changes induced by the binding of XP280. Electrophoresis in the presence of calcium and magnesium shows very little difference in the migration behavior of untreated or XP280-treated protein (data not shown). Thus, the net charge and hydrodynamic radius are not greatly affected by ligand binding. It is interesting to note that, although EDTA reverses formation of GP IIb-IIIa*, the less vigorous removal of divalent cations under the native electrophoresis conditions does not. This suggests that the binding affinity for at least some of the calcium and magnesium binding sites is greater upon XP280 binding.

The Conformation of GP IIb-IIIa* Is Maintained after Dissociation of Antagonist-- To assess whether the presence of ligand is necessary for maintenance of the GP IIb-IIIa* conformation, analysis was carried out to detect ligand in the gel-shifted band in SDS-PAGE. SDS-PAGE analysis was carried out using tritiated XP280 and GP IIb-IIIa at a 1:1 molar ratio. Gel slices were cut out and measured for tritium post-staining. The results are presented in Fig. 2A. If an equimolar amount of ligand was retained in GP IIb-IIIa*, ~1 × 106 cpm would be expected to be found in the gel-shifted band. However, less than 0.15% of this amount was found in that region of the gel (light panels). This suggests that the conformation of the gel-shifted form is stable in the absence of the antagonist, a conclusion supported by time-course experiments (see below). It may be argued that the ligand was present until staining and destaining of the gels. In order to access this, similar experiments were carried out with a fluorescein labeled ligand, XL086, at 1:1 and 1:100 molar ratios (Fig. 2B). For this derivative, only partial gel-shifting is observed as shown in the left panel. The right panel of Fig. 2B shows a fluorescence image of the gel immediately after electrophoresis. No fluorescence was observed in the protein band even before staining and even in the presence of 100-fold excess ligand. Thus, under two independent assessments, no ligand is detectable above background levels in the GP IIb-IIIa* band. This strongly suggests that this conformation is maintained even after dissociation of the ligand. This point will be pursued further below.


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Fig. 2.   The ligand is dissociated from GP IIb-IIIa*. Panel A, distribution of tritium from gel slices. The number of counts are plotted on a logarithmic scale. The total panel indicates the total number of counts used in the experiment. The two light gray panels indicate where GP IIb-IIIa* migrates. Panel B, left side, SDS-PAGE analysis of a fluorescein-labeled ligand, XL086, at 1:1 and 1:100 molar ratio (left and right lanes, respectively); right side, fluorescent intensity of left panel prestaining. In both panels, the migration front is indicated.

GP IIb-IIIa* Has a Different Conformation than Native and RGDS-bound GP IIb-IIIa-- The changes in electrophoretic behavior discussed above result from changes in stability of the protein. In order to access directly changes in protein conformation, the protease susceptibility in the presence and absence of ligand was examined. Fig. 3A shows the SDS-PAGE analysis of GP IIb-IIIa digestion with subtilisin in the presence of no ligand (lane 2), RGDS peptide (lane 3), or XP280 (lane 4). The rate of digestion and resultant pattern are clearly dependent upon the presence and identity of ligands. To quantitate this effect, the amount of protein in the band migrating at the Mr 97,000 marker was measured by densitometry and these results are presented in Fig. 3B. Errors bars represent 1 standard deviation from four independent experiments. This measurement likely reflects the remaining amount of intact IIIa, but the possibility of IIb breakdown products comigrating with intact IIIa cannot be ruled out. Addition of RGDS peptide results in a small but significant protection from proteolysis (open squares) relative to the untreated protein (open circles). It has previously been shown that RGD-containing peptides induce a conformational change in GP IIb-IIIa (9, 11). Addition of XP280 results in much more pronounced protection (filled squares). The results suggest that both RGDS and XP280 induce conformational changes in the receptor but that the conformations are different and the change larger for XP280. The IIb chain is more susceptible to proteolysis than the IIIa chain with subtilisin, and quantitation is therefore more difficult. This has also been observed with Arg-C digestions (7, 8). Control experiments using a chromogenic substrate did not show any inhibition of subtilisin by XP280 or RGDS peptide at the concentrations used in these experiments. The difference in proteolysis patterns therefore reflects altered conformations in the presence of ligands and not inhibition of subtilisin.


