From the Department of Vascular Biology, The Scripps
Research Institute, La Jolla, California 92037 and the
¶ Department of Biochemistry, University of Oxford, South Parks
Road, Oxford, OX1 3QU, United Kingdom
Received for publication, March 2, 2001, and in revised form, April 11, 2001
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ABSTRACT |
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Previous evidence suggests that interactions
between integrin cytoplasmic domains regulate integrin activation. We
have constructed and validated recombinant structural mimics of the
heterodimeric The integrin family of adhesion receptors is essential for the
development and functioning of multicellular animals (1). Integrin-mediated adhesion is rapidly and precisely regulated, a
process that is often central to integrin functions (2). One important
regulatory mechanism is cellular modulation of integrin affinity for
ligand (activation). These affinity changes arise from both changes in
the conformation of the extracellular domain and lateral clustering of
integrins in the plane of the membrane (3). Integrin activation has
been widely documented among members of this receptor family and
controls cell adhesion, migration, and the assembly of the
extracellular matrix (4, 5). Thus, integrin activation plays an
important role in mediating integrin functions.
Integrins are noncovalent heterodimers of type I transmembrane protein
subunits termed In addition to a role for the membrane-distal portion of the We previously used a synthetic strategy to produce a model of the
cytoplasmic domain of integrin In the present work, we utilized these model proteins as a tool to
probe the potential role of integrin Model Protein Synthesis and Peptide Production--
Polymerase
chain reaction as described (12) was used to create a cDNA
encoding the modified GCN4 heptad repeat protein sequences reported by
John et al. (25). The cDNAs were ligated into a NdeI-HindIII-restricted modified pET15b vector
(12) (Novagen).
Synthetic peptides were prepared by the Scripps Microchemistry Core
using a Gilson AMS 422 or ABI 430A Peptide Synthesizers. One
Antibodies--
Antibodies to the
Enzyme-linked Immunosorbent
Assays--
Immunoprecipitations--
The generation of Chinese hamster
ovary cells expressing recombinant integrins
NMR Spectroscopy--
The Fibrinogen Binding Assays--
Fibrinogen binding to platelets
was performed as described previously (30). Briefly, 100 nM
125I-labeled fibrinogen and the indicated
concentration of peptide inhibitor were preincubated for 30 min at
22 °C with a suspension of washed platelets (2 × 108/ml) in Tyrode's solution containing added 10 mM Hepes, 2 mg/ml bovine serum albumin, 2 mg/ml glucose,
and 2 mM CaCl2. 20 µM ADP and 20 µM epinephrine were added to this suspension, and the
resulting mixture was incubated for an additional 30 min. Bound
125I-labeled fibrinogen was separated from free by
centrifugation through a 20% sucrose cushion and was quantified by Construction and Characterization of Recombinant Heterodimer
Integrin Cytoplasmic Domain Model Proteins--
To test potential
interactions between the
A dimer was formed in which the Jun-like helix was fused to the
Immunochemical Characterization of the
JG3
Having established that antibodies raised against
JG3
To exclude potential solubility artifacts, we examined the binding of
an antibody against a linear The Interaction of
The foregoing result suggested that the A Heptapeptide Sequence in the Cell Permeable Peptides Containing the Previous evidence suggests that interactions between integrin
cytoplasmic domains regulate integrin activation. In the present work,
we have constructed and validated recombinant structural mimics of the
heterodimeric Antibodies raised against the The presence of combinatorial epitopes in intact
The interaction of When the The results presented here and in previous literature suggest a
unifying hypothesis for the activation of integrins. The receptor is
proposed to be restrained in a resting state through an interaction involving the membrane-proximal region of the IIb
3 cytoplasmic
domain. The mimics elicited polyclonal antibodies that recognize a
combinatorial epitope(s) formed in mixtures of the
IIb
and
3 cytoplasmic domains but not present in either isolated tail. This epitope(s) is present within intact
IIb
3, indicating that interaction between
the tails can occur in the native integrin. Furthermore, the
combinatorial epitope(s) is also formed by introducing the
activation-blocking
3(Y747A) mutation into the
3 tail. A membrane-distal heptapeptide sequence in the
IIb tail (997RPPLEED) is responsible for
this effect on
3. Membrane-permeant palmitoylated
peptides, containing this
IIb sequence, specifically blocked
IIb
3 activation in platelets.
Thus, this region of the
IIb tail causes the
3 tail to resemble that of
3(Y747A) and suppresses activation of the integrin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
. Each subunit has a large (>700 residues)
N-terminal extracellular domain, a single membrane-spanning domain, and
a generally short (13-70 residues) cytoplasmic domain (5). In
general, integrin
and
subunits contain a remarkably conserved
7-10-residue motif near the junction of the transmembrane and
cytoplasmic domains (membrane-proximal segment) (6). The remainders of
the cytoplasmic domain sequences are generally less well conserved.
Integrin
cytoplasmic domains are required for activation because
mutations or truncations of specific membrane-distal sequences in
cytoplasmic tails can disrupt activation (7-10). A particularly
sensitive site is a highly conserved NPX(Y/F) motif in the
cytoplasmic domain where substitution of the Tyr with Ala
(e.g.
