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INTRODUCTION |
The integrin ligand binding event is a dynamic process that is key
to cellular function. Most integrins are displayed in at least two
distinct conformations on the cell. These conformations have varying
affinities for ligand. The transition between one affinity state and
another is typically referred to as "activation," and such
transitions are brought about by "inside-out" signaling (for
reviews, see Refs. 1-5). The activation of integrins is crucial to
events like morphogenesis, cell migration and invasion, and platelet
aggregation (6-8).
Platelet integrin 
IIb
3 has served as a
paradigm of many aspects of integrin structure and function. This
receptor is a particularly good model of an integrin whose activation
is of immense physiologic significance (9). Integrin
IIb3 is pivotal to the control of
bleeding because it mediates platelet aggregation. Integrin IIb3 is activated when platelets are
stimulated by agonists like ADP or thrombin. Activation endows
IIb3 with the ability to bind soluble
fibrinogen (Fg)1 present in
the plasma. Because Fg is a dimer, its binding leads to platelet
aggregation and ultimately halts the loss of blood. Because the
concentrations of both Fg and platelets are so high in the blood,
IIb3 must be maintained in a resting, or
inactive state, to ensure proper blood flow. Therefore, an
understanding of how IIb3 is activated is
of great importance.
The precise mechanism by which the ligand binding affinity of
IIb3, and other integrins, is modulated
by cellular stimulation is still not completely understood. Several
pathways to activation have been put forth. Many of these have focused
on the role of the integrin cytoplasmic domains in activation (10). One
hypothesis suggests that changes in the conformation of the cytoplasmic
tails can release a conformational constraint or open an "integrin
hinge" (11). This hinge could potentially be opened by proteolysis of
the integrin cytoplasmic domains (12-14). Alternatively, the hinge
could be released by phosphorylation of the integrin. The cytoplasmic
tail of 3 contains several potential phosphorylation sites (15, 16). One of these phosphorylation sites, an NPXY motif
within the tail of 3, is essential for integrin
activation (17, 18). Therefore, phosphorylation must also be considered as a potential route to activation. Another pathway to activation may
involve proteolysis within the ectodomain of the integrin. In fact,
there is evidence that IIb3 can be
activated in this manner (19). Still other work indicates that divalent
ions, which bind to the integrin and control ligand affinity, may
activate the integrin (20-22). The physical association of integrins
with regulatory proteins (23-25) could also enact conformational
changes that lead to activation. Intracellular signaling pathways like those controlled by Ha- and R-Ras (26, 27) have also been implicated in
the activation of integrins.
Despite this progress, there is still no unifying hypothesis on exactly
what biochemical changes occur within an integrin during activation. We
reasoned that one way to gain insight into this issue was to compare
the differences in the structure and function of the resting and active
integrin. We have used Surface Plasmon Resonance (SPR) to compare the
kinetic behavior of the active and resting form of
IIb3 and mass spectrometry to probe the
structural differences between the two conformers. The study reveals
that the active and resting integrin are virtually identical in
chemical structure but reveals important conformational distinctions. These observations lay the fundamental groundwork required to properly
interpret other studies aimed at deciphering how inside-out signaling
alters the ligand binding function of integrins.
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MATERIALS AND METHODS |
Purification of IIb3--
The two
forms of integrin IIb3, AS-1 and AS-2,
were purified according to previously published procedures (28-30)
with minor modifications. Briefly, washed, outdated platelets were
lysed in a 20 mM Tris buffer, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, 5 mM
phenylmethylsulfonyl fluoride, 1 mM CaCl, and
10
5 leupeptin. The lysate was centrifuged at 50,000 × g and stored at 
80 °C until further use. The lysate
was rapidly thawed at 37 °C, centrifuged at 50,000 × g, and passed over Con-A-Sepharose (Amersham Pharmacia
Biotech). Proteins were eluted with Buffer A (20 mM Tris
buffer, pH 7.0, 150 mM NaCl, 1 mM
CaCl2, 1 mM MgCl2, 0.1% Triton
X-100, and 0.05% NaN3) containing 200 mM
methyl -D-mannopyranoside (Sigma) and 105
M leupeptin (Sigma). Fractions containing
IIb3 were pooled and then depleted of the
active form of the integrin (AS-2) by circulating over a column of
KYGRGDS-Sepharose. To ensure that the flow through was depleted of
AS-1, the lysate was passed over two RGD affinity columns in series.
The AS-2 form of the integrin was eluted from these columns using
soluble RGD peptide. AS-2 was concentrated, dialyzed against buffer A,
and stored at 80 °C. The flow through from the KYGR6DS-Sepharose
column, containing the resting form of the integrin (AS-1) was
circulated over heparin-agarose column to remove the thrombospondin.
The effluent was concentrated and AS-1 further purified by gel
filtration on Sephacryl S-300. The integrin was concentrated, dialyzed
against Buffer A, and stored at 80 °C. The purity and integrity of
each form of the integrin was assessed by SDS-PAGE.
Peptides, Ligands, and Antibodies--
Human fibrinogen was
purchased from Enzyme Research Laboratories. Fab-9, a recombinant
antibody containing the RGD motif (31, 32), was purified by affinity
chromatography on a column of goat anti-human IgG-Sepharose. Synthetic
peptides were purchased from Coast Scientific and from Anaspec.