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Fig. 3.   Subtilisin digestion of GP IIb-IIIa in the presence and absence of ligands. Panel A, SDS-PAGE analysis. Lane 1, undigested; lane 2, no compound; lane 3, RGDS peptide; lane 4, XP280. Molecular size markers are indicated at left. Panel B, quantification of the 97-kDa band by densitometry: no compound (open circles), RGDS peptide (open squares), and XP280 (filled squares). Error bars indicate one standard deviation for quadruplicate measurements.

GP IIb-IIIa* Forms on the Platelet Surface-- To test for the possibility of forming the GP IIb-IIIa* on the platelet surface, fresh platelets were incubated with ligands under standard conditions, SDS-lysed, and probed by Western analysis using a mixture of the monoclonal antibodies SZ21 and SZ22, which are specific for the IIIa and IIb chains, respectively. Platelets not incubated with any compound prior to analysis show the expected banding pattern (Fig. 4, lane 1). Addition of XP280 causes the appearance of a band at a molecular weight corresponding to GP IIb-IIIa*, while at the same time the non-gel-shifted IIb and IIIa bands virtually disappear (lane 2). The band for GP IIb-IIIa* partially reverses after removal of the ligand (lane 3, see "Discussion"). These results strongly suggest that GP IIb-IIIa* can be formed on the platelet surface by treatment with XP280. It may be possible that the gel-shifted conformation is only formed after the platelets are SDS-lysed. However, control experiments with purified protein have shown that once GP IIb-IIIa is SDS-denatured, addition of antagonist does not induce formation of the gel-shifted complex (data not shown). Additionally, GP IIb-IIIa* is stable on the platelet surface after removal of XP280 (see below).


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Fig. 4.   Western analysis of platelets using monoclonal antibodies for GP IIb and GP IIIa. Lane 1, platelet control; lane 2, platelets incubated with XP280; lane 3, platelets after removal of XP280. The migration of molecular size markers is indicated to left of the figure.

Formation of GP IIb-IIIa* Is Reversible-- Fig. 1A (lane 4) shows a competition experiment between XP280 and RGDS peptide. As described above, lanes 1-3 show no ligand, RGDS peptide, and XP280, respectively, mixed with GP IIb-IIIa prior to electrophoresis. For the sample in lane 4, RGDS peptide and XP280 were mixed prior to addition of the protein. Clearly, RGDS peptide is able to partially reverse the conformational change caused by XP280. In additional competition experiments, one compound was added to the protein and incubated for 1 h. The second compound was then added and with an additional 1-h incubation. The amount of GP IIb-IIIa* observed was not affected by the order of addition, suggesting that equilibrium is reached within the 1-h incubation (data not shown). The RGDS peptide was used at a concentration of 10 mM in these experiments. Higher concentrations of peptide began to distort the electrophoresis. RGDS peptides have been reported to have an affinity to GP IIb-IIIa in the 1-30 µM range (13, 19). In this competition experiment, both ligands are present at ~5000 times the Kd for their interaction with GP IIb-IIIa. The roughly 50% reversal of GP IIb-IIIa* formation that is observed is therefore within the expected range. It is clear from these results that RGDS peptides can compete with XP280 and reverse the formation of GP IIb-IIIa*.

To evaluate if GP IIb-IIIa* would convert back to the native conformation in the absence of any ligand, a time-course study after ligand removal was conducted. The presence of GP IIb-IIIa* in the absence of ligand was measured by following the amount of gel-shifted protein by SDS-PAGE for purified protein or Western analysis for platelet GP IIb-IIIa following removal of the ligand. The results are presented in Fig. 5. When time-course experiments are pursued with purified protein, there is essentially no conversion over a 40-h time period when the sample is incubated under native conditions (open squares). When the purified protein is incubated in SDS solutions, GP IIb-IIIa* has a half-life of ~3 h (filled squares). When GP IIb-IIIa is incubated in the presence of XP280 and SDS, no conversion of GP IIb-IIIa* to GP IIb-IIIa is observed for several days (data not shown). A similar time course for the conformational change from GP IIb-IIIa* to GP IIb-IIIa on the platelet surface following removal of XP280 is also shown (filled diamonds). Approximately 35% of the protein converts back to the native conformation within 3 h, the first time point taken in these experiments. The remaining protein has a much longer half-life, as evidenced by the negligible additional conversion in the following 22 h. The data suggest at least two populations of GP IIb-IIIa* on the platelet surface.


