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INTRODUCTION |
Integrins are 
heterodimeric cell-surface receptors that
promote not only adhesion to components present within the
extracellular matrix or on the surface of opposite cells but also
transfer information into and out of a cell (1). The adhesive functions
of integrins can be regulated by intracellular processes referred to as
"inside-out signaling." Conversely, ligand binding to the
extracellular domain of integrins initiates a cascade of intracellular
events termed "outside-in signaling" that generate a large spectrum
of cellular responses, such as cell migration, proliferation,
differentiation, and gene expression (2). Integrin cytoplasmic tails
appear to be key elements in these bidirectional signaling pathways, despite their short size as compared with other signaling receptors and
the absence of any demonstrable catalytic activity (3, 4). Integrin
and
cytoplasmic domains are thought to mediate signaling events
through modifications of their own structural and spatial organization
and/or through interactions with specific cytoplasmic components.
Various proteins have been identified that bind, at least in
vitro, to the cytoplasmic tail of
and
subunits and are
likely to play a role in regulating integrin signaling functions. These
include cytoskeletal components such as talin and
-actinin, as well
as several signaling or regulatory proteins such as integrin-linked
kinase p59ILK, focal adhesion kinase pp125FAK,
Grb2,
3-endonexin, cytohesin-1, integrin cytoplasmic
domain-associated protein ICAP-1, calreticulin and calcium- and
integrin-binding protein CIB1
(reviewed in Refs. 5 and 6).
Recently used methods for studying protein-protein interactions, such
as the two-hybrid system, have allowed the identification of
integrin-specific intracellular ligands (7-12). These methods are
based on the use of a unique linear amino acid sequence as a bait and
consequently do not take into account the secondary and tertiary
structural features of the interacting molecules. However, numerous
studies tend to demonstrate that
and
cytoplasmic domains adopt
a defined conformation and that the preservation of these structural
constraints is crucial to maintain the functional properties of
integrin receptors (13-20).
One of the best studied integrins is the platelet fibrinogen receptor,
integrin
IIb
3, that undergoes
conformational changes necessary for receptor function. In order to
elucidate further the structural relationship of the cytoplasmic tails
of
IIb and
3, we have used surface
plasmon resonance biosensor technology to monitor real time assembly of
the integrin
IIb and
3 cytoplasmic tails
and to investigate their ligand binding capacity.
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EXPERIMENTAL PROCEDURES |
Antibodies and Synthetic Peptides--
The anti-
3
monoclonal antibody (mAb) 4D10G3, the anti-
3 cytoplasmic
domain mAb C3a.19.5, and the anti-
IIb mAb S1.3 were kindly provided by Dr. D. R. Phillips (Cor Therapeutics, South San
Francisco, CA), the anti-
3 mAb D3GP3 by Dr. L. K. Jennings (University of Tennessee, Memphis, TE), and the
anti-
IIb
3 complex-specific mAb 10E5 by
Dr. B. S. Coller (Mount Sinai School of Medicine, New York, NY).
The anti-
IIb
3 mAb PAC-1 was from
Becton-Dickinson (San Jose, CA), and the anti-human
v
mAb VNR 139 was from Life Technologies, Inc. (Merelbeke, Belgium).
Polyclonal anti-
IIb
3 antibodies were
raised in rabbits against purified human platelet
IIb
3. Synthetic peptides corresponding to
either the wild type
IIb cytoplasmic domain
(
IIb Lys989-Gln1008), the
IIb cytoplasmic sequence with a R995A substitution
(
IIb R995A), or the
IIb sequence deleted
of the 989KVGFFKR995 motif (
IIb
Asn996-Gln1008) were all purchased from
Neosystem (Strasbourg, France).
Platelets and Cell Lines--
Outdated platelet concentrates
were kindly provided by Dr. J.-C. C. Faber (Luxembourg Red Cross Blood
Transfusion Center). The stable transfected CHO cell line A06,
expressing high levels of human
v
3
integrin (21), was grown in Iscove's modified Dulbecco's medium, and
HEL-5J20 cells in RPMI medium (Life Technologies) (22). Culture medium
was supplemented with glutamine, penicillin, and streptomycin, and 10%
heat-inactivated fetal calf serum. The adherent CHO cells were
routinely passaged with EDTA buffer, pH 7.4 (1 mM EDTA, 126 mM NaCl, 5 mM KCl, 50 mM Hepes).
Construction of pGEX-4T-2 Expression Plasmids--
The cDNA
encoding the wild type or S752P mutant human
3 integrin
cytoplasmic tail (Lys716-Thr762) was generated
by the polymerase chain reaction (PCR) using full-length pBJ1-
3 plasmids as templates (21). The upstream (sense)
primer was a 28-mer with a BamHI site (G
GATCC)
corresponding to the
3 nucleotide sequence 2245-2266,
5'-GGATCCAAACTCCTCATCACCATCCACG-3'. The downstream (antisense) primer
was a 30-mer corresponding to the
3 nucleotide sequence
2365-2388 and comprising an SmaI restriction site
(CCC
GGG) followed by a stop codon
5'-CCCGGGTTAAGTGCCCCGGTACGTGATATT-3'. PCR amplification was performed
using the Takara PCR kit (Shiga, Japan). The full-length cDNA
encoding human CIB was obtained by reverse transcriptase-PCR (RT-PCR)
of HEL-5J20 cell mRNA. Briefly, total RNA was isolated from 5 × 106 cells according to the method of Chomczynski and
Sacchi (23), and RT-PCR was performed using the RNA-PCR kit from
Promega (Madison, WI). The sense primer was a 36-mer corresponding to
the published CIB nucleotide sequence 1-30 (12) with an additional
BamHI restriction site (G
GATCC),
5'-GGATCCATGGGGGGCTCGGGCAGTCGCCTGTCCAAG-3'. The downstream antisense
primer was a 32-mer corresponding to the CIB nucleotide sequence
551-576 followed by a stop codon and an EcoRI restriction
site (G
GATTC), 5'-GGATTCTCACAGGACAATCTTAAAGGAGCTGG-3'. All the
primers used to generate cDNA fragments were obtained from Life
Technologies, Inc. PCR and RT-PCR products were purified using the PCR
Preps DNA Purification System from Promega. They were digested with the
corresponding restriction enzymes and inserted into the glutathione
S-transferase (GST) vector pGEX-4T-2 (Amersham Pharmacia
Biotech, Uppsala, Sweden) containing a thrombin cleavage site. The
expected nucleotide sequence was confirmed for each construct by direct
sequencing using the T7 sequencing kit from Amersham Pharmacia Biotech.