Antibodies against the C-terminal domain of IIb,
including PM1-1, anti-V41 (33) which are specific for C terminus of
the heavy chain of IIb and the N terminus of the light
chain of IIb, respectively, were generous gifts from Dr.
Mark Ginsberg (Scripps Research Institute). Cells expressing
recombinant variants of Fg lacking the RGD motifs were generously
provided by Dr. David Farell (Milton Hershey Medical Center, Penn State University).
Construction of Ligand Affinity Resins--
For affinity
purification of IIb3, the peptide KYGRGDS
was coupled to CNBr-Sepharose (Amersham Pharmacia Biotech) according to
the manufacturer's specifications. To analyze the depth of the
integrins ligand binding pocket, affinity resins were constructed in
which the RGD peptide was coupled to the resin with an extended spacer.
This was accomplished using a procedure reported by Yan et
al. (34) with some modifications. Briefly, Sepharose was activated
with epichlorohydrin at pH 12. Then 1,6-hexanediamine was added and
incubated with the resin at 45 °C for 2 h with stirring. After
washing with water, the resin was incubated with epichlorohydrin again
for an additional hour. The activated resin was washed extensively with
distilled water, and the peptide with sequence KYGRGDS was added at a
concentration of 1 mM. The peptide was allowed to couple to
the resin for 18 h, and free peptide was removed by extensive washing with 20 mM Tris buffer, pH 7.0, 150 mM
NaCl, 1 mM CaCl2, 1 mM
MgCl2, containing 0.1% Triton X-100.
Surface Plasmon Resonance--
The kinetic parameters
(association and dissociation rate constants, k1
and k1, respectively) and the affinity
constant (Kd) between Fg and
IIb3, or Fab-9 and
IIb3, were measured by SPR using methods
we have described previously (35, 36). To eliminate conformational
changes, the ligands, either Fg or Fab-,9 were coupled to the sensor
chip. Then, a solution containing integrin was applied to the surface
and binding was measured as a function of time. Briefly, a CM5 sensor
chip was activated by 15 µl
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide and 40 µl of Fg at 50 µg/ml in sodium acetate buffer, pH 5.87, was
coupled to the chip. The surface was then treated with 10 µl of
ethanolamine to block uncoupled carboxylate groups on the chip. Fab-9
was linked to the sensor chip in the same manner except that the pH of
the immobilization buffer was set at 4.5. Following binding, chips were
regenerated with a vast excess of RGD peptide, or with 10 mM CaCl2. For all the reactions the working
buffer was Tris-buffered saline, pH 7.4, 0.005% surfactant P20 and
included divalent cations. Integrin was diluted from concentrated
purified stocks into this working buffer just prior to use. Kinetic
constants were fitted to a model that assumes a one-to-one binding
relationship using the Langmuir equations in the BIAcore 3000 software package.
Peptide Mapping--
Limited digestion of
IIb3 was accomplished with Asp-N protease
(Roche Molecular Biochemicals, sequencing grade). Asp-N was added to
purified IIb3 (1 mg/ml) of 100 µg in
100 µl of 20 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 1 mM CaCl2, 1 mM
MgCl2, 0.1% Triton X-100. Protease was added in a ratio of
1:300 (weight to weight) and incubated with integrin at 37 °C for
4 h. The reaction was stopped by chilling to 4 °C and
immediately analyzing the sample on 12% SDS-PAGE.
Protein and Peptide Mass Fingerprinting--
Peptide mass
fingerprinting (37, 38) was used to define the composition of
IIb and 3 and the composition of their
proteolytic fragments. Proteins or peptides were separated on 12%
SDS-PAGE under non-reducing conditions. Proteins were visualized by
silver staining. Gel slices containing protein were excised and then reduced and alkylated by performing the following procedure three times. Gel slices were dehydrated with acetonitrile and subjected to
reduction with 20 mM dithiothreitol (DTT) at 56 °C for
1 h. Proteins embedded in the gel were then alkylated with 60 mM iodoacetamide at room temperature for 30 min. Gel slices
were washed with 100 mM NH4HCO3,
again dehydrated with acetonitrile. Gel slices containing reduced and
alkylated integrin were dried in a vacuum centrifuge and then
re-hydrated in 20 mM NH4HCO3
containing 20 µg/ml of trypsin (Roche Molecular Biochemicals,
modified sequencing grade). Each gel slice was incubated with enzyme in
an ice-bath for 1 h, and then at 37 °C for 18 h. Gel
slices were washed with 20 mM
NH4HCO3 and then tryptic peptides were
extracted with three changes of a solution of 5% formic acid, 50%
acetonitrile, and 25% isopropanol. The solution containing peptides
was evaporated to dryness. Dry peptide mixtures were dissolved in 0.1%
trifluoroacetic acid. To detect some domains within IIb
and 3 by mass spectrometry, it was necessary to
deglycosylate the protein. Deglycosylation was carried out in 20 mM sodium phosphate buffer, pH 7.5 with O-glycanase, N-glycanase, and sialidase (Roche
Molecular Biochemicals) at 37 °C for 40 h.