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Fig. 5.   Time course for reversion of GP IIb-IIIa* to GP IIb-IIIa following removal of XP280. Figure shows purified protein under native conditions (open squares), purified protein in SDS (filled squares), and platelets (filled diamonds).

alpha vbeta 3 Conformational Change-- To test whether other family members display the same type of conformational behavior, alpha v beta 3 was examined for similar ligand-induced conformational changes. SDS-PAGE analysis is shown in panel A of Fig. 6. Protein not treated with any compound shows two bands near the expected molecular weight (lane 1). The beta 3 chain migrates as a fuzzy doublet. When the protein is incubated with SS496, an alpha vbeta 3 antagonist, a single band is observed of higher molecular weight (lane 2). This is very similar to the pattern observed for GP IIb-IIIa when treated with XP280. Addition of XP280 does not result in any change (lane 3). alpha vbeta 3* is stable to treatment with reductants and is also partially reversed by treatment with RGDS peptide (data not shown). Pretreatment of SS496 with reductants or boiling did not alter the compounds ability to induce formation of alpha vbeta 3*.


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Fig. 6.   Gel shift of alpha vbeta 3 by ligands. Panel A shows SDS-PAGE analysis of alpha vbeta 3 incubated with no compound (lane 1), SS496 (lane 2), and XP280 (lane 3). Panel B shows Western analysis using an alpha v monoclonal antibody. Shown are 10 ng of purified alpha vbeta 3 incubated without compound (lane 1) or with SS496 (lane 2), or platelets incubated without compound (lane 3) or with SS496 (lane 4).

Western blot analysis of the alpha v chain is shown in Fig. 6B. Lane 1 shows 10 ng of purified alpha vbeta 3 untreated with compound, and lane 2 shows the result of pretreatment with SS496. The alpha v chain migrates at the expected molecular weight when not treated with antagonist. When the protein is treated with SS496, alpha v now migrates with a molecular weight corresponding to the gel-shifted band observed in the SDS-PAGE results presented in panel A. Interestingly, the band intensity is much lower in the gel-shifted form, indicating that the epitope detected by this monoclonal antibody is less exposed upon treatment with antagonist. In contrast to the LIBS sites, which become more exposed upon ligand binding, this site may become less exposed when ligand-bound. Further experimentation will be needed to characterize this possibility. alpha vbeta 3* also forms on the platelet surface as shown in panel B of Fig. 6. Lane 3 shows Western analysis of untreated platelets. Lane 4 shows platelets treated with SS496. Once again, alpha v migrates at the expected molecular weight when platelets are not treated with compound. When treated with SS496, no distinct alpha v band is detected. The disappearance of the alpha v band is likely the result of formation of the gel-shifted form of alpha vbeta 3 on the platelet surface and the lower reactivity of the monoclonal antibody used with the conformation of alpha vbeta 3 induced by SS496 binding. Detection of the gel-shifted form of alpha vbeta 3 was limited by the number of platelets that could be analyzed without distortion of the electrophoresis. These results show that an SDS stable conformation of alpha vbeta 3 is induced by ligand binding for both purified protein and alpha vbeta 3 on the surface of platelets.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The data presented here show that ligand binding to GP IIb-IIIa can induce a conformation that is stable to treatment with SDS and suggests that noncovalent inter-chain interactions are maintained in these solutions. SDS is considered to be a "strong" denaturant and is expected to unfold proteins to a near-random coil ensemble of conformations in addition to disrupting all protein-protein interactions. This provides the basis for the common procedure of estimating molecular weight by electrophoretic mobility during SDS-PAGE. Thus, the stability to SDS treatment observed here is highly unusual. It should be noted, however, that there are reports of structure being maintained in SDS solutions for peptides (20-23), membrane proteins and membrane peptides (24), and even for the maintenance of protein-protein (25-28) and protein-DNA interactions (29).

