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INTRODUCTION |
Interaction of adhesive proteins with transmembrane integrin
adhesion receptors is essential for diverse biological processes including embryogenesis, angiogenesis, immune response, and hemostasis (1, 2). It is generally agreed that inside-out signaling processes
regulate the affinity state of integrins for binding extracellular
ligands (2, 3). Thus, upon activation of blood platelets by
physiological stimuli (e.g. thrombin or ADP) at sites of
vascular injury, the prototype integrin
IIb
3 undergoes a conformational change
from an inactive to an active state competent to bind soluble
fibrinogen (1-5). The cytoplasmic domain of
IIb
3 appears to play a regulatory role in
IIb
3 activation, since truncations of the
entire cytoplasmic sequence of either the
IIb or
3 subunit, including their membrane-proximal regions,
were found to increase the ligand binding affinity of the mutant
receptor expressed on Chinese hamster ovary
(CHO)1 cells (6, 7). More
recently, a potential salt bridge hinge formed between the
IIb and
3 cytoplasmic sequences has been suggested to maintain
IIb
3 in a default
low affinity state; disruption of this structure may result in receptor
activation (8).
It is well established that binding of adhesive ligands to integrins
initiates outside-in signaling processes that mediate post-ligand
binding events including cytoskeleton reorganization, receptor
clustering, and gene transcription (2, 3, 9). Although the mechanisms
regulating outside-in signaling of integrins remain elusive, the
binding of cytoskeletal proteins and signaling molecules to the
receptor's cytoplasmic domain as well as the receptor's
conformational state have been implicated to play an important role in
this process. In this regard, it has been suggested that ligand
occupancy of the
5
1 integrin may induce a
transmembrane conformational change of the receptor, thereby unmasking
specific regions in the receptor cytoplasmic domain mediating
cytoskeletal attachment, which ultimately leads to receptor
localization to focal contacts (10). However, to date, ligand-induced
transmembrane conformational changes of an integrin receptor have not
been demonstrated.
It has previously been shown that ligand binding to
IIb
3 induces further conformational
changes of the receptor extracellular domain, resulting in the exposure
of neoantigenic sites termed ligand-induced binding sites (LIBS), which
are recognized by anti-LIBS monoclonal antibodies (mAbs) (11-16).
Furthermore, certain anti-LIBS mAbs were found to activate
IIb
3 to bind soluble fibrinogen (14, 15).
In this study, we postulated that bidirectional conformational changes
of
IIb
3 transducing through the
receptor's transmembrane segment occur as a result of cellular
activation and ligand binding. To test this possibility, we examined
whether extracellular ligand binding induces the exposure of LIBS
epitope(s) in the cytoplasmic domain of
IIb
3. In addition, we evaluated the
functional role of a putative cytoplasmic LIBS epitope in regulating
IIb
3 ligand binding affinity.
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MATERIALS AND METHODS |
Peptides and Antibodies--
Peptides, represented as sequences
of single letter amino acid codes (17), were synthesized by solid-phase
synthesis using an ABI model 431 peptide synthesizer or were purchased
from Research Genetics, Inc. (Huntsville, AL). The amino acid
composition of each peptide was consistent with its desired sequence.
The anti-
IIb
3 antibodies PMI-1 (18),
anti-V41 (19), anti-LIBS1 (13), and mAb 15 (13) were from Dr. M. H. Ginsberg, and AP-2 (20) was from Dr. T. J. Kunicki of the
Scripps Research Institute (La Jolla, CA). Anti-
IIbC, an
antipeptide polyclonal antibody raised in rabbits against the
IIb cytoplasmic sequence
Phe992-Glu1008, was a generous gift of Dr. X. Du of the University of Illinois (Chicago, IL).
Production of Anti-LIBScyt1--
For the production
of antipeptide mAbs against the
IIb cytoplasmic
sequence, the full-length P2b peptide (CKVGFFKRNRPPLEEDDEEGE) was
coupled to keyhole limpet hemocyanin using
m-maleimidobenzoic acid N-hydroxysuccinimide
ester and used as immunogen for BALB/c mice. Isolated splenocytes were
fused with P3-X63Ag8.653 myeloma cells. Hybridomas were grown in
selective media (hypoxanthine/aminopterin/thymidine), and their
supernatants were tested in an ELISA for the presence of antibodies
reactive with RGD affinity-purified
IIb
3
(13, 21). A positive hybridoma 3F5, which secreted
anti-
IIb antibodies belonging to the IgG1
subclass, was subcloned twice at limiting dilutions of 0.5 cell/well.