In some cases proteins were digested in solution rather than in
acrylamide gel slices. Because Triton X-100 suppresses peptide detection in MALDI mass spectrometry, it was necessary to remove this
detergent. This was accomplished with the following procedure. Methanol
(140 µl) was added to 35 µg of integrin in a volume of 35 µl. The
sample was vortexed and centrifuged at 9000 × g for 10 s. Then, chloroform (35 µl) was added to the solution and
vortexed, and the sample was centrifuged again. Finally, water (90 µl) was added and the sample vigorously vortexed. Phase separation
was enacted by centrifugation for 1 min. The upper phase was discarded. The lower phase, containing chloroform, and the interface, comprised of
precipitated IIb3, were retained.
Methanol (90 µl) was added to the remainder of the sample to
precipitate the IIb3. Precipitated integrin was recovered by centrifugation for 2 min at 9,000 × g. The supernatant was discarded and the protein pellet was
air dried. Then the protein was resuspended in 20 mM
NH4HCO3 containing 0.2% octylglucoside. Lys-C
protease (Roche Molecular Biochemicals, modified sequencing grade) was
added to a final ratio of 1:10 (weight to weight), and the sample was
incubated at 37 °C for 18 h.
Mass Spectrometry--
MALDI-MS spectra were obtained with a
Voyager DE-RP MALDI-TOF mass spectrometer (PerSeptive Biosystems,
Framingham, MA) equipped with a nitrogen laser (337 nm, 3-ns pulse).
Spectra were collected in reflector mode. The accelerating voltage in
the ion source was 20 kV. Data were acquired with a transient recorder
with 2-ns resolution. The matrix used in this work was
-cyano-4-hydroxycinnamic acid dissolved in water/acetonitrile (1:1,
v/v) to give a saturated solution at room temperature. To prepare the
sample for analysis, 1 µl of the peptide solution (containing 1-10
pmol of protein in 0.1% trifluoroacetic acid) was added to 1 µl of
the matrix solution and applied to a stainless steel sample plate. The
mixture was then allowed to air dry on the sample plate before being
introduced into the mass spectrometer. Each spectrum was produced by
accumulating data using 128 laser pulses. Mass assignments were
assigned with an accuracy of approximately ± 0.1% (± 1 Da/1000
Da). The computer program called Peptide Mass on the ExPASy Molecular
Biology Server was used to calculate the masses of all possible peptides.
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RESULTS |
Purification of the Active and Resting Forms of
IIb3 from Platelet Lysates--
The
ability to purify two forms of IIb3 from
platelet lysates is well documented in the literature (28, 29). Our
procedure for obtaining the resting (AS-1) and active (AS-2)
conformations of IIb3 essentially
paralleled the procedures established by others. With these methods,
approximately 2.5 mg of AS-2 and 20 mg of AS-1 could be obtained from
50 units of outdated platelets. The purity of each form of the integrin
was judged to be at least 90% by Coomassie staining of SDS-PAGE (Fig.
1A.) We found no evidence of
the reproducible co-purification of any low Mr
proteins that could be associated with
IIb3 in a stoichiometric manner.
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Fig. 1.
Comparing the depth of the RGD binding pocket
on purified AS-1 and AS-2. The resting and active conformers of
IIb3 were purified from outdated platelet
lysates as described under "Materials and Methods." The purity of
AS-1 (panel A, lane 1) and AS-2 (panel
A, lane 2) was assessed by Coomassie staining of 7%
acrylamide gels run under non-reducing conditions. The depth of the RGD
binding site on AS-1 (panel B, lanes 1 and
2) and AS-2 (panel B, lanes 3 and
4) was measured by testing the ability of the integrin to
bind to either KYGRGDS-Sepharose, (lanes 1 and 3)
or to KYGRGDS-hexanediamine-Sepharose (lanes 2 and
4). Integrin (20 µg) was mixed with 20 µl of settled
affinity resin and incubated for 18 h. The affinity resins were
washed extensively to remove unbound integrin. Bound integrin was
removed from the resin by incubation in SDS sample buffer. The eluted
integrin was analyzed on SDS-PAGE. This experiment is representative of
three repetitions, making use of three different batches of purified
integrin in which identical results were obtained.
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One of the hallmark differences between the resting and active forms of
IIb3 on the platelet surface is the depth
of the RGD binding pocket. This depth has been gauged by measuring the ability of resting and activated platelets to adhere to beads coupled
to RGD peptides via spacers of varying length (39). To verify that the
purified forms of IIb3 are similar to the conformers observed on the platelet surface, we assessed the depth of
the RGD binding pocket on AS-1 and AS-2. We measured the binding of
each conformer to affinity columns in which the RGD motif was linked to
the resin in different ways. In one approach, the RGD peptide was
linked directly to the resin, providing a short spacer. In another
approach, the RGD peptide was bridged to the resin with a hexanediamine
spacer of about 18 Å. Only AS-2 bound the RGD peptide linked directly
to Sepharose (Fig. 1B), but both AS-1 and AS-2 bound the
beads in which the RGD was displayed on an extended linker. These
observations indicate that the differences in the exposure of the RGD
binding pocket on purified AS-1 and AS-2 are similar to those observed
on the platelet surface.