The increase in stability upon ligand binding is likely resultant from a significant conformational change in GP IIb-IIIa. Such a change would be consistent with the exposure of parts of the protein that are normally not accessible to solvent or other macromolecules such as proteases. This would potentially cause a difference in protease susceptibility for the altered protein. We observe changes in the protease digestion pattern and rate when the protein is treated with subtilisin, a nonspecific protease. Clearly, the nature and extent of exposure of subtilisin susceptible sites changes upon ligand binding. It is difficult to speculate on the nature of the conformational change induced by these ligands. The GP IIb-IIIa structure has been modeled using electron microscopic and other biochemical data (11, 30). The model suggests a very broad interface between the two subunits, any part of which may be involved in the interactions which govern SDS stability. We have shown that the IIb light chain is not necessary for formation of this species. The light chain contains the transmembrane domain, and, therefore, the dimerization is not mediated through the transmembrane moieties on IIb and IIIa. This is in contrast to dimerization of the glycophorin transmembrane domains, which are also stable to SDS treatment (25). Interestingly, addition of RGDX peptides promote an opening of the structure and less interaction between the two subunit chains (11). However, the conformations induced by RGDX peptides and XP280 appear to be different based on 1) the stability to SDS treatment, 2) the rate of proteolysis in the presence of compound, 3) the stability of the altered conformation in the absence of ligand, and 4) the reversibility of SDS stability by RGDS peptides. Similarly, in the more open conformation induced by RGDX peptides, the majority of interchain interactions are maintained by the IIb light chain, the part of IIb shown not to be necessary for SDS stability (11). It is possible that the conformational change described here entails the exposure of new epitopes, i.e. LIBS. Future investigation will be needed to examine this possibility.

Our results show that the altered conformation of GP IIb-IIIa* is stable in the absence of the antagonist. The SDS stability in the absence of ligand suggests a conformational free energy minimum discrete and separate from that of the native structure. Conversion back to the native conformation does not occur over several days with the purified protein. This indicates that the altered conformation is more stable than the native conformation and/or that it is separated by a high energy barrier. On the surface of a platelet, ~65% of the GP IIb-IIIa shows similar behavior while the remaining 35% converts back within a few hours after antagonist removal. The presence of at least two rates for conversion back to the native conformation indicates conformational subpopulations on the platelet surface. The difference between these subpopulations may be related to cytoskeletal interactions with the IIb and/or IIIa cytoplasmic domains. These have been shown to physically interact, and this interaction could play a role in regulating the affinity of the receptor, presumably by altering the protein conformation (31, 32). Interactions with other platelet surface components can also not be ruled out. Phosphorylation state of the IIIa chain has also been shown to affect the conformation of GP IIb-IIIa as assessed by the exposure of ligand-binding sites (33). The fraction of activated GP IIb-IIIa on resting platelets would be expected to be far less than either population, so it is unlikely that the two rates represent activated and resting GP IIb-IIIa. The stability of GP IIb-IIIa* in the absence of ligand and long-lived nature of this conformation make it possible that platelets could circulate with extended time periods with this conformation of GP IIb-IIIa on the surface. The consequences of this toward platelet function and hemostasis could be quite profound. Since RGDS peptide is able to at least partially reverse the formation of GP IIb-IIIa*, it is possible that ambient RGDS containing proteins such as fibrinogen and von Willebrand factor could reverse GP IIb-IIIa* formation on the surface of a circulating platelet. Future studies will be needed to examine aspects of platelet function after formation of GP IIb-IIIa*.

The integrin alpha vbeta 3 also shows similar increases in stability, and presumably changes in conformation, when treated with antagonist. The changes take place with purified protein or in the native environment of the platelet surface. This result implies that the conformational changes observed here are conserved throughout the integrin superfamily.