The antibody was produced as ascites and purified by chromatography on
protein A-Sepharose CL-4B (Amersham Pharmacia Biotech).
Immunoprecipitation--
Gel-filtered platelets were
surface-labeled with Na125I and solubilized in lysis buffer
containing 50 mM octyl glucoside (21). Cell lysates were
incubated with GRGDSP, GRGESP, or vehicle buffer for 30 min at
37 °C. Antibodies were then added and incubated overnight at
4 °C. The immunoprecipitated proteins were collected on protein
G-Sepharose, electrophoresed on SDS-7% polyacrylamide gels under
nonreducing conditions, and analyzed by autoradiography.
Indirect Immunofluorescent Microscopy--
Washed human
platelets resuspended in Tyrode's solution (2.5 × 108 cells/ml) were incubated with the indicated reagents
(see legend of Fig. 2) at 37 °C for 30 min and subsequently fixed
with 1% paraformaldehyde on ice for 1 h. After blocking unreacted
aldehyde with Tris-buffered saline (30 mM Tris, 120 mM NaCl, pH 7.4) containing 0.5 M
NH4Cl, cells were allowed to settle onto polylysine-coated glass coverslips and incubated with 0.2 mg/ml lysophosphatidylcholine (LPC) for 5 min to render them permeable. Permeabilized cells were
rinsed with Tris-buffered saline containing 0.1% bovine serum albumin
and incubated with anti-LIBScyt1 followed by
rhodamine-conjugated goat anti-mouse IgG. Samples were mounted with a
droplet of FITC guard, and platelets were viewed with a Jenaval
phase/fluorescence microscope (Jenoptik Jena GmbH) and photographed
with Eastman Kodak Tri-X panchromatic film (22).
Competitive ELISA--
Microtiter wells were coated with the
full-length P2b peptide (5 µg/well) and blocked with 3% bovine serum
albumin. Anti-LIBScyt1 was incubated with 10 µM inhibitory peptides at 37 °C for 30 min and added
to the P2b-coated wells. Antibody binding to the adsorbed P2b proceeded
at 37 °C for 1 h. The wells were washed, and bound antibody was
detected with horseradish peroxidase-conjugated goat anti-mouse IgG
using o-phenylenediamine as substrate (12). Absorbance at
490 nm (A490) was measured, and percentage
inhibition was calculated relative to control without inhibitor.
Site-directed Mutagenesis--
The expression constructs
encoding wild type
IIb (CD2b) and
3
(pc3A) have been previously described (23, 24). To generate the pc2b
construct encoding wild type
IIb, a 3.3-kilobase
fragment of
IIb containing the entire coding sequence
and the 3'-untranslated region was excised from CD2b by digestion with
XbaI and ligated into the expression vector pcDNA3. The
resultant construct was designated as pc2b. Both pc2b and pc3A were
kindly provided by Dr. J. C. Loftus at the Mayo Clinic
(Scottsdale, AZ). The P998A/P999A mutation in
IIb was
generated by splice overlap extension mutagenesis (25). Overlapping
fragments containing this mutation were first made by polymerase chain
reaction amplifications on pc2b using the following
oligonucleotide pairs: (a)
5'-CACAAGCGGGATCGCAGACAGATCTTCCTGCCAGA-3' and (b)
5'-CTTCTTCCAGGGCTGCCCGGTTCCGCTTG-3'; (c)
5'-CAAGCGGAACCGGGCAGCCCTGGAAGAAG-3' and (d)
5'-GGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAA-3' as primers. The
overlapping fragments were combined, denatured by heating at 94 °C
for 5 min, and reannealed by cooling to 55 °C. The ends were filled
in with Pfu, and the double-stranded fragments were then
amplified by polymerase chain reaction using the oligonucleotide pair
a and d. The amplified product was digested with
BamHI and XbaI and reinserted into a
BamHI-XbaI-digested pc2b vector fragment. The
mutant construct was identified by automated DNA sequencing, purified
by chromatography on QIAGEN Tip-100, and co-transfected with the wild
type
3 construct (pc3A) into CHO-K1 cells (ATCC, Rockville, MD) by liposome-mediated transfection as described (7).