Expression and Purification of Recombinant Fusion
Proteins--
Native GST and in-frame GST fusion proteins were
expressed in Escherichia coli JM105 (Amersham Pharmacia
Biotech) according to the manufacturer's instructions. Bacterial
pellets were suspended in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
1.8 mM KH2PO4, pH 7.4) containing
10 mM EDTA and 50 µM
aminoethylbenzenesulfonyl fluoride and were incubated for 30 min at
room temperature in the presence of 200 µg/ml lysozyme. The bacterial
suspension was submitted to short bursts of sonication and further
treated with 1% Triton X-100 for 30 min at 4 °C. The insoluble
material was pelleted by a 20-min centrifugation at 10,400 × g at 4 °C. Supernatants were filtered on a 0.8-µm
membrane and submitted to affinity chromatography on a
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) column (100 × 20-mm inner diameter) previously equilibrated with PBS. Bound fusion
proteins were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0. GST-
3 and
GST-
3(S752P) fusion proteins were further purified by
immunoaffinity chromatography using the anti-
3
cytoplasmic tail mAb C3a.19.5 immobilized on CNBr-activated Sepharose
CL4B (Amersham Pharmacia Biotech) in order to eliminate free GST.
Proteolytic Cleavage of GST Fusion Proteins and Purification of
Recombinant Products--
Cleavage of recombinant CIB or wild type
3 cytoplasmic tail peptide from GST was achieved by
incubating 2 NIH units of thrombin protease (Amersham Pharmacia
Biotech) per mg of purified material under gentle stirring for 5 h
at room temperature.
3 peptide was purified by
preparative reverse-phase high performance liquid chromatography (HPLC)
using a C4 column (100 × 20-mm inner diameter) with a 0-40%
linear gradient of acetonitrile in 0.05% trifluoroacetic acid. The
amino acid sequence of the recombinant peptide was checked by
microsequencing on an Applied Biosystems Procise sequencer. The
purified peptide was stored dessicated at 4 °C. For CIB
purification, the thrombin hydrolysate was extensively dialyzed against
PBS and then passed through a glutathione-Sepharose column in order to
remove GST. The flow-through fraction containing CIB was kept frozen at
20 °C until use.
Preparation of
IIb
3- or
v
3-enriched Glycoprotein
Concentrates--
For
IIb
3-enriched
glycoprotein concentrates, outdated platelets were washed and then
lysed in 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM Tris-HCl (TBS), pH
7.4, containing 1% Triton X-100, 10 µM leupeptin, 500 µM PMSF, 2 mM N-ethylmaleimide,
0.02% NaN3, according to the procedure described by
Fitzgerald et al. (24). The lysate was applied to a
concanavalin A (ConA)-Sepharose 4B (Amersham Pharmacia Biotech) column
(100 × 20-mm inner diameter) equilibrated with TBS, pH 7.0, 0.1%
Triton X-100 (TBS/ConA), and bound glycoproteins were eluted with 100 mM
-methyl-D-mannose in running buffer. For
v
3-enriched glycoprotein concentrates,
v
3-expressing CHO cells (cell clone A06)
were detached with EDTA buffer, pH 7.4, for 10 min at 37 °C and then
washed in cold PBS. Cells (5-9 × 106) were lysed for
30 min in 500 µl of ice-cold lysis buffer, pH 7.5 (150 mM
NaCl, 50 µM aminoethylbenzenesulfonyl fluoride, 1% Triton X-100, 10 mM Tris-HCl), and the lysate was
precleared by centrifugation at 10,000 × g for 10 min
at 4 °C. The supernatant was incubated for 2 h at 4 °C with
1 volume of 50% slurry ConA-Sepharose 4B suspension. After extensive
washes with cold TBS/ConA, bound glycoproteins were eluted as described
above and kept on ice until use.
IIb
3 Binding to Immobilized Human
Fibrinogen--
IIb
3 binding to
fibrinogen was determined according to Kouns et al. (25)
with some modifications. 96-well microtiter plates (Costar, Cambridge,
MA) were coated overnight at 4 °C with 100 µl/well purified human
fibrinogen (Sigma, Bornem, Belgium) at 5 µg/ml in TBS. The plates
were then saturated with TBS containing 3.5% bovine serum albumin and
0.05% NaN3 (125 µl/well) overnight at 4 °C. The
ConA-enriched platelet glycoprotein fraction was serially diluted in
TBS/ELISA alone (TBS containing 1% bovine serum albumin, 0.035%
Triton X-100) or in TBS/ELISA containing either 2 µg/ml D3GP3 mAb or
10 mM MnCl2. 100-µl aliquots were added to
the wells and incubated 4 h at room temperature. After three
washes with TBS, 1 µg/ml polyclonal rabbit
anti-
IIb
3 antibodies in TBS/ELISA were
added (100 µl/well) for 2 h at room temperature. The wells were
washed three times with TBS, followed by 90 min incubation at room
temperature with 100 µl/well donkey anti-rabbit Ig antibodies
conjugated to horseradish peroxidase (Amersham Pharmacia Biotech).
After three washes with TBS, 100 µl of 0.1 mg/ml
3,3',5,5'-tetramethylbenzidine, 0.01% H2O2 in
140 mM sodium acetate-citrate buffer, pH 6.0, were dispensed into each well. The enzymatic reaction was stopped by addition of 25 µl of 2 M H2SO4,
and the absorbance was measured at 450 nm.
In Vitro Liquid Phase
IIb
3-CIB
Binding Assays--
ConA-purified platelet glycoproteins (250 µg) or
v
3-enriched CHO-A06 cell glycoproteins (1 mg) were incubated with 50 µg of purified GST-CIB or GST alone for
2 h at 4 °C under gentle stirring. Experiments were carried out
in ice-cold 0.1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, containing either 2 mM
CaCl2, 2.5 mM EGTA, or 1 mM
CaCl2, 1 mM MgCl2 ± 10 mM MnCl2. In Mn2+-related assays,
platelet glycoproteins were first preincubated for 30 min at room
temperature in 10 mM MnCl2 or in
Mn2+-free buffer before incubation with GST fusion
proteins. For
IIb
3 capture, the mixtures
were incubated with 5 µg of the 10E5 mAb for 2 h at 4 °C or,
alternatively, with 4 µg of the mAb PAC-1 for 2 h at room
temperature followed by 8.5 µg of rabbit anti-mouse µ-chain-specific antibodies (Jackson Immunoresearch, West Grove, PA)
for an additional 2 h at 4 °C. Protein A-Sepharose CL 4B beads (80 µl of a 50% slurry suspension) were added and incubated for 2 h at 4 °C. For GST fusion protein capture, the mixtures were incubated for 2 h at 4 °C with 80 µl of 50% slurry
glutathione-Sepharose 4B suspension. Control experiments were performed
using non-substituted Sepharose CL4B. The adsorbents were washed three
times with 500 µl of the respective ice-cold incubation buffer, and
the captured proteins were recovered by boiling the beads in 30-50
µl of 5%
-mercaptoethanol, 2% SDS, 10% glycerol, 25 µg/ml
bromphenol blue in 15.625 mM Tris-HCl, pH 6.8. Each sample
was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed
by immunoblotting as described below.