    Addendum

During the review of this article, a report appeared suggesting that other members do in fact populate ligand-induced SDS-stable conformations (34). Thibault shows that echistatin and other disintegrins form SDS-stable complexes with several integrins including alpha vbeta 3, alpha 5beta 1, alpha vbeta 1, and alpha 8beta 3. These SDS-stable complexes also show lability to EDTA and heat and reversibility with RGDS peptides as observed here for GP IIb-IIIa. However, there is a higher susceptibility to reduction, and, interestingly, echistatin remains bound in the SDS-stable form while we observe that the ligand used here is not present in the SDS-stable form of GP IIb-IIIa. More recently, it has been reported that echistatin also forms SDS-stable complexes with GP IIb-IIIa (35). It is likely that the conformations formed by echistatin are highly similar to those described here. Finally, the work cited above and this report strongly suggest that SDS-stable conformations are available to many or all members of the integrin superfamily.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: DuPont Pharmaceutical Co., Experimental Station, E336/241B, P.O. Box 80336, Wilmington, DE 19880-0336. E-mail: richard.wynn@dupontpharma.com.

Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M009627200

1 The abbreviations and trivial names used are: GP, glycoprotein; LIBS, ligand-induced binding site; XP280, 3-[[[(5S)-3-[4-(aminoiminomethyl)phenyl]-4,5-dihydro-5-isoxazolyl]acetyl]amino]-N-(butoxycarbonyl)-L-alanine monobenzenesulfonate; SS496, 3-[(4-[2-(2-amino-1 6-dihydro-6-oxo-4-pyrimidinyl)ethyl]benzoyl]amino]-N-[(2,4,6-trimethylphenyl)sulfonyl]-L-alanine monosodium salt; XL086, D-lysine thiourea fluorescein adduct of cyclic[D-Lys-N2-methyl-L-arginyl-glycyl-L-aspartyl-3-(aminomethyl-benzoic acid)]; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve]
2. Pelletier, A., Kunicki, T., Ruggeri, Z., and Quaranta, V. (1995) J. Biol. Chem. 270, 18133-18140[Abstract/Free Full Text]
3. Gille, J., and Swerlick, R. (1996) Ann. N. Y. Acad. Sci. 797, 93-106[Abstract]
4. Horton, M. A. (1996) in Adhesion Receptors as Therapeutic Targets (Horton, M. A., ed) , pp. 1-8, CRC Press, New York
5. Bazzoni, G., and Hemler, M. (1998) Trends Biochem. Sci 23, 30-34[CrossRef][Medline] [Order article via Infotrieve]
6. Calvete, J. (1995) Proc. Soc. Exp. Biol. Med. 208, 346-360[Abstract]
7. Calvete, J., Mann, K., Schafer, W., Fernandez-Lafuente, R., and Guisan, J. (1994) Biochem. J. 298, 1-7[Medline] [Order article via Infotrieve]
8. Kouns, W., Hadvary, P., Haering, P., and Steiner, B. (1992) J. Biol. Chem. 267, 18844-18851[Abstract/Free Full Text]
9. Parise, L., Helgerson, S., Steiner, B., Nannizzi, L., and Phillips, D. (1987) J. Biol. Chem. 262, 12597-12602[Abstract/Free Full Text]
10. Du, X., Plow, E., Frelinger, A. L., III, O'Toole, T. E., Loftus, J., and Ginsberg, M. (1991) Cell 65, 409-416[Medline] [Order article via Infotrieve]
11. Hantgan, R. R., Paumi, C., Rocco, M., and Weisel, J. W. (1999) Biochemistry 38, 14461-14474[CrossRef][Medline] [Order article via Infotrieve]