Comparison of the Fibrinogen Binding Properties of AS-1 and
AS-2--
In equilibrium binding studies, Kouns et al. (28)
found that only AS-2 could bind to Fg. No binding between AS-1 and Fg could be detected. We sought to determine whether the failure to detect
binding between AS-1 and Fg is a consequence of a slow association rate
or a rapid dissociation rate between the two proteins. This distinction
has important implications for understanding how cellular signaling
events regulate integrin activation.
SPR was used to measure k1 for the binding of
AS-1 and AS-2 to Fg (Fig. 2). The
association between AS-1 and fibrinogen could not be detected, even at
high concentrations of integrin. Under the conditions of this
experiment, the lowest association rate that can be observed with the
BIACore was 1 × 103 M1
s1. Therefore, the rate at which AS-1 associates with Fg
is below this value. In contrast, AS-2 bound to Fg with an association rate constant of 1.3 × 105
M1 s1. In related experiments,
we found that the association rate between AS-2 and the model
RGD-ligand Fab-9 was also about 7-fold faster than the rate of
association between AS-1 and Fab-9 (2.3 × 105
M1 s1 versus
3.2 × 104 M1
s1). Therefore, a primary difference between AS-1 and
AS-2 is the rate at which ligands associate with their respective
binding pockets.
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Fig. 2.
Binding of AS-1 and AS-2 to fibrinogen.
The binding of AS-1 and AS-2 to Fg-linked plasmon resonance sensor
chips was measured with the BIACore 3000. Binding was performed in
buffer containing 1 mM Ca2+ and 1 mM Mg2+. From a series of such sensorgrams in
which the amount of integrin included in the analyte was varied, the
association rate constant (k1) between AS-2 and
Fg was derived as described (35, 36).
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Testing the Role of Mn2+ as an Activator--
The
divalent ion Mn2+ has been linked to the activation of
integrins because it increases ligand binding affinity and promotes cell adhesion (20, 40, 41). It is widely thought that Mn2+
mimics physiologic activation of integrin. To test this idea, we
measured the ability of Mn2+ to convert AS-1 to AS-2. AS-2
binds to Fg when Ca2+ is the only available divalent ion,
and this binding function is stable to prolonged dialysis. Therefore,
these characteristics were set as criteria for the conversion of AS-1
to AS-2.
To perform the conversion test, AS-1, purified in
Ca2+-containing buffer, was dialyzed into buffer containing
200 µM Mn2+. Then, the ability of the
integrin to bind to fibrinogen was measured by SPR. As shown in Fig.
3, Mn2+ increased the
association between AS-1 and Fg to the point where it could be detected
by SPR. However, when the Mn2+-loaded AS-1 was returned to
buffer containing only Ca2+, its ability to bind Fg was
lost. This cation-exchange procedure could be repeated through several
cycles, each resulting in an increase in k1 for
Fg when Mn2+ was present, and a subsequent ablation of
binding to Fg when only Ca2+ was present. Thus, whereas
Mn2+ increases the association rate of AS-1 for ligand, it
does not convert AS-1 to AS-2.
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Fig. 3.
Effect of Mn2+ on the
binding of AS-1 and AS-2 to fibrinogen. To determine whether
Mn2+ could convert AS-1 to AS-2, the AS-1 form of the
integrin was dialyzed into buffer containing 200 µM
Mn2+. A sample of this material was then dialyzed back into
buffer containing 1 mM Ca2+. Then, the binding
of AS-2 ( ), AS-1 ( ), AS-1 dialyzed into Mn2+ ( ),
and AS-1 dialyzed into Mn2+ and then back into
Ca2+ ( ) to fibrinogen was measured with SPR. The
resulting sensorgrams are shown. This experiment is one of three
repetitions, making use of two separate batches of purified AS-1 and
AS-2, each of which yielded nearly identical results.
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Probing the Chemical Structure of AS-1 and AS-2 by Peptide Mass
Fingerprinting--
There is no detailed information on how the
structures of AS-1 and AS-2 differ. This leaves open the possibility
that they are alternatively spliced forms of the integrin or differ in
some key posttranslational modification. A comprehensive mass
fingerprinting study was undertaken to probe for differences between
AS-1 and AS-2. Because of the accuracy of mass spectrometry, this type of analysis is capable of revealing differences in amino acid sequence
and in post-translational modification. Mass fingerprinting was
performed by separating the IIb and 3
subunits (derived from AS-1 and AS-2) by SDS-PAGE under reducing
conditions. Bands corresponding to each subunit were excised and
subjected to in-gel digestion with trypsin as described under
"Materials and Methods." Then, the resulting mixtures of peptides
were analyzed by MALDI-TOF mass spectrometry. From this analysis we
were able to detect peptides comprising 86% of the entire sequence of
IIb (Table I, sections A
and B), and 85% of the sequence of 3 (Table I, section
C). Perhaps surprisingly, no differences were observed in the peptides that were detected from AS-1 and AS-2. The majority of the undetected tryptic fragments were of very low mass (<300 daltons) and are not
amenable to detection by MALDI-TOF. The only larger fragments that
remained undetected by MALDI included tryptic fragments corresponding to residues 166-276 from IIb and a fragment
corresponding to residues 151-181 of 3. These fragments
were not observed in either AS-1 or AS-2. We also failed to detect a
peptide encompassing the amino-terminal 12 residues of the
IIb light chain, which is not unexpected because
alternative processing of this terminus of the light chain of
IIb has been reported before. Amino acid sequencing of
this chain of IIb showed Val-13 to be the amino-terminal residue (42, 43). We were also unable to Western blot
IIb with the anti-V41 antibody which binds to this
region of IIb (33).