Surface expression of mutated
IIb
3 was
analyzed by flow cytometry using FITC-conjugated AP-2. Stable cell
lines were selected in medium containing 0.75 mg/ml G418 (Sigma), and
single cell sorting was performed to obtain stable clonal lines, which
were high expressors of the mutant
IIb
3.
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RESULTS AND DISCUSSION |
Ligand Binding Induces A Transmembrane Conformational Change of
IIb
3--
To examine the possibility
that ligand binding induces the exposure of LIBS epitopes in the
cytoplasmic domain of
IIb
3, we developed
anti-peptide mAbs against the receptor's cytoplasmic sequences and
screened for antibodies that preferentially bind to the ligand-occupied
conformer of
IIb
3. In the present study, we focused on the
IIb cytoplasmic tail. Initially, mAbs
reactive with RGD affinity-purified
IIb
3
in an ELISA system were further characterized by immunoblotting and
immunoprecipitation studies. The mAb obtained from clone 3F5 recognizes
a ligand-induced binding site in the cytoplasmic domain of
IIb
3 (LIBScyt), and therefore this mAb is designated as anti-LIBScyt1. Fig.
1A shows that
anti-LIBScyt1 specifically immunoblotted the 140-kDa
nonreduced
IIb subunit in RGD affinity-purified
IIb
3 (lane 1) and
in a detergent extract of platelet proteins (lane
2). Upon reduction of purified
IIb
3 and proteins in the platelet lysate,
anti-LIBScyt1 immunoblotted the 27-kDa light chain of
IIb, which contains its cytoplasmic sequence
(lanes 3 and 4). To determine whether
the interaction of anti-LIBScyt1 with nondenatured
IIb
3 is dependent on ligand occupancy, we
performed immunoprecipitation experiments using lyates of
surface-radioiodinated platelets in the presence and absence of an RGD
peptide. As judged by densitometric scanning of the immunoprecipitated
125I-labeled protein bands, incubation of platelet lysates
with GRGDSP caused a 7.2-fold increase in the amount of
IIb
3 immunoprecipitated by
anti-LIBScyt1 (Fig. 1B). In contrast, the
variant GRGESP peptide was much less effective (1.5-fold). As controls,
we used the well characterized anti-LIBS1 mAb (13), which demonstrated
a similar effect in RGD-dependent immunoprecipitation of
IIb
3. However, using the control mAb 15, whose binding to
IIb
3 is not markedly influenced by ligand occupancy (13), we observed that GRGDSP incubation
induced only a slight (1.5-fold) increase in the immunoprecipitation of
IIb
3. Thus, these results suggest that
interaction of GRGDSP with the extracellular ligand binding site of
IIb
3 induces a conformational change in
the receptor's cytoplasmic domain. In support of our finding that the
cytoplasmic domain of
IIb
3 can exist in
different conformational states, the cytoplasmic sequences of
IIb and
3 have been shown to interact
with each other, and at least two docking models with different
tertiary structures of the
IIb
3
cytoplasmic domain have been proposed (26-28).

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Fig. 1.
Immunoblot and immunoprecipitation
characterization of anti-LIBScyt1. A, RGD
affinity-purified IIb 3 (lanes
1 and 3) and octyl glucoside extracts of platelet
proteins (lanes 2 and 4) were resolved
by SDS-polyacrylamide gel electrophoresis, transferred onto
nitrocellulose membranes, and probed with anti-LIBScyt1
followed by detection with 125I-labeled goat anti-mouse
IgG. Positions of molecular mass markers in kDa are indicated on the
right. B, lysates of surface-radioiodinated
platelets were incubated with vehicle buffer (lane
1), 1 mM GRGESP (lane 2),
or 1 mM GRGDSP (lane 3) for 30 min at
37 °C. Immunoprecipitation with the indicated antibodies proceeded
overnight at 4 °C. The immunoprecipitated proteins were
electrophoresed on SDS-7% polyacrylamide gels under nonreducing
conditions and analyzed by autoradiography.