12. Mazurov, A., Khaspekova, S., Byzova, T., Tikhomirov, O., Berndt, M., Steiner, B., and Kouns, W. (1996) FEBS Lett. 391, 84-88[CrossRef][Medline] [Order article via Infotrieve]
13. Frelinger, A. L., III, Cohen, I., Plow, E., Smith, M., Roberts, J., Lam, S., and Ginsberg, M. (1990) J. Biol. Chem. 265, 6346-6352[Abstract/Free Full Text]
14. Frelinger, A. L., III, Du, X., Plow, E., and Ginsberg, M. (1991) J. Biol. Chem. 266, 17106-17111[Abstract/Free Full Text]
15. Mondoro, T., Wall, C., White, M., and Jennings, L. (1996) Blood 88, 3824-3830[Abstract/Free Full Text]
16. Kouns, W., Kirchhofer, D., Hadvary, P., Edenhofer, A., Weller, T., Pfenninger, G., Baumgartner, H., Jennings, L., and Steiner, B. (1992) Blood 80, 2539-2547[Abstract]
17. Mousa, S., Bozarth, J., Forsythe, M., Tsao, P., Pease, L., and Reilly, T. (1994) Life Sci. 54, 1155-1162[Medline] [Order article via Infotrieve]
18. Tsao, P., Bozarth, J., Jackson, S., Forsythe, M., Flint, S., and Mousa, S. (1995) Thromb. Res. 77, 543-556[CrossRef][Medline] [Order article via Infotrieve]
19. Cierniewski, C., Byzova, T., Papierak, M., Haas, T., Niewiarowska, J., Zhang, L., Cieslak, M., and Plow, E. (1999) J. Biol. Chem. 274, 16923-16932[Abstract/Free Full Text]
20. Kloosterman, D., Scahill, T., and Friedman, A. (1993) Pept. Res. 6, 211-218[Medline] [Order article via Infotrieve]
21. Najbar, L., Craik, D., Wade, J., Salvatore, D., and McLeish, M. (1997) Biochemistry 36, 11525-11533[CrossRef][Medline] [Order article via Infotrieve]
22. Parker, W., and Song, P. (1992) Biophys. J. 61, 1435-1439[Abstract]
23. Zhang, S., Lockshin, C., Cook, R., and Rich, A. (1994) Biopolymers 34, 663-672[Medline] [Order article via Infotrieve]
24. Pervushin, K., Arseniev, A., Kozhich, A., and Ivanov, V. (1991) J. Biomol. NMR 1, 313-322[Medline] [Order article via Infotrieve]
25. Lemmon, M., Flanagan, J., Hunt, J., Adair, B., Bormann, B., Dempsey, C., and Engelman, D. (1992) J. Biol. Chem. 267, 7683-7689[Abstract/Free Full Text]
26. Neefjes, J., and Ploegh, H. (1992) EMBO J. 11, 411-416[Abstract]
27. Chang, T., and Abraham, C. (1996) Ann. N. Y. Acad. Sci. 777, 183-188[Abstract]
28. Bayer, E., Ehrlich-Rogozinski, S., and Wilchek, M. (1996) Electrophoresis 17, 1319-1324[Medline] [Order article via Infotrieve]
29. Glaser, T., Rothbarth, K., Stammer, H., Kempf, T., Spiess, E., and Werner, D. (1997) FEBS Lett. 413, 50-54[CrossRef][Medline] [Order article via Infotrieve]
30. Rocco, M., Spotorno, B., and Hantgan, R. (1993) Protein Sci. 2, 2154-2166[Abstract/Free Full Text]
31. Fox, J., Lipfert, L., Clark, E., Reynolds, C., Austin, C., and Brugge, J. (1993) J. Biol. Chem. 268, 25973-25984[Abstract/Free Full Text]
32. Bennett, J., Zigmond, S., Vilaire, G., Cunningham, M., and Bednar, B. (1999) J. Biol. Chem. 274, 25301-25307[Abstract/Free Full Text]
33. van Willigen, G., Hers, I., Gorter, G., and Akkerman, J. (1996) Biochem. J. 314, 769-779[Medline] [Order article via Infotrieve]
34. Thibault, G. (2000) Mol. Pharmacol. 58, 1137-1145[Abstract/Free Full Text]
35. Thibault, G., Tardif, P., and Lapalme, G. (2001) J. Pharmacol. Exp. Ther. 296, 690-696[Abstract/Free Full Text]


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