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Table I
Detection of tryptic peptides from AS-1 and AS-2
Integrin IIb3 was separated on SDS-PAGE, and the
heavy chain of IIb (section A), the light chain of
IIb (section B) or the 3 subunit (section C) was
excised and subjected to in-gel tryptic digestion as described under
"Materials and Methods." Peptides were extracted from the gel slice
and analyzed by MALDI-TOF mass spectrometry. The spectra between 800 daltons and 6000 daltons was examined for peptides. The monoisotopic
mass of the predicted tryptic peptides are compared to the masses of
peptides observed in the MALDI profile. The peptides observed in the
spectra are in the column designated as "Native
IIb3." Some peptides could not be detected due
to their glycosylation, so IIb3 was enzymatically
deglycosylated and analyzed in the same manner (column noted as
"Deglycosylated"). Peptide fragments that were not observed due to
size or detection limitations are noted as ND. The change in the mass
of a fragment containing an N-linked sugar chain after
deglycosylation (Asn  Asp) is an increase of 1 dalton.
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Because so much attention has focused on the role of the integrin
cytoplasmic tails in the process of activation, particularly the
cytoplasmic domain of the subunit, the MALDI spectra containing tryptic peptides derived from the tail of 3 are shown in
Fig. 4. In fingerprints generated with
trypsin, peptides encompassing 3 residues 717 to 762, all but the two most C-terminal amino acids, were observed in MALDI
profiles (Fig. 4B). The presence of the carboxyl-terminal
two residues on 3 was confirmed by digestion with Lys-C
protease, yielding the predicted fragment with mass of 1561 daltons
(Fig. 4, B and C, insets).
Phosphorylation of the cytoplasmic tails would be evident as an
increase in the mass of tryptic peptides by 80 daltons per phosphate
group. There was no reproducible evidence of phosphorylation of the
peptides derived from either AS-1 or AS-2. This observation was
supported by the inability to Western blot either form of
IIb3 with anti-phosphotyrosine antibodies
(not shown). Similar observations were made with the cytoplasmic domain
of IIb (Table I, section B). Based on these mass
fingerprints, we conclude that the two conformers of
IIb3 have the same primary structure over
the vast majority of their sequence.
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Fig. 4.
Detection of tryptic peptides derived from
the cytoplasmic domain of 3 from
AS-1 and AS-2 with MADLI-TOF mass spectrometry. A, the
amino acid sequence of the cytoplasmic domain of 3 is
shown along with the predicted cleavage points for trypsin
(top arrows) and Lys-C protease (bottom arrows).
The monoisotopic mass of the predicted fragments are shown above each
peptide. B, a portion of the mass spectra from
3 derived from AS-1 and digested with trypsin is shown.
The peptide peaks corresponding to each of the peptides from within the
cytoplasmic tail are labeled (arrows). A smaller section of
the spectra from 3 derived from AS-1 but digested with
Lys-C protease is shown in the inset, revealing a peptide
fragment corresponding to the extreme C terminus of 3.
C, mass spectra derived from the AS-2 form of
3 treated in the same manner as described for
panel B are shown.
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A Difference in the Sensitivity of Disulfide Bonds in AS-1 and AS-2
to Reduction--
Given that the chemical structure of AS-1 and AS-2
appeared to be identical, effort was focused on identifying the
conformational distinctions between the two molecules. In one approach,
we examined the sensitivity of disulfide bonds to reduction (Fig.
5). AS-1 was largely insensitive to mild
reduction with DTT (Fig. 5A, lane 2). In
contrast, the disulfide bond connecting the heavy and light chain of
IIb was reduced using only mild reductant (Fig.
5B, lane 2). Therefore, a major conformational
distinction between AS-1 and AS-2 is the exposure of the disulfide bond
joining the heavy and light chains of IIb. With an
increase in the amount of DTT, and with a prolonged incubation time,
the disulfide bond connecting the heavy and light chain of
IIb in AS-1 was partially reduced (Fig. 5A,
lane 3). The addition of RGD peptide stabilized both forms
of the receptor to reducing agent (Fig. 5, A and
B, lanes 4), indicating a conformational
connection between the labile disulfide bonds and the ligand binding
pocket.
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Fig. 5.
Measuring the lability of disulfide bonds in
AS-1 and AS-2. The susceptibility of disulfide bonds in AS-1
(panel A) or AS-2 (panel B) to reduction was
measured. The integrin was analyzed on SDS-PAGE under non-reducing
conditions (lane 1), following a 5-min treatment with 3 mM DTT (lane 2), following a 45-min treatment
with 20 mM DTT (lane 3), or after a treatment
with 20 mM DTT in the presence of "protecting" RGD
peptide (lane 4). Arrows show the positions of
IIb and 3.
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Mapping Conformational Differences in the 3 Subunit
of AS-1 and AS-2--
To examine the conformational differences
between AS-1 and AS-2 in more detail, the two proteins were subjected
to a peptide mapping study. This procedure differed from the mass
fingerprinting study because the proteins were subjected to limited
digestion with Asp-N protease in solution. Then the resulting fragments were subjected to mass fingerprinting to determine their composition.