Protein Assay, Electrophoresis, and Western Blot
Analysis--
Protein concentration was determined using the Bio-Rad
Protein assay reagent. SDS-PAGE was performed using the mini-Protean II
electrophoresis system (Bio-Rad), and Tris-Tricine SDS-PAGE was carried
out according to the method described by Schagger and von Jagow (26).
Electrophoresed samples were transferred onto Hybond-C nitrocellulose
membrane (Amersham Pharmacia Biotech) using a semi-dry transblot
apparatus (Amersham Pharmacia Biotech). The membranes were blocked
overnight in blotting buffer (5% dry milk, 0.1% Tween 20, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4), and incubated
for 2 h with the anti-
3 4D10G3 mAb mixed with
either the anti-
IIb S1.3 mAb or the
anti-
v VNR 139 mAb diluted in blotting buffer. After
three washes in blotting buffer, membranes were incubated for 1 h
with diluted sheep anti-mouse Ig conjugated to horseradish peroxidase
(Amersham Pharmacia Biotech). Membranes were again washed three times
in blotting buffer and then in 137 mM NaCl, 20 mM Tris-HCl, pH 7.4 (TBS/WB), and developed using the
chemiluminescence ECL kit (Pierce) according to the manufacturer's instructions. The membranes were then stripped by successive washes in
TBS/WB containing 100 mM
-mercaptoethanol, 2% SDS, 52.5 mM Tris-HCl, pH 6.7, for 30 min at 50 °C, and again in
TBS/WB. After an overnight incubation in blotting buffer, the membranes
were reprobed for 2 h with polyclonal goat anti-GST antibodies
(Amersham Pharmacia Biotech), and antibody binding was detected as
described above with horseradish peroxidase-conjugated rabbit anti-goat IgG (Jackson Immunoresearch).
Surface Plasmon Resonance Binding Studies--
Real time
biomolecular interaction analysis was performed using the
BialiteTM or Biacore XTM instruments (Biacore,
Uppsala, Sweden). Purified proteins were covalently attached to
carboxymethyl dextran (CM5) chips (Biacore) previously activated with a
mixture of N-hydroxysuccinimide and N-ethyl-N'-dimethylaminopropyl carbodiimide
according to the manufacturer's instructions. Experiments were
performed at 25 °C using as running buffers either Biacore HBS (150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20, 10 mM Hepes, pH 7.4) or TBS/Bia (150 mM NaCl,
0.005% Tween 20, 50 mM Tris-HCl, pH 7.4) ± 2 mM CaCl2. The sensorchips were regenerated with
a short pulse of either 200 mM glycine HCl, pH 2.2 (anti-GST antibody-coated chip), or 10 mM HCl (GST fusion protein-derivatized chip). The amount of analyte bound to the immobilized ligand was monitored by measuring the variation of the
surface plasmon resonance angle as a function of time. Results were
expressed in resonance units (RU), an arbitrary unit specific for the
Biacore instrument (1000 RU correspond to approximately 1 ng of bound
protein/mm2 and are recorded for a change of 0.1° in
resonance angle) (27). The transformation of crude data, the
preparation of overlay plots, and the determination of kinetic
parameters of the binding reactions were performed using the
Biaevaluation 3.0 software. The association rate constant
(kon) and the dissociation rate constant
(koff) were determined separately from
individual association and dissociation phases, respectively, assuming
a one-to-one interaction. The affinity constant KD
was calculated as
koff/kon. Experimental values from the first 20 s at the beginning of each phase were not
considered in the fitting to avoid distortions due to injection and mixing.
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RESULTS |
Generation of Recombinant Wild Type or Mutant
3
Integrin Cytoplasmic Peptides and of CIB--
In order to perform
in vitro studies of
IIb and
3
cytoplasmic tail association, we generated recombinant GST fusion
proteins containing a thrombin cleavage site and corresponding to the
entire wild type or S752P mutant cytoplasmic domain of the
3 integrin (residues 716-762) and to the
IIb-binding protein CIB (residues 1-191). The fusion
proteins were isolated from bacterial cell lysates by glutathione
affinity chromatography, and the GST-
3 proteins were
further immunopurified using the anti-
3 monoclonal antibody (mAb) C3a.19.5. Thrombin-released wild type
3
cytoplasmic tail peptide and CIB were purified free of GST using
reverse-phase HPLC and glutathione affinity chromatography,
respectively. The accurate amino acid sequence of the
3
peptides was confirmed by microsequencing. SDS-PAGE analysis of
isolated proteins revealed a purity greater than 98%, as evaluated by
densitometric scanning of the gel (Fig.
1). The apparent molecular masses of both
GST-
3 and GST-
3 (S752P) (34 kDa), GST-CIB
(48 kDa), CIB (25 kDa), and GST (29 kDa) were in good agreement with
the predicted mass deduced from their amino acid composition. When
analyzed with higher resolutive SDS-PAGE and on a C18 HPLC column, the
5.6-kDa
3 peptide appeared homogeneous with only slight
impurities (Fig. 2).

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Fig. 1.
SDS-PAGE analysis of purified GST fusion
proteins. Proteins were electrophoresed under reducing conditions
in a 12.5% Tris glycine SDS gel, and stained with Coomassie Brilliant
Blue: lane 1, immunoaffinity purified GST- 3,
and lane 2, GST- 3(S752P); lane 3, thrombin digest of GST- 3; lane 4, purified
recombinant 3 cytoplasmic domain peptide; lane
5, native GST; lane 6, affinity isolated GST-CIB
before, and lane 7, after thrombin proteolysis; lane
8, purified CIB after removal of GST on a glutathione-Sepharose
affinity column; lane 9, molecular mass standards.
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Fig. 2.
Analysis of purified wild type
3 cytoplasmic domain peptide. The
purity of the recombinant 3 peptide was checked by
reverse-phase HPLC using an analytical C18 column (50 × 4.6-mm
inner diameter) with a 0-40% linear gradient of acetonitrile in
0.05% trifluoroacetic acid and by 15% Tris-Tricine SDS-PAGE. The
position of the 5.6-kDa 3 cytoplasmic peptide is
indicated by an arrow. AU, absorbance arbitrary
units (214 nm).