3(Y747A)) blocks activation (11). This
region of the
tail is important for binding to cytoskeletal
proteins, such as talin because a Tyr
Ala substitution in the
NPX(Y/F) motif also disrupts talin binding to
tails
in vitro (12, 13). Moreover, overexpression of an
integrin-binding fragment of talin in cells activates integrin
IIb
3 (14). Thus, interactions of membrane
distal portions of the
cytoplasmic domain with proteins such as
talin appear to be important in integrin activation.
cytoplasmic domain, interactions between integrin
and
subunit cytoplasmic tails may regulate activation. Deletion of the
membrane-proximal region of either
or
tail activates integrins
(7, 10, 15-17). In addition, specific point mutations in this segment
of both the
and
subunits promote constitutive bidirectional
signaling in integrin
IIb
3 (18) and other
integrins (19). Complementary mutations in the
and
subunits
suggest that these activating mutations disrupt an interaction between
the highly conserved membrane-proximal portions of the
and
cytoplasmic tails (18). In vitro integrin
and
cytoplasmic domain interactions have been reported by surface plasmon
resonance analysis (20, 21) and by alterations in circular dichroism
and intrinsic fluorescence (22). Furthermore, replacement of the
and
cytoplasmic domains with acidic and basic peptides that form an
-helical coiled-coil caused inactivation of integrin
L
2 (23). In contrast, replacement of
these cytoplasmic domains with two basic peptides that do not form an
-helical coiled-coil activated
L
2.
Thus, there is evidence to suggest that an interaction between integrin
and
tails regulates activation.
IIb
3 (24).
The integrin cytoplasmic domains are tethered at their N termini to
membrane spanning presumptive
-helices. Moreover, they are laterally
constrained because the subunits interact with each other. More
importantly, they have vertical constraints, because they are initially
parallel to each other and are in a specific vertical stagger as they
exit the membrane. The model protein was made by the covalent ligation of two synthetic polypeptides (called "minisubunits") consisting of
an integrin cytoplasmic tail at the C terminus, connected to a
28-residue stretch comprised of four heptad repeats derived from
tropomyosin. The parallel orientation and stagger of the two tails is
defined by the formation of the covalent linkage and by a noncovalent
helical coiled-coil structure between heptad repeats on each
minisubunit. We now describe a completely recombinant model of the
IIb
3 cytoplasmic domain. By using
recombinant proteins, we avoided limitations of polypeptide length and
modest yield encountered in the initial synthetic approaches. Moreover,
we modified the design of the model protein to contain four heptad repeats derived from the GCN4 transcription factor in place of those
derived from tropomyosin. We used two variant sequences that
preferentially heterodimerize (25) to obviate an inherent 50% loss in
yield of heterodimer.
tail interaction in
regulating activation. Antibodies raised against these model proteins
revealed the presence of combinatorial epitopes formed by a mixture of
the
IIb and
3 tails but not present in
either isolated tail. These combinatorial epitopes were present in
intact integrin
IIb
3 isolated from
platelets, suggesting that the
IIb and
3
tail can interact in the intact receptor. Moreover, the same set of
epitopes was present in the
3 tail bearing a
Tyr747
Ala mutation, even in the absence of the
IIb tail. This result indicates that
IIb
cytoplasmic domain causes the
3 tail to resemble the
activation-defective
3(Y747A) mutant. Furthermore, we
mapped the
IIb residues required for this interaction
into a minimal heptapeptide sequence and found that palmitoylated
peptides containing this sequence specifically blocked the
agonist-induced activation of integrin
IIb
3 in platelets. Thus, our data provide
evidence that the
IIb and
3 tails can
interact in the native integrin and indicate that the interaction
opposes integrin activation by altering the structure of the
3 cytoplasmic domain.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
IIb and
3 integrin
cytoplasmic domain cDNAs were generated by polymerase chain
reaction from appropriate cDNAs using forward oligonucleotides
introducing a 5'-HindIII site and reverse oligonucleotide creating a 3'-BamHI site directly after the stop codon.
Integrin cytoplasmic domains were joined to the helix as
HindIII-BamHI fragments and verified by
sequencing. Recombinant expression in BL21(DE3)pLysS cells (Novagen)
and purification of the recombinant products was performed as described
with an additional final purification step on a reverse phase
HPLC1 column (Vydac) (12).
Typical yields were ~10 mg/liter of bacterial culture. To form
heterodimers, a mixture of 45 µM
3 and 90 µM
IIb cytoplasmic tail model proteins
were denatured for 20 min at 100 °C in the presence of 10 mM dithiothreitol. The dithiothreitol was removed by gel
filtration through a PD 10 column (Bio-Rad), and the mixture was then
air-oxidized by stirring overnight. The disulfide-bonded products were
purified by reverse phase C18 HPLC. The products were analyzed by
electrospray ionization mass spectrometry using an API-III quadrupole
spectrometer (Sciex). Typically we recovered ~30 mg of
heterodimer/preparation. The identity of each model protein is
specified by the nature of the GCN4 helix (Jun-like = J,
Fos-like = F), the number of Gly spacers (G0-G4), and the identity of the integrin tail (
IIb and
3), e.g. JG3
IIb.
3 cytoplasmic domain peptide
(
3(Ile719-Thr762)) was a
generous gift of Drs. Ed Plow and Tom Haas (Cleveland Clinic). The
peptides were routinely >90% homogeneous as judged by reverse phase
HPLC using an analytical Vydac C-18 column. Their mass varied by less
than 0.1% from that predicted from their sequence as judged by
electrospray ionization mass spectroscopy. Peptides were desalted prior
to use, and peptide concentrations were verified by quantitative amino
acid analysis.