Peptide maps that revealed fragments that distinguish AS-1 and AS-2
were generated by limited proteolysis. The Asp-N fragments unique to
the digests of AS-1 and AS-2 were evident on SDS-PAGE (Fig.
6A). Each of the unique Asp-N
fragments was excised from the SDS gel and subjected to in-gel
digestion with trypsin and peptide mass fingerprinting as described
under "Materials and Methods." From the masses of the peptides
present in these MALDI spectra, the composition of these peptides and
their location within the sequences of IIb and
3 was surmised. The Asp-N-generated fragments can be
separated into two groups that are discussed below.
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Fig. 6.
Peptide maps of AS-1 and AS-2 digested with
Asp-N protease. A, AS-2 (lane 1) and AS-1
(lane 2) were digested with Asp-N protease at an
enzyme:protein ratio of 1:300 (w/w). Digestion was performed for 6 h at 37 °C. Then, samples were separated on 12% acrylamide gels,
and peptide fragments were visualized by Coomassie staining.
B, peptides 1, 7, and 9 generated by Asp-N digestion of
IIb3 were excised from the gel shown in
panel A and subjected to in-gel tryptic digestion as
described under "Materials and Methods." Their composition was
determined by peptide mass fingerprinting with MALDI-TOF mass
spectrometry. This figure contains structural information required to
interpret results of these peptide mass fingerprints from peptides 1, 7, and 9 (A). The amino-terminal domain of the
3 subunit (boxed) is linked to the
cysteine-rich domain (boxed) via a long-range disulfide bond
between Cys-5 and Cys-435 (arrows). Other known disulfide
bonds within the domain are also noted with arrows. Peptides
1, 7, and 9 were generated by cleavage with Asp-N protease, which
cleaves on the amino-terminal side of aspartic acid residues. The Asp-N
fragments were then excised from the gel and subjected to peptide mass
fingerprinting using trypsin, which cleaves at lysine and arginine
residues. Therefore, the cleavage points for both Asp-N and trypsin are
shown as vertical lines above and below the amino acid sequence. In
addition, the monoisotopic mass of all fragments that were detected
within the mass fingerprints by MALDI-TOF mass spectrometry is noted
above the sequence. A key distinction between AS-1 and AS-2
is the presence of the tryptic peptide extending from residues 412-423
(bold text) in the mass fingerprints. This tryptic peptide
is detected in mass fingerprints of Asp-N fragments 1 and 7 (AS-1) but
is not detected in fragment 9 (AS-2). This tryptic peptide must be
derived from the slightly larger Asp-N fragment, which was generated
from the original digestion of IIb3 and
that extends Asp-393 to Asp-423 (underlined).
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Fragments 1 and 7 from AS-1 and fragment 9 from AS-2 are derived from
the 3 subunit (refer to Fig. 6B for a visual
representation of this domain). These Asp-N fragments contain segments
of a domain encompassing the amino terminus of 3 linked
to the cysteine-rich domain through a long-range disulfide bond. The
amino acid sequence of these domains, along with the Asp-N and trypsin
cleavage points are shown in Fig. 6B. Peptides corresponding
to the amino terminus of 3 (residues 1-62), along with
peptides that correspond to residues 413-448 were evident in the mass
fingerprint of fragment 1 (Table II,
section A). Similarly, fragment 7 gave rise to tryptic peptides
corresponding to the amino terminus of 3 (residues
1-62) along with peptides corresponding to 3 residues
413-578 (Table II, section B). Therefore, we conclude that fragments 1 and 7 contain segments of 3 that are connected by the
disulfide bond that connects Cys-5 and Cys-435 (44). Fragment 1 migrates on SDS-PAGE at a mass consistent with the composition proposed
in Table II, section A. However, fragment 7 migrates much higher than
predicted based on the peptides detected in the mass fingerprint. This
may indicate that fragment 7 contains additional peptide chain on its C
terminus which is not detected by mass fingerprinting or indicate that
it migrates anomolously on SDS-PAGE. The latter possibility is not
entirely unexpected because the fragment is cysteine-rich and may
remain partially folded during electrophoresis.
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Table II
Mass fingerprints of fragments 1, 7, and 9 derived from 3
AS-1 and AS-2 were digested with Asp-N protease, and the resulting
fragments were separated on SDS-PAGE. Peptide fragments 1 and 7 that
are unique to AS-1 (Figure 6A) and fragment 9 that is unique
in AS-2 were excised and subjected to in-gel trypsin digestion to
determine their composition. The mass fingerprint of each fragment was
determined using MALDI-TOF mass spectrometry. The mass range extending
from 800 to 6000 Da was examined. The predicted mass of each peptide
and the corresponding fragment observed in MALDI are shown. Some
tryptic peptides fall outside of the detectable mass range.
Consequently, the peptides that are observed do not make up the
contiguous sequence of each Asp-N fragment.