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In Vitro Complex Formation of
IIb and
3 Cytoplasmic Domains as Monitored by SPR
Analysis--
In vitro complex formation of wild type
IIb and
3 integrin cytoplasmic tails was
investigated by surface plasmon resonance (SPR) in order to monitor
real time biomolecular interactions. We first examined whether the
synthetic
IIb peptide was able to bind to the
3 cytoplasmic tail fused to GST. Purified anti-GST polyclonal antibodies were immobilized on a sensor chip through amine
coupling and were allowed to stably capture native GST or the
GST-
3 fusion protein before injection of the
IIb peptide. As shown in Fig.
3A, a characteristic binding
signal was monitored when the peptide was brought into contact with
captured GST-
3 but not with either an uncoated surface,
immobilized antibodies alone, or GST-antibody complexes. In these
latter control experiments, the rapid change in the resonance signal
was due to a dilution buffer-induced, nonspecific change in the bulk
refractive index. The maximum response monitored at the end of the
peptide injection phase was about 60-70 RU for 1100 RU of initially
captured GST-
3 protein. The corresponding molar ratio
was estimated at ~0.8 mol/mol and was consistent with a 1:1
interaction. Further studies showed that
IIb binding was
dose-dependent and could be almost completely inhibited by
soluble GST-
3 protein but not by GST alone (Fig. 3,
B and C). Taken together, these data demonstrate
that
IIb specifically interacts with the
3 cytoplasmic tail and that this interaction can be
monitored using SPR technology.

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Fig. 3.
SPR analysis of the interaction between
IIb and
3 cytoplasmic tails. The
experimental sensorgrams were recorded on a Bialite apparatus using
TBS/Bia as running buffer and a flow rate of 5 µl/min (except for
B which is 50 µl/min). Data are given as absolute
responses. A, response curves recorded during and after
injection of 41.7 µM IIb peptide on an
uncoated surface (curve 1) or on a chip with
immobilized polyclonal anti-GST antibodies before (curve 4)
and after capture of ~1100 RU of GST (curve 3) or
GST- 3 (curve 2). GST/antibody interaction was
very stable over time with a decrease in signal of only 1-2 RU/min.
B, overlaid dose-response binding curves obtained with
various concentrations of IIb peptide injected over a
GST- 3-coated chip surface (~12,500 RU). C,
interaction of covalently immobilized GST- 3 (~12,500
RU) with 41.7 µM of IIb peptide
preincubated with 1.5 mg/ml free purified GST (curve 2) or
GST- 3 (curve 1). The binding curve obtained
in the absence of the competitor is shown as a reference (curve
3).
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The
IIb
3 Cytoplasmic Domain Complex
Is Stabilized by Divalent Cations--
In order to determine the role
of divalent cations in integrin
IIb
3
cytoplasmic domain association, we investigated the binding of
IIb to immobilized GST-
3 in the presence
of 2 mM CaCl2 or MgCl2. The
influence of Ca2+ on the interaction was apparent from the
slopes of the sensorgrams in Fig. 4.
Interestingly, the rates of association of
IIb with GST-
3 were similar, independent of the presence or
absence of Ca2+. In contrast, the dissociation of
IIb was more rapid in the absence of Ca2+,
as demonstrated by a greater slope in the curve. Similar results were
obtained with a low cation concentration (50 µM) or with a Mg2+-containing buffer (data not shown). To characterize
further the dynamic parameters of the interaction, we determined
binding isotherms in the presence or absence of Ca2+ by
injecting
IIb peptide solutions ranging from 4.2 to 83.3 µM over a GST-
3 fusion protein-coated chip
(Fig. 3B). From these curves, the association and
dissociation rates and the apparent KD of the
binding were determined as indicated under "Experimental
Procedures." As shown in Table I, the
on rates with or without Ca2+ were very similar. In
contrast, the peptide dissociation was slower when Ca2+
ions were present in the flow as compared with Ca2+-free
buffer. This resulted in an increased affinity, although the
KD value was still in the range of weak
interactions. Taken together, these data suggest that divalent ions
have a different effect on the association and dissociation of
IIb and
3 cytoplasmic tails.

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Fig. 4.
Effect of Ca2+ on the interaction
of soluble IIb peptide with
immobilized GST- 3 fusion
protein. Sensorgrams were collected during and after the injection
of 41.7 µM IIb peptide at a flow rate of 5 µl/min using a Bialite apparatus and Ca2+-free TBS/Bia as
running buffer. Peptide dilutions were prepared either in running
buffer or in TBS/Bia complemented with 2 mM
CaCl2. In Ca2+-related experiments, the
Ca2+-containing buffer was injected during the dissociation
phase to replace the running buffer (arrow). Data are
expressed as absolute responses. The sensor chip used was the same as
in Fig. 3, B and C.
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Table I
Kinetics of IIb peptide interaction with GST- 3
fusion protein as a function of [Ca2+]
Experiments were performed on a Bialite instrument using a flow rate of
50 µl/min and TBS/Bia buffer ± 2 mM CaCl2
as running and dilution buffer. The association
(kon) and dissociation (koff)
rate constants were generated using the Biaevaluation analysis
software, from data recorded for a range of synthetic IIb
peptide concentrations (4.2-83.3 µM) injected over a
GST- 3 fusion protein-coated surface (~12,500 RU). The
affinity constants (KD) were calculated as
koff/kon from curve-fitting
analysis as described under "Experimental Procedures." The kinetic
data presented are the mean values ± S.D. of at least two
separate experiments.
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In Vitro
/
Heterodimerization Is Impaired by the R995A
Substitution or the KVGFFKR Deletion within the
IIb
Cytoplasmic Tail but Not by the S752P Substitution in the
3 Cytoplasmic Domain--
Several mutations or
deletions within the
IIb and
3
cytoplasmic tails have been shown to disturb
IIb
3-mediated signaling, such as the
IIb (R995A) substitution, the
IIb
membrane-proximal GFFKR truncation, or the
3 (S752P)
point mutation (15, 28, 29). To investigate the influence of these
mutations on the in vitro
·
complexation, a chip
coated with GST-
3 was used. The binding curves obtained
following the injection of equimolar solutions of
IIb
(R995A) or
IIb (Asn996-Gln1008)
peptides were strongly reduced as compared with the curve monitored with wild type
IIb (Fig.