IIb
3 cytoplasmic domain heterodimer model
protein (see Fig. 1) (anti-
IIb
3-Cyt) were
raised in 2.5-kg New Zealand White rabbits. Antibodies were also
produced against the
3(Y747A) model protein
(anti-
3(Y747A)). 100 µl of a water solution containing
1 mg/ml of the model protein was emulsified in 1 ml of incomplete
Freund's adjuvant and administered subcutaneously to the rabbits. Two
additional injections at 2-week intervals were followed by bleeding
(~50mls) at monthly intervals until the rabbits were sacrificed. The
blood was permitted to clot at room temperature, and the serum was
recovered by centrifugation. Serum was heat-inactivated at 56 °C for
30 min and stored at
20 °C. A preimmune serum, obtained prior to
immunization, was used as a control. Antibodies against a synthetic
peptide containing the last 20 residues of the
3
cytoplasmic domain (anti-
3-Cyt, rabbit 8275; Ref. 15)
and the extracellular domain of
IIb
3 (D57; Ref. 26) have been described.
IIb
3 was purified by gel
filtration as described previously (26) with omission of the heparin
and Con A affinity chromatography steps. The final product was greater
than 95% homogenous as judged by SDS-PAGE. In the enzyme-linked
immunosorbent assay (ELISA) the
IIb
3 was
used at a concentration of 5 µg/ml in a coating buffer containing 0.1 M NaHCO3 and 0.05% NaN3. 50 µl/well was used to coat Immulon II microtiter wells at 4 °C
overnight. After removal of the coating solution, 150 µl of blocking
buffer (coating buffer containing 5% bovine serum albumin) was added.
After an additional 1-h incubation at 4 °C, the blocking buffer was
removed, and the plates were washed three times with wash buffer (0.01 M Tris, 0.15 M NaCl, 0.01% thimerisol, 0.05%
Tween 20, pH 8.0). Preliminary titration experiments established a
dilution of anti-model protein antibody resulting in 75% maximal
binding. 25 µl of the competitor was added to each well followed by
25 µl of this dilution of the anti-model protein antibody. Following
mixing, the plate was covered for 1 h at 37 °C and washed four
times with wash buffer. To quantify bound antibody, 50 µl of
horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) was
diluted to a concentration of 1:1000 in wash buffer containing 1 mg/ml
bovine serum albumin. These plates were then incubated at 37 °C for
1.5 h. After four washes, bound antibody was assayed by measuring
peroxidase activity with O-phenylenediamine as a substrate
and quantifying reaction product by its optical density at 490 nm. The
data were expressed as B/B0 where
B = A490 in the presence of
competitor and B0 = A490
in its absence. In some experiments, varying concentrations of
IIb peptides were added to a fixed, saturating quantity
of the
3 model protein (10-50 nM).
Competition was again expressed as
B/B0; however,
B0 was A490 in the
presence of the
3 protein and no added
IIb peptide. EC50 was defined as the dose of
IIb resulting in B/B0 = 0.5.
IIb
3 and
IIb
996
3
717 has been described (10).
Chinese hamster ovary cell lines were cell surface-labeled with
sulfo-NHS-Biotin (Pierce) following the manufacturer's instructions. Cells were lysed on ice for 30 min in an immunoprecipitation buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 mM benzamidine HCl, 0.02% sodium
azide, 1% Triton X-100, 0.05% Tween 20, 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin). After clarification by centrifuging at 12,000 rpm for 20 min at 4 °C, cell lysate was then incubated with protein G-Sepharose
(Amersham Pharmacia Biotech) coated with antibody overnight at 4 °C.
The beads were washed with the immunoprecipitation buffer four times,
and the precipitated polypeptides were extracted with SDS sample
buffer. Precipitated cell surface biotin-labeled polypeptides were
separated by SDS-PAGE under nonreducing conditions and detected with
streptavidin-peroxidase followed by ECL (Amersham Pharmacia
Biotech).
3 minisubunit of the
IIb
3 heterodimer model protein was
biosynthetically labeled with 15N as
described.2 NMR signal
intensities were taken from a two-dimensional
15N-1H heteronuclear single-quantum
coherence (28, 29) spectrum of 1.2 mM heterodimer
model protein in 10 mM acetic acid-d3, pH 4.5, 37 °C on a home-built spectrometer operating at a 1H
frequency of 750 MHz. Backbone assignment has been described previously, and 3JHN
coupling
constants for the tails have been reported previously2 and
were determined now for the coiled-coil region from the same data set
as described previously.
scintillation spectrometry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
IIb and
3
cytoplasmic tails, we first produced model proteins designed to mimic their normal arrangement in integrin
IIb
3. Recombinant
-helical coiled-coils were previously used to model dimerized integrin
cytoplasmic domains (12); this strategy was employed to produce heterodimeric integrin cytoplasmic domains. Heptad repeats of identical
length derived from GCN4 were used to form the nearly symmetrical,
parallel coiled-coil. To favor heterodimer formation, Lys residues were
placed in the in the g1 and e2 positions of the first and second heptad
repeats to make a "Jun-like" helix (25). A complementary
"Fos-like" helix was prepared by introducing Glu residues in these
positions. These complementary helices produce preferentially
heterodimerizing parallel coiled-coils (25). We also introduced a
unique Cys residue N-terminal of each helix so that the formation of a
cystine bridge would ensure parallel orientation and correct
stagger of the coiled-coil. (Fig.
1A).
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Fig. 1.
