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
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ABSTRACT |
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Integrins are a large family of cell surface
receptors that are involved in a wide range of biological processes.
The integrin 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 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,
Materials--
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%
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- Gel Shift of
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.
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.
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,
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.
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.
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).
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.
Western blot analysis of the 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 IIb
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
v
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
and
subunits. To date, 16
and 8
subunits have been identified (5). Pairing of subunits is
semispecific, with some subunits being specific for a single partner
(
IIb pairs only with
3) while others pair
less strictly (
v pairs with
1,
3,
5,
6, and
8) (3).
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.
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.
v
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
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
v
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).
-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.
-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.
v
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
v
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
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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.
v
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.
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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.
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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.
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[in a new window]
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.
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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).
v
3 Conformational Change--
To
test whether other family members display the same type of
conformational behavior,
v
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
3 chain migrates as a fuzzy doublet.
When the protein is incubated with SS496, an
v
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).
v
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
v
3*.
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[in a new window]
Fig. 6.
Gel shift of
v
3
by ligands. Panel A shows SDS-PAGE analysis
of
v
3 incubated with no compound
(lane 1), SS496 (lane 2),
and XP280 (lane 3). Panel B
shows Western analysis using an
v monoclonal antibody.
Shown are 10 ng of purified
v
3 incubated
without compound (lane 1) or with SS496
(lane 2), or platelets incubated without compound
(lane 3) or with SS496 (lane
4).
v chain is shown in Fig.
6B. Lane 1 shows 10 ng of purified
v
3 untreated with compound, and lane 2 shows the result of pretreatment with
SS496. The
v chain migrates at the expected molecular
weight when not treated with antagonist. When the protein is treated
with SS496,
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.
v
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,
v migrates at the
expected molecular weight when platelets are not treated with compound.
When treated with SS496, no distinct
v band is detected.
The disappearance of the
v band is likely the result of
formation of the gel-shifted form of
v
3
on the platelet surface and the lower reactivity of the monoclonal
antibody used with the conformation of
v
3 induced by SS496 binding. Detection of the gel-shifted form of
v
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
v
3 is induced by ligand binding for both
purified protein and
v
3 on the surface of platelets.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
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
v
3,
5
1,
v
1, and
8
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.
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.
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