5A). Residual binding
calculated from these curves 20 s after the end of the sample
injection, using wild type
IIb peptide binding as a
100% reference, were only ~15% for the substituted mutant and
~20% for the deleted mutant. In contrast, sensorgrams obtained when
wild type
IIb peptide was injected over
GST-
3 or GST-
3 (S752P) were
superimposable (Fig. 5B). The presence of 2 mM
Ca2+ in the running buffer only slightly improved
IIb mutant binding to GST-
3 (22%
residual binding for both). Calcium stabilized the interaction of the
wild type
IIb peptide with GST-
3 (S752P) in the same way as with GST-
3, and kinetic parameters
determined for the
IIb/GST-
3 (S752P)
interaction in the absence or presence of 2 mM
CaCl2 were essentially the same as those obtained with wild
type
3 (data not shown). These results demonstrate that the
IIb membrane-proximal KVGFFKR sequence and the
IIb residue Arg995 are crucial for the
formation of
·
complexes, as well as their subsequent
stabilization by divalent cations. In contrast, the S752P mutation in
the
3 cytoplasmic tail does not affect
·
dimerization and stabilization, suggesting that the
3
C-terminal part is not involved in these processes.

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Fig. 5.
SPR analysis of the binding properties
of IIb and
3 cytoplasmic tail mutants.
Sensorgrams were monitored on a Biacore X instrument using
Ca2+-free TBS as running and dilution buffer and a flow
rate of 20 µl/min. Data are expressed as relative responses after
subtraction of the background signal recorded on a reference surface
made up of ethanolamine-substituted dextran matrix. A, 100 µM peptide solutions of either wild type
IIb (curve 1), IIb (R995A)
(curve 2), or IIb
(Asn996-Gln1008) (curve 3) were
passed over a chip with immobilized GST- 3 (~7300 RU).
B, 50 µM wild type IIb peptide
solution was injected over a chip with ~2000 RU of antibody-captured
GST- 3 (curve 1) or GST- 3
(S752P) (curve 2).
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Calcium-independent Interaction of CIB with the
IIb
Cytoplasmic Tail--
We have used purified recombinant CIB as a
control reporter protein to monitor
IIb cytoplasmic tail
ligand binding functions. As shown in Fig.
6A, a binding signal was
recorded when the
IIb peptide was passed over
sensorchips with antibody-captured GST-CIB but not with anti-GST
antibodies alone, GST, or ethanolamine-substituted dextran, indicating
that the interaction occurred specifically between
IIb
and CIB. The molar ratio calculated from these curves was ~0.7
mol/mol and was consistent with a 1:1 stoichiometry. Binding
experiments performed in the presence or absence of 2 mM
CaCl2 in the running buffer showed that CaCl2
did not significantly modify either the association or the dissociation
phase (Fig. 6B), demonstrating that the
IIb
interaction with CIB was Ca2+-independent, despite the fact
that CIB is a Ca2+-binding protein (12). The on and off
rates, and the apparent KD, calculated as described
previously from overlaid sensorgrams, are summarized in Table
II. As predicted from the sensorgrams in
Fig. 6B, all the dynamic parameters were essentially the
same, independent of the presence or absence of Ca2+, and
revealed a weak affinity of 12 µM.

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Fig. 6.
Characterization by SPR analysis of the
interaction between IIb
cytoplasmic domain and CIB. The experiments were performed on a
Biacore X instrument using TBS/Bia as running and dilution buffer and a
flow rate of 20 µl/min. Data are given as relative responses.
A, IIb peptide (41.7 µM) was
passed over a chip with immobilized polyclonal anti-GST antibodies
before (curve 2) or after capture of ~1300 RU of GST
(curve 3) or GST-CIB (curve 1). B,
sensorgrams recorded during the interaction of 41.7 µM of
IIb peptide with GST-CIB (~1200 RU) previously
captured by immobilized anti-GST antibodies. The experiments were
performed with TBS/Bia either alone or complemented with 2 mM CaCl2.
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Table II
Kinetics of the interaction of IIb peptide with GST-CIB
fusion protein as a function of [Ca2+]
Sensorgrams were collected for a range of IIb peptide
concentrations (4.2-83.3 µM) interacting with
antibody-captured GST-CIB fusion protein (1200-1400 RU). Measurements
were carried out on a Biacore X instrument at a flow rate of 50 µl/min using TBS/Bia ± 2 mM CaCl2 as
running and dilution buffer. On and off rates (with
KD = koff/kon)
were evaluated using Biaevaluation software as described under
"Experimental Procedures." Kinetic data presented are the mean
values ± S.D. of three separate experiments.
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The
IIb Cytoplasmic Peptide Can Bind Simultaneously
to the
3 Cytoplasmic Tail and to CIB--
We further
examined whether the binding of the
IIb peptide to
antibody-captured GST-CIB was influenced by the
3
cytoplasmic tail. Samples of each of the two isolated peptides were
injected sequentially or simultaneously after an in vitro
incubation in the presence or absence of CaCl2, in order to
allow
·
complex formation. Experiments were performed in a
Ca2+-free running buffer. As expected from previous
results, the
IIb peptide always bound to captured
GST-CIB when injected alone (Fig. 7A). In contrast, the purified
3 peptide did not. Injection of the
IIb
and
3 peptides preincubated in a Ca2+-free
buffer led to a response almost similar to that monitored with the
IIb peptide alone, whereas the
/
mixture
preincubated in the presence of Ca2+ supported an enhanced
resonance signal (Fig. 7B). Comparable results were obtained
when the experiments were carried out with running buffer containing 2 mM CaCl2 (data not shown). Considering that the
SPR response is directly related to the mass of the analyte adsorbed on
the chip surface, we conclude from these results that binding of
preformed
IIb·
3 complexes was
responsible for the difference observed with the reference signal
recorded with the
IIb peptide alone. These data indicate
that
IIb and
3 cytoplasmic tails can form
a multimolecular complex together with CIB, suggesting that the regions
in the
IIb amino acid sequence implicated in the
interaction with
3 and CIB are distinct. Since the
IIb membrane-proximal region has been shown to contain
the key contact sites for
3 binding, CIB interaction is
likely to involve the
IIb C terminus.

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Fig. 7.
Interaction of the
IIb· 3
cytoplasmic tail complex with GST-CIB fusion protein. Experiments
were carried out on a Biacore X apparatus at a flow rate of 20 µl/min. TBS/Bia without CaCl2 was used as running and
dilution buffer. Data are expressed as relative responses. Both
IIb and 3 peptides were injected at a
concentration of 40 µM. Sensorgrams were obtained using
an antibody-captured GST-CIB chip surface (~1700-1800 RU) after
sequential injection (A) of each of the peptides
(curve 1, IIb followed by 3;
curve 2, 3 followed by IIb) or
after a single injection (B) of the two peptides previously
incubated together for at least 15 min at room temperature with
(curve 3) or without 2 mM CaCl2
(curve 4). Sample injections are indicated by
arrows.