Structure and properties of
IIb
3
cytoplasmic domain model protein. A, strategy for
cytoplasmic domain mimics. Depicted is the amino acid sequence of the
recombinant minisubunit. An N-terminal polyhisitidine
(His-Tag) is used to immobilize and purify the construct. A
thrombin cleavage site permits release of the His tag, and a unique
cysteine is used to form covalent parallel dimers in a defined vertical
stagger. Heptad repeats, from GCN4, nucleate the formation of a
parallel dimeric coiled-coil. The presence of Lys residues at the g1
and e2 positions of the heptad repeats (bold type) render
the helix Jun-like. Glu substitutions at this position result in a
Fos-like helix that preferentially forms heterodimers with the Jun-like
helix (25). Cytoplasmic domains, such as that of integrin
IIb depicted here, were joined to the coiled-coil. Three
Gly residues were inserted between the coiled-coil and the integrin
tail to disrupt induced helical structure in the cytoplasmic domain
(12). At the bottom of the panel is a list of the
integrin-specific sequences of all recombinant constructs used in this
study. All integrin peptides correspond to published human integrin
sequences in Swissprot. To preserve a HindIII site, a Val
residue in the cytoplasmic domain of the human integrin
IIb chain was replaced by Leu. The membrane-promixal
conserved segment of the
IIb and
3
cytoplasmic domains are underlined and in bold
type. B, schematic of the
JG3
IIb-FG3
3 model protein. The
heterodimeric model protein was formed from a JG3
IIb and
FG3
3 minisubunit. The hexahistidine tag was removed from
the
IIb subunit to facilitate its separation from the
heterodimer during purification. C, ion spray mass spectrum
of an
IIb
3 model protein. The predicted
mass based on the expected covalent structure was 19008.2 Da (assuming
no 13C) and the measured mass was 19013 + 3.1 Da.
D, SDS-PAGE analysis of
JG3
IIb-FG3
3 model protein. Purified
JG3
IIb-FG3
3 was separated on a 4-20%
SDS-PAGE in the presence (R) or absence (NR) of
10 mM dithiothreitol. The positions of the
JG3
IIb (J-
IIb) and
FG3
3 (F-
3) minisubunits are
indicated. E, verification of the coiled-coil. NMR signal
intensities from a two-dimensional 15N-1H
heteronuclear single-quantum coherence spectrum at 37 °C and
750 MHz of 1.2 mM JG3
IIb-FG3
3
in which the FG3
3 minisubunit is
15N-labeled. The heterodimeric
JG3
IIb-FG3
3 was dissolved in 10 mM acetic acid-d3, pH 4.5. The GCN4 coiled-coil
element exhibits uniform intensity, whereas the
3 tail
exhibits more intense signals with intensities increasing toward the C
terminus. This behavior and the low
3JHN
coupling constants of the
coiled-coil (<5.5 Hz) are indicative of the coiled-coil being well
folded in a helical conformation.
IIb cytoplasmic domain and a Fos-like helix was fused to the
3 cytoplasmic domain (Fig. 1B)
(JG3
IIb-FG3
3) model protein. A three-Gly
spacer was inserted between the helices and the integrin
or
cytoplasmic domains to prevent the propagation of helical structure
into the integrin tails (12).2 The mass of the heterodimer
differed by less than 0.1% from that predicted for the desired
covalent structure (Fig. 1C), and SDS-PAGE confirmed that
disulfide bonds covalently linked the heterodimers (Fig.
1D). Furthermore, NMR analysis confirmed formation of the symmetrical, parallel coiled-coil domain by the heptad repeats and that
the helical structure of this domain did not propagate into the
3 tail as will be discussed in full detail
elsewhere.2 In particular, the coiled-coil behaved as a
rigid, folded unit, whereas the motions of the
3 tail
residues appear largely uncoupled from each other as judged from NMR
signal intensities of the FG3
3 subunit of
JG3
IIb-FG3
3 (Fig. 1E). Because
of the symmetry of the coiled-coil all properties of the Fos-like
heptad-repeats can be inferred to be present the Jun-like heptad
repeats. The value of the 3JHN
coupling constant reflects the backbone conformation (ranging from
helical to extended conformations) at the residue in question (31). For
the Fos-like heptad repeats, values below 5.5 Hz show distinctly
helical (and folded) conformations. Thus, the recombinant
JG3
IIb-FG3
3 model protein had the
expected properties.
IIb-FG3
3 Protein--
As noted above,
there is substantial mutational and in vitro data to suggest
that integrin
IIb and
3 cytoplasmic
domains interact with each other. An immunochemical approach was used to determine whether such interactions could occur within the intact
receptor. Antibodies were raised against
JG3
IIb-FG3
3 protein
(anti-
IIb
3-Cyt). As expected, those
antibodies reacted with native
IIb
3
as judged by immunoprecipitation of the intact receptor. Furthermore,
the capacity of these antibodies to immunoprecipitate the receptor
depended on the presence of the cytoplasmic domains, because deletion
of both cytoplasmic domains abolished reactivity (Fig.
2A).
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Fig. 2.
Immunochemical analysis of the
anti- IIb
3
model protein antibody. A, the antibody reacts with the
cytoplasmic domains of native integrin
IIb
3. Surface
biotinylated Chinese hamster ovary cells expressing recombinant
integrin
IIb
3
(
IIb
3) or
IIb
3 lacking its cytoplasmic domains
(
IIb(
996)
3(
717))
were lysed in immunoprecipitation buffer. The lysates were
immunoprecipitated with anti-model protein antibody
(
IIb
3-Cyt), an antibody (rabbit
8275) directed against the cytoplasmic domain of
3
(
3-Cyt), or D57, an
anti-
IIb
3 monoclonal antibody
(
IIb
3). Lysates were also
precipitated with normal rabbit serum (NRS). The
immunoprecipitates were fractionated by SDS-PAGE, transferred to
nitrocellulose filters, and developed with avidin peroxidase.