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To examine whether RGD occupancy also induces transmembrane
conformational changes of
IIb
3 in
situ, we performed indirect immunofluorescence microscopy using
whole platelets preincubated with or without GRGDSP followed by
paraformaldehyde fixation and LPC permeabilization to allow antibody
access. As shown in Fig. 2A,
incubation of platelets with GRGDSP (panel a)
resulted in significant intracellular staining of
anti-LIBScyt1 as opposed to control platelets incubated
with vehicle buffer (panel b) or GRGESP
(panel c). Furthermore, the observed rim staining
pattern with GRGDSP-treated permeabilized platelets is suggestive of
anti-LIBScyt1 localization to the inner face of the plasma
membrane, since minimal staining was observed with nonpermeabilized
cells (not shown). To investigate whether binding of the physiological
ligand fibrinogen to
IIb
3 on activated
platelets also induces the exposure of LIBScyt1, we
performed indirect immunofluorescence studies with ADP-stimulated
platelets in the presence and absence of exogenous fibrinogen. Again,
anti-LIBScyt1 staining was performed following fixation and
cell permeabilization. The addition of fibrinogen to ADP-stimulated
platelets dramatically increased anti-LIBScyt1 staining as
compared with activation of platelets with ADP alone (Fig.
2B, panels a and b). In
control samples, resting platelets failed to stain for
anti-LIBScyt1 in the presence and absence of fibrinogen
(Fig. 2B, panels c and d).
Therefore, these results demonstrate that anti-LIBScyt1
recognizes the ligand-occupied but not the activated unoccupied
conformer of
IIb
3. By immunogold staining
with AP6, an anti-LIBS mAb directed against the
3
extracellular domain, Nurden et al. (16) previously reported
that a pool of
IIb
3 in the
-granules
of unactivated platelets exists in the ligand-occupied state. However,
using anti-LIBScyt1, we failed to detect immunofluorescent
staining of
IIb
3 in the
-granules of
resting platelets. This may be due to the association of
IIb
3 with cytoskeletal components that
mediate internalization and transport of the
fibrinogen-
IIb
3 complex to the platelet
-granules (22, 29, 30), thus blocking interaction of
anti-LIBScyt1 with the cytoplasmic domain of
ligand-occupied
IIb
3 in the
-granule membranes. Nonetheless, the observation that fibrinogen binding to
IIb
3 on the platelet surface induces a
transmembrane conformational change of the receptor provides a possible
mechanism by which ligand occupancy of
IIb
3 mediates a variety of post-ligand
binding function of blood platelets including clot retraction, receptor internalization, and cytoskeletal attachment.

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Fig. 2.
Indirect immunofluorescent microscopy studies
to detect the exposure of the anti-LIBScyt1 epitope in
ligand-occupied
IIb 3
of permeabilized platelets. A, platelets were treated
with 1 mM GRGDSP, 1 mM GRGESP, or vehicle
buffer prior to fixation and permeabilization with LPC. B,
resting or activated platelets (10 µM ADP) were incubated
with or without fibrinogen (3 µM), fixed, and
permeabilized with LPC (original magnification, × 400).
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To identify specific residues within the
IIb cytoplasmic
sequence mediating interaction with anti-LIBScyt1, we
performed competitive ELISA analyses using peptides corresponding to
the full-length or partial sequences of the
IIb
cytoplasmic tail. As shown in Table I,
the full-length P2b peptide, as well as the truncated 15-mer
(KVGFFKRNRPPLEED) effectively blocked anti-LIBScyt1 binding
to immobilized P2b peptide. Moreover, using two overlapping peptides,
we further localized the anti-LIBScyt1 epitope to the KRNRPPLEED sequence. Molecular modeling suggests that this region in
both
IIb and
v subunits would form a
tight
-turn (28, 31). Since Pro998-Pro999
may facilitate this
-turn formation, we tested the ability of KRNRAALEED to inhibit anti-LIBScyt1 binding. The inhibitory
effect of the peptide was found to be significantly diminished by
substitution of the two proline residues with alanine, indicating that
anti-LIBScyt1 recognizes a structural motif dependent on
these two proline residues.