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The Membrane-proximal KVGFFKR Domain and the Arg995
Residue of
IIb Cytoplasmic Tail Are Also Required for
Optimal
IIb Binding to CIB--
To delineate further
the contact site of CIB within the
IIb cytoplasmic tail,
we investigated the binding of
IIb (R995A) and
IIb (Asn996-Gln1008) peptides
to GST-CIB. Interestingly, the sensorgrams obtained with both mutants
were markedly reduced, as compared with the binding curve monitored
with the wild type
IIb peptide (Fig. 8), although residual binding of ~30%
persisted for the two mutants independent of the presence or absence of
calcium. These results indicate that CIB binding to
IIb
does not rely exclusively on the C-terminal part of
IIb
and that the KVGFFKR sequence or the Arg995 residue within
IIb are required for optimal binding.

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Fig. 8.
Interaction of the
IIb mutant peptides with GST-CIB fusion
protein. Experiments were carried out on a Biacore X instrument
using TBS/Bia as running and dilution buffer and a flow rate of 20 µl/min. Data are given as relative responses. The sensorgrams were
recorded using 50 µM peptide solutions of either wild
type IIb (curve 1), IIb(R995A)
(curve 2), or IIb
(Asn996-Gln1008) (curve 3) injected
over a chip with antibody-captured GST-CIB (~1600 RU).
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Calcium-independent Binding of CIB to Intact Platelet
IIb
3--
To confirm further the
calcium-independent interaction of CIB with the
IIb
cytoplasmic domain, we performed in vitro liquid phase
binding assays using intact
IIb
3. For
this purpose, an
IIb
3-rich glycoprotein
fraction was prepared from platelets by ConA affinity chromatography.
Similarly, an
v
3-enriched glycoprotein concentrate was prepared from CHO cells expressing human
v
3 for control experiments. SDS-PAGE and
Western blot analysis demonstrated that these samples contained
appreciable levels of
3 integrins (Fig.
9A, lanes 2 and 3).
Platelet or CHO-A06 cell glycoproteins were incubated with GST-CIB or
GST alone in the presence of 2 mM CaCl2 or 2.5 mM EGTA. GST fusion proteins were recovered using glutathione-Sepharose beads, and bound proteins were analyzed by
SDS-PAGE and Western blotting using anti-
IIb,
anti-
v, anti-
3, and anti-GST antibodies.
As shown in Fig. 9B (panel 1), GST-CIB was able
to retain
IIb
3 independent of the
presence or absence of Ca2+, whereas GST alone was not. In
contrast, no
v
3 integrin was detected in
either GST-CIB- or GST-containing samples incubated with the
v
3-enriched fraction (Fig. 9B,
panel 2). In competitive inhibition experiments, an excess of free
IIb cytoplasmic peptide abrogated
IIb
3/GST-CIB coprecipitation (Fig.
9B, panel 3). Taken together, these results demonstrate that
CIB binds specifically to intact platelet
IIb
3 in a Ca2+-independent
manner.

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Fig. 9.
In vitro binding of GST-CIB to
platelet
IIb 3.
A, ConA-purified glycoproteins from either nontransfected
CHO cell lysate (100 µg, lane 1), human
v 3-expressing CHO-A06 cell lysate (100 µg, lane 2), or IIb 3-rich
platelet extract (25 µg, lane 3) were resolved by 7.8%
reducing SDS-PAGE (upper panel) and subjected to Western
blot analysis using specific anti- 3 (4D10G3),
anti- IIb (S1.3), or anti- v (VNR 139) mAbs
(lower panel). Considering the chemiluminescence signal
obtained using an equivalent dilution of anti- 3 mAb and
the same film exposure time, the level of
IIb 3 in the platelet glycoprotein extract
was estimated to be ~4 times higher than that of
v 3 in the CHO-A06 glycoprotein fraction.
The slight difference in the electrophoretic mobility of
IIb and v is not apparent in this
mini-gel. B, ConA-purified glycoproteins from platelet
lysate (250 µg, panel 1) or
v 3-expressing CHO-A06 cell lysate (1 mg,
panel 2), both containing approximately equal amounts of
3 integrins, were incubated with 50 µg of purified GST
or GST-CIB in the presence of 2 mM CaCl2 (+) or
2.5 mM EGTA ( ). Binding inhibition experiments were
performed by adding 25 nmol of IIb cytoplasmic peptide
to the platelet glycoproteins/GST-CIB or GST mixture (panel
3). Proteins were captured onto glutathione-Sepharose beads and
were analyzed by 7.8% reducing SDS-PAGE and Western blot. The membrane
was first probed with anti- 3 (4D10G3) mAbs combined with
either (panels 1 and 3)
anti- IIb (S1.3) or (panel 2)
anti- v (VNR 139) mAbs (upper panel), stripped
and reprobed with anti-GST antibodies (lower panel). The
weak band observed in some GST immunoblot analysis corresponds to an
unidentified protein distinct from GST-CIB, as demonstrated by slightly
different electrophoretic mobilities.
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CIB Binds Preferentially to Mn2+-activated
IIb
3--
Since both inactive and active
IIb
3 conformers are isolated from
platelet lysate by ConA affinity chromatography (30), we determined the
activation state of the
IIb
3 heterodimers present in our ConA-purified platelet extract by examining the binding
capacity of
IIb
3 to immobilized
fibrinogen. Fig. 10A shows
that specific
IIb
3 binding was
significantly weaker with crude sample than with fractions incubated
either with the activating anti-
3 mAb D3GP3 or with 10 mM Mn2+, demonstrating that the ConA-purified
platelet extract contained essentially inactive
IIb
3 and minor amounts of activated
IIb
3.

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Fig. 10.
In vitro binding of
ConA-purified
IIb 3
to immobilized fibrinogen and GST-CIB as a function of
MnCl2. A, various concentrations of crude
ConA-purified platelet glycoproteins were incubated for 4 h at
room temperature with 2 µg/ml D3GP3 mAb ( ), 10 mM
MnCl2 ( ), or buffer alone ( ) in 96-well microtiter
plates coated with 5 µg/ml human fibrinogen. After washing, bound
IIb 3 was detected by ELISA using rabbit
anti- IIb 3 antibodies as stated under
"Experimental Procedures." The figure is representative of two
independent experiments, each data point corresponding to the mean of
duplicates. B, ConA-purified platelet glycoproteins (250 µg) were incubated with 50 µg of purified GST-CIB or GST in the
presence (+) or absence ( ) of 10 mM MnCl2.