B, rationale for immunochemical analysis. On the
left are depicted linear integrin cytoplasmic tails, and on
the right are depicted the tails in their assumed
relationship in the intact receptor. The classes of epitopes expected
are those present in the linear peptide and the intact receptor
(1). If the cytoplasmic tails of the receptor interact, then
additional classes of potential epitopes are those manifest in the
linear peptide but lost in the native receptor (2) and
combinatorial epitopes formed in the native receptor but absent from
the linear peptides (3). Note that epitope classes 1 and 2 could be present on either the
or
subunit cytoplasmic domain
peptides. C, immunochemical analysis of the anti-model
protein antibody. A competitive ELISA was used to measure the binding
of the polyclonal anti-model protein antibody
(anti-
IIb
3-Cyt) to purified platelet
IIb
3. The results are expressed as
B/B0, where B = A490 in the presence of competitor and
B0 = A490 in the absence
of competitor. The data depict competition with full-length
IIb cytoplasmic domain peptide
(
IIb),
3(Ile719-Thr762) cytoplasmic
domain peptide (
3), or an equimolar mixture of
the
IIb and
3 peptides
(
IIb+
3). Depicted are the means
of triplicate determinations. The curves represent best fits
of the data to B/B0 = 1
(E*C/(K + C)), where
E is the fraction of antibodies that can bind the
competitor, C is the concentration of the competitor, and
K is the average apparent dissociation constant for the
binding of the antibodies to the competitor. For the
IIb
peptide E = 0.15 ± 0.01, for
3
E = 0.63 ± 0.007, and for
IIb +
3 E = 1.00 ± 0.01. D,
immunochemical analysis of the anti-
3 cytoplasmic domain
antibody. ELISA was used to measure binding of an antibody directed
against a linear
3 cytoplasmic domain peptide
(anti-
3-Cyt, rabbit 8275) to purified platelet
IIb
3. The data depict competition with
full-length
IIb cytoplasmic domain peptide
(
IIb) or
3(Thr720-Thr762) cytoplasmic
domain peptide (
3).
IIb-FG3
3 reacted with the cytoplasmic
domain of the native receptor, we sought to analyze potential
interactions between the tails. We reasoned (Fig. 2B) that
if the
and
cytoplasmic domains interact with each other, then
this interaction could create novel, combinatorial epitopes. To detect
potential combinatorial epitopes, we assayed the effect of linear
peptides comprising the
IIb or
3
cytoplasmic domains on the binding of
anti-
IIb
3-Cyt to integrin
IIb
3 purified from human platelets. A
full-length
IIb cytoplasmic domain peptide produced negligible
inhibition (Fig. 2C). The
3 cytoplasmic
domain peptide competed to a maximum of 63 ± 0.7%. Thus,
antibodies reactive with intact integrin
IIb
3 were resistant to competition by
either of the linear peptides (Fig. 2C). These residual
antibodies, however, could be competed by a mixture of the
IIb and
3 peptides (Fig. 2C).
Consequently, these antibodies recognize epitopes expressed in the
native
IIb
3 and formed by the interaction
of the
IIb and
3 cytoplasmic domains.
3 cytoplasmic domain
peptide to the native
IIb
3 integrin (Fig.
2D). In contrast to the result with
anti-
IIb
3-Cyt, the
3
peptide could completely inhibit antibody binding. Furthermore, an
IIb peptide completely inhibited the binding of an
anti-
IIb cytoplasmic domain to
IIb
3 (data not shown). These
combinatorial antibodies were produced by only two of the four New
Zealand White rabbits immunized with
JG3
IIb-FG3
3. They may represent an
unusual specificity because immunization of several strains of inbred
mice (e.g. Balb C, C57Bl 6) failed to elicit such antibodies
(data not shown). Thus, these immunochemical results indicate that the
IIb and
3 cytoplasmic domains can form a
combinatorial epitope(s) in the intact integrin.
IIb and
3
Cytoplasmic Domains Alters the Antigenicity of the
3
Cytoplasmic Domain--
In the presence of the
IIb tail
there is reduced targeting of
3-containing integrins to
cytoskeletal structures and a suppression of bidirectional signaling
(15, 32, 33). Similar effects result from changing a Tyr in the first
NPXY motif of
3 to an Ala
(
3(Y747A)); Refs. 11, 34, and 35). These functional similarities prompted us to test the capacity of the
3(Y747A) mutant to compete for the set of antibodies
recognizing combinatorial epitopes in the cytoplasmic domain of
IIb
3. The
3(Y747A) mutant competed nearly completely for the binding of
anti-
IIb
3-Cyt to integrin
IIb
3 (Fig.
3A). Furthermore, addition of
the
IIb cytoplasmic domain peptide to the
3(Y747A) produced little increase in competition. In
contrast, as noted previously, a population of antibodies was resistant
to inhibition by the wild type
3 cytoplasmic domain.
Thus, a point mutation in the
3 tail that disrupts its
signaling function and its capacity to interact with the cytoskeleton
causes the
3 tail to exhibit combinatorial epitopes formed in the presence of the
IIb tail.
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Fig. 3.
The IIb
cytoplasmic domain alters the antigenicity of the
3 cytoplasmic domain.