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Fragment 9, which is found only in the Asp-N digest of AS-2, is related
to fragments 1 and 7 even though it migrates differently on SDS-PAGE
(Fig. 6A). Tryptic mass fingerprinting of fragment 9 revealed the peptides shown in Table II, section C, and like peptides 1 and 7, it contains domains within the amino terminus linked to the
cysteine-rich domain of 3. However, fragment 9 differs
from fragments 1 and 7 with regard to a tryptic peptide with a mass of
1123.8 daltons. This peptide, which is absent in the fingerprint of
fragment 9, is consistently observed in the mass fingerprints of both
fragments 1 and 7. This peptide corresponds to residues 413-422 (Fig.
6B, boldface). The presence of this tryptic
peptide in fragments 1 and 7 indicates that they contain residues
393-423 which correspond to the Asp-N fragment that encompasses the
tryptic peptide (Fig. 6B, underlined). The
inability to detect this tryptic peptide in fragment 9 cannot be taken
as proof of its absence. Nevertheless, the reproducible detection of
the same tryptic peptide in several fingerprints of fragments 1 and 7, and from several distinct batches of purified AS-1, its absence in the
fingerprint of fragment 9 is striking. We suggest that Asp-393 and
Asp-423, the Asp-N cleavage points that flank this peptide are exposed
to solvent in AS-2 and are liberated by Asp-N protease. In contrast,
this fragment in AS-1 remains connected to the amino terminus during
Asp-N digestion, indicating that it is buried. This distinction,
combined with the simple fact that peptides 1,7, and 9 all migrate at
different positions on SDS-PAGE, show that the conformation of AS-1 and
AS-2 differ at the disulfide knot that connects the N terminus of
3 to the cysteine-rich domain.
Mapping Conformational Differences in the IIb
Subunit of AS-1 and AS-2--
The other group of unique fragments
generated by digestion with Asp-N protease arise from cleavage of the
divalent cation binding domains within IIb. These
include fragments 2-6 and 8 which are observed only in AS-1 (Fig.
6A). Mass fingerprinting of these fragments showed that they
each contained peptides derived from the cation binding domains of
IIb (Table III). No Asp-N
generated fragments encompassing these domains of IIb
were detected in AS-2. Consequently, we conclude that the divalent
cation binding domains within AS-2 are highly sensitive to proteolytic
attack by Asp-N protease. This is consistent with the fact that the
divalent ion binding domain is rich in aspartate residues, a property
that probably leads to the complete digestion of this domain to small peptide fragments.
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Table III
Mass fingerprints of fragments 2-6 and 8 derived from IIb
AS-1 and AS-2 were digested with Asp-N protease, and the resulting
fragments were separated on SDS-PAGE. Peptide fragments 2-6 and 8 that
are unique to AS-1 (Figure 6A) were excised and subjected to
in-gel trypsin digestion to determine their composition. The mass
fingerprint of each fragment was determined using MALDI-TOF mass
spectrometry. The mass range extending from 800 to 6000 Da was
examined. The predicted mass of each peptide and the corresponding
fragment observed in MALDI are shown. Some tryptic peptides fall
outside of the detectable mass range. Consequently, the peptides that
are observed do not make up the contiguous sequence of each Asp-N
fragment. Fragments 2-4 migrate as a triplet on SDS-PAGE but show the
same mass fingerprint.
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DISCUSSION |
Despite advances in our understanding of the signaling pathways
that enact activation of integrins, little attention has been given to
the changes in the kinetics of ligand binding that lead to activation.
Similarly, the identity of chemical modifications, or positions of
conformational change, that accompany activation have not been mapped.
Here, we address these issues by using the platelet integrin
IIb3 as a model.
As an initial step toward understanding the differences between AS-1
and AS-2, we compared their ligand binding properties. Prior study
shows that IIb3 contains two ligand
binding sites, one for Fg and another site that binds to RGD (35, 45).
These ligand binding sites are physically separate but are linked
kinetically. For example, RGD ligand blocks the association of Fg with
its binding site and has the ability to dissociate pre-bound Fg (35). Our finding that the RGD binding site is present on AS-1 and AS-2 confirms the prior observation by Kouns (28). The fact that AS-1 and
AS-2 have RGD binding sites with different degrees of exposure, or of
depth, is also consistent with the observation that the depth of this
site is regulated by activation on the platelet surface (39). The fact
that the RGD site is more accessible to ligand on AS-2 also provides a
structural basis for the more rapid rate of association with the model
RGD ligand Fab-9.
No study has measured the association and dissociation rates between
AS-1 and Fg. Therefore, the lack of binding between AS-1 and Fg under
equilibrium conditions could have resulted from a high dissociation
rate rather than a lack of association. In the present study, no
association between As-1 and Fg could be detected, even at very high
concentrations of Fg. This observation suggests that the Fg binding
site may be entirely absent on resting integrin and may come to exist
only when conformational rearrangements create such a site. This is in
contrast to the RGD binding site, which can be detected on both AS-1
and AS-2 and which appears to be displayed at different depths on the
two conformers.