Binding inhibition experiment was performed using 25 nmol of
IIb cytoplasmic peptide. Proteins were recovered using
glutathione-Sepharose beads and were analyzed by 7.8% reducing
SDS-PAGE and by Western blot. The membrane was first probed with
anti- 3 (4D10G3) and anti- IIb (S1.3) mAbs
(upper panel), stripped and reprobed with anti-GST
antibodies (lower panel). Two nonspecific bands of high
electrophoretic mobility were observed with the GST-CIB-containing
samples.
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To investigate further whether Mn2+-induced activation of
platelet
IIb
3 was able to influence
IIb
3/CIB interaction, we performed in vitro binding studies in the presence or absence of
MnCl2. Interestingly, the binding of
IIb
3 to GST-CIB was more pronounced in
the presence of 10 mM MnCl2 than in
Mn2+-free buffer (Fig. 10B). Also,
IIb
3 heterodimers were undetectable in
competitive assays using an excess of
IIb cytoplasmic
peptide or in control experiments performed with purified GST alone,
demonstrating that the specificity of the interaction was not modified
by Mn2+. To confirm further these results, we studied the
binding of CIB to platelet
IIb
3 as a
function of Mn2+ by immunoprecipitation experiments. Crude
or Mn2+-treated platelet glycoproteins were first incubated
with purified GST-CIB and then with the
anti-
IIb
3 complex-specific mAb 10E5 to
retain total
IIb
3, or with the
anti-
3 fibrinogen-mimetic mAb PAC-1 to capture activated
IIb
3. The immunoprecipitates were
analyzed by SDS-PAGE and Western blotting using
anti-
IIb, anti-
3, and anti-GST
antibodies. As shown in Fig. 11,
IIb
3 binding to PAC-1 was weak in the
absence of Mn2+ (B, lane 1) and was
significantly enhanced in 10 mM MnCl2 buffer (B, lane 2), demonstrating that the activated
IIb
3 conformers were increased in the
Mn2+-treated platelet glycoprotein concentrate. In
Mn2+-free buffer, few GST-CIB coimmunoprecipitated with
10E5-captured
IIb
3 (A, lane
3), whereas GST-CIB was not detectable with PAC-1-bound
IIb
3, probably on account of the small
recovery in
IIb
3 obtained with this
sample, containing a weak proportion of active heterodimers (B,
lane 3). Conversely, Mn2+ treatment of the platelet
glycoproteins led to a marked increase in GST-CIB coprecipitation in
experiments performed with either 10E5 or PAC-1 mAb (A and
B, lane 4). This effect was particularly apparent from the
level of GST-CIB coprecipitated in 10E5 assays, which was significantly
greater in the presence of 10 mM Mn2+ than in
Mn2+-free buffer, although the amount of captured
IIb
3 remained unchanged (A, lanes 3 and 4). As expected from our previous assays, addition of
IIb cytoplasmic peptides to the incubation mixture almost completely inhibited GST-CIB binding to platelet
IIb
3 (A and B, lane
5), and no GST was coprecipitated with
IIb
3 (A and B, lane
6). Taken together, these results demonstrate that CIB binds
preferentially to the Mn2+-treated, activated form of
IIb
3 integrin. These data further suggest
that CIB is unlikely to have a regulatory effect on
IIb
3 ligand binding function, since its
interaction with
IIb
3 does not stimulate
PAC-1 binding to inactive
IIb
3 nor
inhibit Mn2+-activated
IIb
3
occupancy by PAC-1.

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Fig. 11.
In vitro GST-CIB binding to
platelet
IIb 3
as a function of MnCl2. ConA-purified platelet
glycoproteins (250 µg) were incubated with GST-CIB (50 µg), and
IIb 3 complexes were immunoprecipitated
using either the anti- IIb 3 mAb 10E5
(A) or the fibrinogen-mimetic mAb PAC-1 (B) and
analyzed by SDS-PAGE and Western blot as described in the legend to
Fig. 10. Samples were as follows: platelet glycoproteins in
Mn2+-free buffer (lane 1) or in 10 mM MnCl2 buffer (lane 2); platelet
glycoproteins incubated with GST-CIB in Mn2+-free buffer
(lane 3), 10 mM MnCl2 buffer
(lane 4), 10 mM MnCl2 buffer
containing 25 nmol IIb cytoplasmic peptide (lane
5); platelet glycoproteins incubated with purified GST in 10 mM MnCl2 buffer (lane 6).
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 |
DISCUSSION |
SPR technology has been successfully used by several authors to
analyze the interaction of structural domains of receptor cytoplasmic
tails with their intracellular targets, such as the interaction of
cytoplasmic tail sorting sequences with the adaptor proteins AP-1,
AP-2, and AP-3 (31-33) or of growth factor receptors with the Src
homology 2 (SH2) or the phosphotyrosine-binding (PTB) domains of
tyrosine kinases (34, 35). SPR has also been used to characterize the
interaction of various integrin receptors with extracellular matrix
proteins (36-38), ligand-mimetic antibodies (39, 40), or with integrin
counter-receptors (41). In this report, we have used SPR technology to
study in vitro complexation of the
- and
-cytoplasmic
tails of integrin
IIb
3, as data from
mutagenesis, spectroscopic, and computer modeling studies indicate this
heterodimerization (14-16, 19). SPR provided a direct and more
informative approach to investigate
·
complexation, since
continuous real time monitoring of the interaction allowed a detailed
characterization of specificity, affinity, and kinetics. Our
experimental data are consistent with a 1:1 interaction that proceeds
transiently according to affinity constants (KD) in
the micromolar range. These affinities are low when compared with those
obtained by SPR for receptor cytoplasmic tails (32, 33, 35) or
signaling molecules (42-45) which usually range from 10
7
to 10
9 M. As our kinetic data are close to
the limits specified for the BiacoreTM instruments (46),
they should be considered as an indication of an order of magnitude
rather than definite numerical data. Nevertheless, the affinities found
here are in agreement with transient and potentially easily modulatory
processes, such as those expected intuitively in signal transduction
pathways, and predict that the interactions actually proceed in
vivo if the binding partners are locally concentrated. This is
easily conceivable for integrin
and
cytoplasmic tails which are
maintained in close proximity at the cytoplasmic face of the plasma
membrane through tight interactions of the integrin extracellular domains.
In contrast to previous studies, in which
or
cytoplasmic tail
peptides were linked to an helical coiled-coil motif mimicking the
transmembrane domain (14, 20), the
·
intersubunit binding shown
here occurs in the absence of any additional structural motifs.