A,
3(Y747A) binds combinatorial antibodies in
anti-
IIb
3-Cyt. A competitive ELISA was
used to measure the binding of the polyclonal anti-model protein
antibody (anti-
IIb
3-Cyt) to purified
platelet
IIb
3, and the data were analyzed
as described in the legend to Fig. 2. The graphs depict
competition with the FG3
3 minisubunit
(
3) or FG3
3(Y747A). Also
depicted is competition with an equimolar mixture of the
FG3
3(Y747A) and full-length
IIb
cytoplasmic domain peptide
(
3(Y747A)+
IIb). Depicted are the
means of triplicate determinations. B,
anti-
3(Y747A) recognizes combinatorial determinants
formed by the interaction of
IIb and
3
tails. A competitive ELISA was used to measure the binding of the
polyclonal antibody against
3(Y747A) to purified
platelet
IIb
3. The graphs depict
competition with FG3
3 cytoplasmic domain
(
3) or FG3
3(Y747A). Also
depicted is competition with an equimolar mixture of the
FG3
3 protein and full-length
IIb cytoplasmic domain
synthetic peptide (
IIb+
3).
Depicted are the means of triplicate determinations.
IIb cytoplasmic
domain causes the
3 tail to resemble the
3(Y747A) mutant. To test this idea, we raised polyclonal
antibodies against the FG3
3(Y747A) model protein and
tested the reactivity of those antibodies with native
IIb
3. The capacity of those antibodies to
bind
IIb
3 was completely inhibited by the
immunogen, FG3
3(Y747A) (Fig. 3B). No
significant increase in competition was observed in the presence of the
IIb cytoplasmic domain peptide (data not shown). In
sharp contrast, the wild type FG3
3 failed to compete
completely (Fib. 3B). The addition of an
IIb cytoplasmic
domain peptide to FG3
3 resulted in complete competition.
Thus, the presence of the
IIb cytoplasmic domain caused
the antigenicity of the
3 cytoplasmic domain to resemble
that of
3(Y747A).
IIb Cytoplasmic
Domain Is Responsible for Its Effect on the
3
Cytoplasmic Domain--
The foregoing studies suggested that the
IIb cytoplasmic domain could change the antigenicity of
the
3 tail. We next sought to evaluate the specificity
of the
IIb cytoplasmic domain effect. The addition of a
synthetic peptide containing the
IIb cytoplasmic domain
sequence to 10 nM FG3
3 protein resulted in
dose-dependent formation of combinatorial epitopes
(EC50 = 15 nM) (Fig.
4A). In sharp contrast, the
cytoplasmic domains of integrin
4 or
5 had no such effect, even though they share a highly conserved N-terminal seven residues with that of the
IIb
cytoplasmic domain (6). We next analyzed a series of nested deletion
mutants from the N and C termini of
IIb in this assay.
To increase sensitivity, these experiments were conducted in the
presence of 50 nM FG3
3. Deletion of the
C-terminal 4 amino acids had no effect on this interaction; however,
deletion of 2 additional residues of
IIb completely
abolished activity (Fig. 4B). A series of N-terminal deletions were performed. The first 7 amino acids were dispensable for
this activity as expected from the results with the
4
and
5 cytoplasmic domains. However, deletion of 3 additional amino acids abolished activity (Fig. 4B).
Consequently, progressively smaller peptides were produced and a
minimal sequence spanning Arg997-Asp1003 had
residual activity. Furthermore, double Ala substitutions in this
sequence at Pro998 and Pro999 blocked activity.
To further confirm our identification of this as the critical site, we
synthesized a loop out peptide deleting both Arg997 and
Pro998
(
IIb(
Lys994-Pro998)) and that
loop out peptide was devoid of activity (Fig. 4B). Thus, the
capacity of the
IIb cytoplasmic domain to alter the antigenicity of the
3 tail exhibits structural
specificity and can be mapped to a minimal heptapeptide sequence.
View larger version (13K):
[in a new window]
Fig. 4.
Mapping the sites in the
IIb tail that interact with the
3 cytoplasmic domain.
A,
IIb peptide dose response. A competitive
ELISA was used to measure the binding of the polyclonal anti-model
protein antibody (anti-
IIb
3-Cyt) to
purified platelet
IIb
3 in the presence of
10 nM FG3
3 model protein. Varying quantities
of full-length
IIb cytoplasmic domain peptide
(
IIb(Lys989-Gln1008)) were
added prior to the addition of the antibody. The results are expressed
as B/B0, where B = A490 in the presence of
IIb
peptide and B0 = A490 in
the absence of
IIb peptide. The results are the means of
triplicate determinations. Note that
B/B0 for
4 and
5 peptides are superimposed. B, mapping the
interactive site. A competitive ELISA was used to measure the binding
of the polyclonal anti-JG3
IIb-FG3
3
(anti-
IIb
3-Cyt) to purified platelet
IIb
3 in the presence of 50 nM
FG3
3 model protein and varying doses of the indicated
synthetic
IIb peptides. Depicted in the column to the
right are the concentrations of
IIb peptides
that produced 50% maximal response (EC50). The sequence of
full length
(
IIb(Lys989-Gln1008)) is shown,
and the boundaries of N- and C-terminally truncated
IIb peptides are
depicted as bars to the left of the
EC50. Note that the minimal active peptide was
997RPPLEED. Furthermore, Ala substitution of
Pro998 and Pro999 and the loop out of
Lys994-Pro998 in abolished activity are
shown.
3-interactive
Site of the
IIb Tail Block Activation of
IIb
3--
The foregoing experiments
identified a short peptide sequence of the
IIb tail that
alters that of
3, mimicking the immunochemical effects
of a Tyr to Ala substitution in the first NPXY motif. The
3(Y747A) mutation blocks the activation of integrin
IIb
3 in vivo (11).