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Table I
Competitive ELISA to localize the anti-LIBScyt1 epitope within
the IIb cytoplasmic sequence
Results of A490 values are means ± S.D. of
triplicate determinations.
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A Site-directed Mutation of the Anti-LIBScyt1 Epitope
Activates
IIb
3 to Bind Extracellular
Ligands--
The ability of certain anti-LIBS mAbs to activate
IIb
3 (14, 15) suggests that these LIBS
epitopes may regulate the ligand binding affinity state of the
receptor. Therefore, we evaluated the functional significance of
Pro998-Pro999 in the regulation of
extracellular ligand binding to
IIb
3. In
these studies, a double P998A/P999A mutation in
IIb was
generated by splice overlap extension mutagenesis (25), and the mutant
IIb construct was co-transfected with a wild type
3 construct into CHO cells. A stable clonal cell line
(G4) expressing the mutant
IIb
3 was
established, and comparative analyses were performed with the control
A5 cell line expressing wild type
IIb
3
(32). As determined by flow cytometry using FITC-conjugated AP-2, a complex-specific anti-
IIb
3 mAb (20), both
G4 and A5 cells expressed similar amounts of
IIb
3 (mean fluorescence intensity: G4,
73.9; A5, 74.9). It has previously been shown that truncation of the
entire
IIb cytoplasmic sequence at residue 991 resulted in an increase of the ligand binding affinity of
IIb
3 (6); therefore, we examined whether
the P998A/P999A mutation might result in proteolytic cleavage of the
IIb cytoplasmic domain, which would lead to receptor
activation. Initially, we compared the molecular mass of the
P998A/P999A mutant
IIb light chain with those of wild
type and truncated
IIb by immunoblotting with anti-V41,
an antipeptide antibody directed against the amino terminus of the
IIb light chain (19). As shown in Fig.
3A, the light chain of the
P998A/P999A mutant migrated with a similar molecular mass as the
inactive wild type
IIb. In contrast, the constitutively active
991 truncation mutant of
IIb (6) migrated with
an increased mobility on SDS-polyacrylamide gel electrophoresis. Furthermore, Fig. 3B shows that both wild type and the
P998A/P999A mutant
IIb reacted with PMI-1, a mAb
directed against an extracellular epitope in the
IIb
heavy chain (18, 33), and with anti-
IIbC, an antipeptide
polyclonal antibody raised against the
IIb cytoplasmic sequence (Phe992-Glu1008). Collectively, these
results indicated that the P998A/P999A mutation did not result in
proteolytic cleavages of the
IIb cytoplasmic tail. As
expected, anti-LIBScyt1 immunoblotted the wild type but not
the mutant
IIb.

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Fig. 3.
Immunoblot characterization of the
P998A/P999A mutant IIb.
Lysates of CHO cells expressing 3 integrins complexed
with wild type, P998A/P999A mutated, or 991 truncated
IIb were subjected to immunoblotting with the indicated
antibodies. A, proteins were resolved on SDS-18%
polyacrylamide gel under reducing conditions and immunoblotted with
anti-V41, an antipeptide antibody directed against the amino terminus
of the IIb light chain. B, proteins were
resolved on SDS-7% polyacrylamide gels under nonreducing conditions
and immunoblotted with PMI-1 directed against an extracellular epitope
of IIb, anti- IIbC directed against the
cytoplasmic sequence of IIb
(Phe992-Glu1008), or
anti-LIBScyt1.
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The affinity state of the mutant
IIb
3 was
then examined by the binding of FITC-conjugated PAC-1, an
activation-specific mAb that, like fibrinogen, preferentially binds to
activated
IIb
3 (34). As reported
previously (32), A5 cells expressing wild type
IIb
3 bound minimal amounts of PAC-1 in
the absence of receptor activation (Fig.
4A). In contrast, we observed
constitutive binding of PAC-1 to the mutant
IIb
3 on G4 cells, and this process was specifically blocked with 1 mM GRGDSP (Fig. 4A).