Furthermore, no homodimerization of
3 peptides was
observed in our study, although this process has been recently
described in cells transfected with a
3 cytoplasmic tail
connected to an
-helical domain (47), suggesting that transmembrane
domains are necessary for integrin oligomerization but not for
·
cytoplasmic tail complexation. Interestingly, our data
demonstrate that the S752P substitution in
3 did not
affect
·
heterodimerization, indicating that the C-terminal
part of the
3 cytoplasmic tail is not involved in this
process. As the
3(S752P) mutation has previously been
shown to disrupt bidirectional signaling in platelets and transfected
CHO cells (28, 29), our results suggest that this defect cannot be
attributed to an alteration in
·
cytoplasmic tail interaction
but rather to a disruption in a specific
3 interaction with intracellular regulatory molecules. Actually, the
3
(S752P) mutation has been shown to markedly reduce
3-endonexin-specific binding to
3 and to
impair its modulating effect on the
IIb
3 affinity state (8, 48). Our data further indicate that the
IIb membrane-proximal region is critical for
·
heterodimer assembly, as both
IIb (R995A) and
IIb (Asn996-Gln1008) peptides
failed to interact efficiently with the
3 cytoplasmic tail. Our data apparently contradict previous biophysical data which
demonstrated that the interactive sites involved in
·
dimerization are located within the
3
Ile721-Asp740 and
IIb
Pro999-Gln1008 sequences (16). However, it
should be noted that their data were primarily derived from terbium
luminescence spectroscopy experiments which did not allow investigation
of an interaction between the N-terminal regions of the
IIb and
3 cytoplasmic tails, since
neither
IIb Leu985-Pro998 nor
3 were able to bind Tb3+ ions necessary for
the generation of a specific fluorescence energy transfer signal. In
contrast, our data are in good agreement with previous mutagenesis
studies (15) and also indicate that the
IIb KVGFFKR motif is
necessary but not sufficient to support
·
heterodimerization.
Indeed, alterations within this sequence did not totally abolish the
IIb·
3 interaction, suggesting that binding sites distinct from the
IIb membrane-proximal
domain are involved in
3 engagement. These additional
contact sites are probably located within the
IIb
C-terminal acidic tail.
We have also provided direct evidence that Ca2+ and
Mg2+ are not required for
IIb·
3 complexation but rather stabilize
the heterodimeric structure by reducing the dissociation rate. Since
·
association proceeded with the same extent, independent of
the presence of cations, the cation coordination sites within
IIb and
3 cytoplasmic tails are likely to
be distinct from those involved in intersubunit binding, namely the
membrane-proximal region of each integrin subunit. Interestingly, our
study indicates that the
IIb membrane-proximal region is
involved in divalent cation-induced
·
complex stabilization, since an alteration in the KVGFFKR sequence impaired the stability of
the heterodimers. In support of these results, a functional cation
binding domain has previously been mapped to the negatively charged
acidic C-terminal region of
IIb (residues 999-1008) and was found to bind divalent cations in coordination with sites located
in the
IIb 985-998 sequence (16, 19). Based on the structural model of
IIb
3, Haas and Plow
(19) speculated that a cation coordination site rearrangement could
occur upon
IIb·
3 cytoplasmic tail
complexation. The faster complex dissociation observed here in the
absence of cations actually suggests that additional intra- and/or
intersubunit cation coordination site(s) probably appear(s) during the
complexation resulting in a more stable structure.
Because CIB is the only intracellular protein identified so far that
specifically interacts with the
IIb integrin subunit cytoplasmic tail, we used recombinant CIB as a reporter protein to
monitor the ligand binding capacity of
IIb either as a
monomer or as a heterodimer in association with
3. Our
data clearly demonstrate that CIB interacts with the
IIb
peptide in a one-to-one, weak affinity reaction (KD = 12 µM) and also binds to the preformed
IIb·
3 cytoplasmic complex, suggesting
that the contact sites within the
IIb amino acid
sequence involved in the interaction with CIB are distinct from those
engaged in
3 binding. As CIB has been shown to interact
only with
IIb and not with
v,
2, or
5 in the yeast two-hybrid system,
Naik et al. (12) concluded that CIB was unlikely to bind to
the highly conserved GFFKR motif common to all
subunits but rather
to the highly acidic C-terminal part of the
IIb
cytoplasmic tail. Our data, however, provide evidence that the KVGFFKR
sequence is necessary for optimal CIB·
IIb interaction,
since both
IIb (R995A) and
IIb
(Asn996-Gln1008) peptides failed to interact
efficiently with CIB. In the three-dimensional model of
IIb
3, the negatively charged C terminus
of the
IIb cytoplasmic tail is predicted to fold back
onto itself and to interact with the positively charged N terminus
through several side chain and backbone contacts (19). Thus, it is
conceivable that the deletion of the KVGFFKR sequence or the R995A
substitution disrupts some of the intrasubunit contacts that stabilize
the
IIb conformation, leading to an alteration in the
capacity of the cytoplasmic tail peptide to bind to CIB. Interestingly,
although CIB has two conserved EF-hand motifs within its protein
structure, and has been found to bind Ca2+ in
vitro (12), our data demonstrate a Ca2+-independent
binding of CIB to the
IIb cytoplasmic peptide. This finding indicates that the potential
Ca2+-dependent regulatory function of CIB,
based on its sequence homology with calmodulin and calcineurin B (12),
is not involved in the association with
IIb but rather
in the activity or in the binding to an as yet unidentified
CIB-associated protein. Most strikingly, when binding studies were
performed with intact
IIb
3, CIB bound preferentially to Mn2+-activated
IIb
3, suggesting that the accessibility
of the CIB-binding site within
IIb is increased in
active
IIb
3 conformers. Since Mn2+ did not affect the binding characteristics of the
GST-CIB·
IIb cytoplasmic peptide complex in SPR studies
(data not shown), our results suggest that Mn2+ activation
of intact
IIb
3 induces a conformational
change that is transmitted from the
IIb
3
extracellular domains to the cytoplasmic tails. Such long range
transmembrane structural alterations have been anticipated from studies
showing that specific epitopes are exposed or disappear within the
cytoplasmic tails of
IIb and
3 subunits
following
IIb
3 activation or ligand
occupancy (49, 50). Alternatively, an increase in
IIb
3 avidity for CIB cannot be excluded,
since the fibrinogen-mimetic mAb PAC-1 used to capture active
IIb
3 is a multimeric IgM antibody and is
thus likely to trigger oligomerization of
IIb
3 complexes that mimic integrin clustering (51). Finally, our data provide evidence that CIB is
unlikely to have a regulatory effect on
IIb
3 ligand binding function, since its
interaction with
IIb does not trigger ligand binding to
inactive
IIb
3 nor inhibit activated
IIb
3 occupancy by a ligand, suggesting
that CIB is most likely involved in
IIb
3 post-receptor occupancy events.