Consequently, we tested the effects of addition of a peptide containing
the
IIb sequence on activation of integrin
IIb
3 in platelets. The peptide was
palmitoylated, a modification that promotes entry of peptides into the
platelet cytoplasm (36). Activation of
IIb
3 was suppressed by a palmitoylated
decapeptide (KRNRPPLEED) that contained the minimal heptapeptide
sequence (Fig. 5A). Nearly
100% inhibition was observed at a peptide concentration of 100 µM. Inhibition was specific, because a scrambled peptide of the same composition was nearly devoid of activity. Furthermore, a
nonpalmitoylated peptide containing the same sequence caused little
inhibition (Fig. 5B). Thus, a palmitoylated
IIb peptide that contains the
3-interactive site inhibits activation of integrin
IIb
3 in platelets.
View larger version (11K):
[in a new window]
Fig. 5.
Palmitoylated
IIb peptides block activation of
platelet integrin
IIb
3.
A, specific inhibition by
3 interacting
peptide. A suspension of washed platelets (3 × 108/ml) was incubated at 22 °C in the presence of the
indicated concentration of palmitoylated
IIb(Lys994-Asp1003)
(P-KRNRPPLEED) or a palmitoylated scrambled peptide of the same
composition (P-NPDKRLEREP) and 300 nM
125I-labeled fibrinogen. Fibrinogen binding was measured 15 min after addition of 10 µM ADP + 20 µM
epinephrine, and the data are expressed as percentages of
inhibition = 100*(B0
B)/B0 where B is
fibrinogen binding in the presence of inhibitor and
B0 is fibrinogen binding in its absence. The
average B0 value was 7320 molecules/platelet.
Depicted are the means ± S.E. of three independent experiments.
B, palmitoylation is required for inhibition. The capacity
of palmitoylated
IIb(Lys994-Asp1003)
(Palmitoylated) or
IIb(Lys994-Asp1003)
(Unmodified) synthetic peptides to inhibit fibrinogen
binding were assayed as described for A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
IIb
3 cytoplasmic domains.
These mimics elicited polyclonal antibodies that recognize a
combinatorial epitope(s) in the presence of a mixture of the
IIb and
3 cytoplasmic domains but not
present in either isolated tail. The presence of this epitope(s) within
intact
IIb
3 indicates that interaction between the tails can occur in the native integrin. Furthermore, the
combinatorial epitope(s) is present in the activation-defective
3(Y747A) mutant. Thus, the
IIb tail
causes the
3 tail to resemble that of
3(Y747A), suggesting that the interaction of the
IIb
3 tails opposes activation of the
integrin. Furthermore, mapping of the site in the
IIb
tail responsible for this activity identified a membrane-distal region
previously implicated in regulation of integrin activation. Finally,
palmitoylated peptides containing the
3-interactive
IIb sequence specifically blocked
IIb
3
activation in platelets. Thus, these studies provide direct evidence
for an interaction of the
IIb and
3
cytoplasmic domains that regulates activation of the integrin in platelets.
IIb
3 model
protein contained a population of antibodies that recognize a
combinatorial epitope formed in the presence of the
IIb
and
3 tails. This polyclonal antibody reacted with the
cytoplasmic domain of native
IIb
3 (Fig.
2). However, its binding to
IIb
3 was only
partially competed by either the isolated
IIb tail or
the
3 tail. The remaining population of antibodies was
inhibited only in the presence of both cytoplasmic tails. These
experiments were conducted at 25-75% saturating antibody
concentrations, and similar results were observed at each
concentration. They were not due to insolubility of the tails, because
the isolated
3 cytoplasmic domain or
IIb
cytoplasmic domains could efficiently compete for the binding of
antibodies specifically directed only against the isolated cytoplasmic
domains. Furthermore, antibodies raised against the
FG3
3(Y747A) protein, which had no reactivity with the
IIb tail, also contained antibodies against these
combinatorial determinants. Thus, immunization with the
JG3
IIb-FG3
3 model protein can elicit the
formation of antibodies against combinatorial epitopes in the
IIb
3 cytoplasmic domain.
IIb
3 suggests that the cytoplasmic
domains can interact in the native receptor. The combinatorial
antibodies react with native
IIb
3 in a
manner dependent on its cytoplasmic domains. However, the
immunochemical assay requires the presence of the antibody to detect
interactions between the
IIb and
3 tails.
Indeed, in detailed NMR analysis, we found that the unpaired
3 tail exhibits essentially the same structural and
dynamic properties as the
IIb-paired
3
tail within the context of the coiled-coil
(JG3
IIb-FG3
3).2 Furthermore,
constructs with different lengths of the glycine linker resulting in
relative vertical tail-tail shifts of
2 to +2 residues did not
uncover any such structural differences. Thus, it is possible that the
antibody is a necessary co-factor that enhanced the interaction. Within
cells, such co-factors, like the antibody, could modify the strength of
the interaction. One such co-factor might be a plasma membrane. Indeed,
Vinogradova et al. (37) reported that when the
IIb tail was myristoylated and inserted into a
phospholipid bilayer, it formed a stable structure. This is in sharp
contrast to the unstructured nature of the
IIb peptide
in aqueous solution (22, 24).2 Furthermore, Leisner
et al. (38) produced an anti-
IIb tail antibody (anti-LIBScyt1) that manifested reduced binding to intact integrin
IIb
3. One interpretation of this
result is that the availability of the epitope for this monoclonal
antibody was influenced by interaction with the
3 tail.
The
IIb sequence implicated here in forming the
combinatorial epitope (RPPLEED) is contained in the region recognized
by the anti-LIBScyt1 antibody (38). Taken together, these results
suggest that interactions between the integrin
and
cytoplasmic
domains occur within cells.