Since the binding of fibrinogen to activated
IIb
3 on platelets and transfected cells
resulted in cell aggregation, we examined the ability of G4 cells to
aggregate in the presence of fibrinogen. Fig. 4B shows that
G4 but not A5 cells aggregated upon fibrinogen addition. Again,
aggregation of G4 cells was specifically blocked with 1 mM
GRGDSP. As a specificity control, we mutated the putative
N744PLY
-turn motif in the
3 cytoplasmic
sequence to QALY. Cells expressing wild type
IIb and
mutated
3 heterodimers failed to bind soluble fibrinogen
and undergo aggregation (not shown). These results indicate that a
structural change in the anti-LIBScyt1 binding site in the
IIb cytoplasmic tail induces a transmembrane conformational change of
IIb
3, mimicking
receptor activation due to inside-out signaling.

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Fig. 4.
Constitutive ligand binding function of G4
cells bearing the
IIb(P998A/P999A) 3
mutant receptor. G4 and A5 cells were harvested with trypsin and
EDTA, washed, and resuspended in Tyrode's buffer. A, the
binding of FITC-PAC-1 (15 nM) to G4 and A5 cells in the
presence and absence of GRGDSP (1 mM) was assayed by flow
cytometry as described by Wencel-Drake et al. (22).
B, spontaneous aggregation of G4 cells in the presence of
fibrinogen. Washed G4 and A5 cells (107/ml) were incubated
with or without 3 µM fibrinogen in 24-well tissue culture
plates and subjected to gyrorotation. In some samples, GRGDSP (1 mM) was added 30 min prior to the addition of
fibrinogen.
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It has previously been shown that ligand binding to integrin
IIb
3 induces long range conformational
changes in the extracellular domains of both
IIb and
3 subunits (12, 35). Our results demonstrated that such
conformational changes transduce through the cell membrane to the
cytoplasmic domain of the receptor. Besides
IIb
3, several other integrins such as
v
3 and
5
1
have been shown to undergo extracellular conformational changes upon
ligand occupancy (13, 36); therefore, it is tempting to speculate that
ligand-induced conformational changes also occur in the cytoplasmic domains of other integrins. Since ligand binding to integrins results
in cytoskeletal rearrangement and the generation of intracellular signals (2, 3, 9), the conformational state of integrin cytoplasmic
domains may play a regulatory role in the assembly of cytoskeletal
proteins and/or signaling molecules. In this regard, it has been shown
that antibody-induced clustering of the
5
1 integrin in the absence of ligand
occupancy is sufficient for the intracellular accumulations of tensin
and at least 20 signal transduction molecules (e.g. RhoA,
Rac1, Ras, Raf, MEK, extracellular signal-regulated kinase, c-Jun
N-terminal kinase, and focal adhesion kinase) (37, 38). In contrast,
both ligand occupancy and clustering of
5
1 are required for transmembrane
accumulations of several cytoskeletal proteins (e.g. talin,
vinculin, and
-actinin). In light of these findings, our present
data suggest that ligand-induced conformational changes of integrin
cytoplasmic domains may play an essential role in the intracellular
assembly of cytoskeletal proteins found in focal adhesions.
Emerging evidence has implicated ligand-induced oligomerization and/or
conformational changes of transmembrane receptor complexes as potential
mechanisms for receptor-mediated signal transduction. Specifically, it
has been demonstrated that following ligand binding and dimerization of
the platelet-derived growth factor receptor, there is a
phosphorylation-dependent conformational change in the receptor
cytoplasmic domain (39-41). Although integrin
IIb
3 on platelets becomes
tyrosine-phosphorylated as a result of ligand binding and cell
aggregation, the monovalent RGD peptide has been shown to block
receptor phosphorylation (42). Inasmuch as GRGDSP binding to
IIb
3 is capable of inducing the exposure
of the anti-LIBScyt1 epitope, receptor phosphorylation is
apparently not required for the observed effect. Thus, our results
provide the first evidence of a direct effect of ligand occupancy on
the conformation of the cytoplasmic domain of an intact integrin
receptor. Additionally, site-directed mutation of the identified
LIBScyt1 epitope resulted in an increase of ligand binding
affinity of
IIb
3, indicating that the
extracellular ligand-binding site and the cytoplasmic LIBS epitope of
the receptor are functionally coupled. In sum, these findings suggest a
bidirectional modulation of
IIb
3
conformations across the cell membrane. Such conformational regulation
may provide a novel mechanism for transmembrane receptor-mediated
signal transduction.