Many artificial stimuli are used to mimic cellular activation in the
study of integrin activation. Among these are antibodies that alter the
integrins affinity state, reducing agents like dithiothrietol and the
divalent ion Mn2+. Mn2+ has been used
extensively as a stimulus because it binds to integrins and increases
the ligand association rate (20, 40, 41). Because of this property,
Mn2+ has come to be considered a "universal" integrin
activator. Given this prevailing notion, we suspected that
Mn2+ might be able to convert AS-1 to AS-2. The
conformations of AS-1 and AS-2 are stable to prolonged dialysis, so we
were able to perform a series of experiments to test the ability of
Mn2+ to convert AS-1 to AS-2. The two parameters that were
considered as benchmarks of conversion were 1) the ability to bind to
Fg in the presence of Ca2+, and 2) the stability of this
property to dialysis. Dialysis of AS-1 into buffer containing
Mn2+ increased the rate of association for Fg, but this
effect was lost when the integrin was dialyzed back into buffer
containing Ca2+. Therefore, we conclude that
Mn2+ enhances the rate of association with ligand but fails
to induce the conformational changes that are equivalent to activation. We have also observed that treatment of AS-1 with mild reductant, like
DTT, will increase the rate of association with ligand. Yet, our
observations indicate that DTT also fails to convert AS-1 to
AS-2.2
Prior study established purification protocols for AS-1 and AS-2, but
no detailed comparison of their chemical structures had been performed.
This left open the possibility that the two variants of
IIb3 were actually alternatively spliced
forms of the integrin, especially because alternative splicing has been observed for both IIb and for 3 (46-48).
By using peptide mass fingerprinting, we were able to detect tryptic
peptide fragments that constitute more than 85% of the sequence of
IIb and 3 in both the AS-1 and AS-2
forms. Most of the peptides that were not detected by MALDI were of
very low mass. These studies showed the amino acid sequence of the two
proteins to be virtually identical, and indicate that AS-2 is not an
alternatively spliced form of the integrin present in platelets at low abundance.
We anticipated that the mass fingerprinting study would reveal some
type of post-transitional modification that would account for the
difference in activity of AS-1 and AS-2. Of particular interest were
potential phosphorylations within the cytoplasmic domain of
3. This subunit contains two consensus tyrosine
phosphorylation sites and several serine and threonine residues that
are candidates for modification by phosphate. We interpret the lack of
phosphorylation on AS-1 or AS-2 to show that phosphorylation is not
necessary to maintain the integrin in an active and stable
conformation. We cannot exclude the possibility that phosphorylation
within the cytoplasmic domains provides the activation energy to guide the integrin through a transition state that ultimately "decays" to
a stable conformation like AS-2. There is certainly precedent for
stepwise activation because some platelet agonists induce reversible
activation of IIb3 but others cause an
irreversible activation (49, 50). Similarly, the activation of the
51 integrin is a two-stage event
(51).
Even though the chemical structure of AS-1 and AS-2 are virtually
identical, they have significant differences in conformation. One
conformational difference centered around the disulfide bond that
connects the heavy and light chain of IIb. Within AS-2, this disulfide bond is sensitive to mild reducing conditions, whereas
the same bond in AS-1 is stable. Interestingly, the binding of RGD
ligand stabilizes this bond to reduction, providing a conformational link to the RGD binding site. The association of this segment of the
integrin with modulation of ligand binding affinity is consistent with
prior work showing that monoclonal antibodies that bind in this
vicinity can activate the ligand binding function of
IIb3 (52). It has also been reported that
the cleavage of IIb in this region by neutrophil
elastase can induce platelet aggregation (19). The association of the
junction between the heavy and light chain of IIb with
activation is also of interest because the junction is generated by
intracellular proteolysis during maturation of the integrin. Most
integrin subunits undergo similar processing at dibasic residues
within the subunits. Although this cleavage is not required for
ligand binding, it may be important for activation. Mutations in the
integrin 6 gene that eliminate the proteolytic
processing site render the 61 integrin
resistant to activation and kept the integrin in a low affinity state
on the cell surface (53). Along with the differences that AS-1 and AS-2
display in this region of the integrin, all of these observations
strongly implicate the domain encompassing the junction of the heavy
and light chains of integrin subunits as a key site where
conformational changes accompany activation.
Peptide mapping also showed that AS-1 and AS-2 exhibit a difference in
conformation in a region of the 3 subunit that can be
considered a disulfide "knot" connecting three regions of the protein that are well separated in the linear sequence. These are the
extreme amino terminus (residues 1 to 62), the cysteine-rich domain
(roughly residues 420 to 620), and a region proximal to the membrane
spanning segment (residues 635 to 663). Within this knotted domain, a
primary difference between AS-1 and AS-2 appears to center on a peptide
extending from residues 393 to 423 that appears to be exposed in AS-2
and buried in AS-1. This segment of 3 is directly
adjacent to Cys-435 which forms the disulfide bond to Cys-5, and it
encompasses Cys-405 which links to Cys-655. Interestingly, the epitopes
for many LIBS antibodies, which can activate the integrin and induce
platelet aggregation, bind to these domains (54, 55). Consequently, the
evidence linking conformational changes within the disulfide knotted
core of 3 to the process of activation is compelling.
Within the limits of detection, the resting and active forms of
IIb3 appear to have the same chemical
structure. However, the two conformers have significant differences in
conformation. These conformational differences primarily effect the
rate of ligand association and, in fact, may create of the Fg binding site. These findings suggest that future effort should focus on understanding the mechanism by which structural transitions within the
ectodomain of the integrin are brought about by inside-out signaling.
IIb
3*
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Program on Cell
Adhesion, The Cancer Research Center at The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3121; E-mail:
jsmith@burnham.org.