IIb and
3 tails alter
the antigenicity of the
3 tail. In particular, we found
that introducing a mutation that blocks integrin activation into the
3 tail resulted in expression of the combinatorial
epitope(s). The
3 tail is largely unfolded in aqueous
solution; however, the
3 Tyr747 is part of
an NPXY motif that has a propensity to form
turns. This
propensity is abolished by the Y747A mutation.2 The Y747A
mutation also abolishes talin binding. The region of talin that binds
to integrin
tails contains a FERM domain (14, 39), a domain
that contains a lobe structurally similar to PTB domains (40).
NPXY motifs, such as that present at
3
Asn744-Tyr747, are often favored binding sites
of PTB domains (41). Thus, binding of talin (or other FERM or PTB
domain-containing proteins) may stabilize a reverse turn at the
NPXY motif in integrin
tails leading to integrin
activation. The presence of the
IIb tail may oppose
formation of this stable turn, thus antagonizing integrin activation.
The
turn propensity at Asn744-Tyr747 is
preserved in the JG3
IIb-FG3
3 model
protein.2 However, the
turn is only present in a small
population of
3 molecules at any time. Consequently, the
IIb tail might oppose stabilization of the turn in the
presence of talin but not appreciably reduce the propensity to form a
turn in the absence of talin. Thus, the
IIb cytoplasmic
domain alters the antigenicity of the
3 tail, causing it
to resemble the structurally and functionally altered
3(Y747A) mutant.
3-interactive region of the
IIb
tail was palmitoylated, it suppressed activation of
IIb
3 in platelets. Platelets are not
readily susceptible to genetic manipulation; however, previous work
from Fitzgerald's laboratory (36) has established that palmitoylated
peptides enter platelets and can regulate activation of
IIb
3. Utilizing their approach, we found
that the identified
3 interactive region of
IIb, when palmitoylated, suppressed
IIb
3 activation in platelets. Specificity
was established by the markedly reduced activity in either a
nonpalmitoylated peptide, presumably because the latter fails to enter
platelets. In addition, a palmitoylated-scrambled peptide also
exhibited greatly reduced activity. Because both the authentic and
scrambled peptides bore the same lipid modification and were of
identical composition, similar distributions within the platelet seem
likely. However, caution is warranted, because definitive proof of a
completely identical subcellular localization for these two peptides is
technically challenging. Nevertheless, a potential explanation of these
results is that the palmitoylated
IIb peptide interacted
with the
3 tail within the cell interior, rendering it
less capable of supporting platelet activation. Such a mechanism would
also account for the observation that a full-length myristoylated
IIb peptide inhibited
IIb
3
activation in platelets (37). In the same work, an
IIb tail peptide inhibited
IIb
3 activation
in vitro; however, it failed to inhibit the activation of a
receptor lacking its
3 cytoplasmic domain. Additional
evidence for the view that the
IIb tail suppresses
activation by interacting with the
3 tail comes from the
present finding that the
IIb(P998A,P999A) mutation blocked formation of the combinatorial epitope. This same mutation abolishes inhibition of activation by the myristoylated
IIb peptide (37) and activates intact recombinant
IIb
3(38). Thus, taken together, these
data strongly suggest that the interaction of this region of
IIb cytoplasmic domain with the
3 tail
opposes activation of
IIb
3 in platelets.
and
cytoplasmic domains (6, 10, 23). The removal of either of these regions results in
a constitutive activated receptor, and that activation is independent
of other interactions of the
cytoplasmic domains or cellular energy
(7, 15, 42). Furthermore, introduction of the lipidated
membrane-proximal sequence of the
IIb tail (KVGFFKR) into platelets results in
IIb
3
activation, presumably by disrupting this membrane-proximal interaction
(36). Thus, the membrane-proximal interaction forms a structural on-off
switch for the receptor. However, normal activation mechanisms require
the distal region of the
3 cytoplasmic domain (7, 9, 17,
27). An interaction of talin with the
3 cytoplasmic
domain can activate the receptor, and this depends on membrane distal
sequences (14). The present results indicate interactions between the
membrane distal
IIb sequence and the
3
tail within the native receptor that block integrin activation.
Disruption of this membrane distal interaction could account for energy
and
tail-dependent activation caused by alterations in
membrane-distal
cytoplasmic domain sequences (7, 42). Furthermore,
the interaction with the
IIb tail may oppose
stabilization of the reverse turn at
3(Asn744-Tyr747) by cytoplasmic
proteins such as talin. Thus, detailed analyses of the structural
effects of interactions of proteins, such as talin, with integrin
cytoplasmic domains are likely be informative in testing this model of
integrin activation. In addition, the identification of physiologic
mechanisms that regulate the interactions of integrin tails with each
other and with proteins such as talin will be central to the
understanding of this important biological process.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge Drs. Ed Plow and Tom Haas for
the generous gift of the
3(Ile719-Thr762) peptide. We
also gratefully acknowledge Drs. Martin Schwartz and Sandy Shattil for
critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This is Publication 13951-VB from the Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by grants from the NHLBI and NIAMS, National Institutes of Health. To whom correspondence should be addressed: Dept. of Vascular Biology, Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7124; Fax: 858-784-7343; E-mail: Ginsberg@scripps.edu.
Supported by the German Academic Exchange Service (Deutscher
Akademischer Austauschdienst) and Biotechnology and Biological Sciences Research Council.
** Supported by the Wellcome Trust and, through the Oxford Center for Molecular Sciences, from the Medical Research Council, Biotechnology and Biological Sciences Research Council, and Engineering and Physical Sciences Research Council.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M101915200
2 T. S. Ulmer, B. Yaspan, M. H. Ginsberg, and I. D. Campbell, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay.
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REFERENCES |
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