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Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2559-2568
A Mutation in the Extracellular Cysteine-Rich Repeat Region of the
3 Subunit Activates Integrins
IIb 3 and V 3
By
Hirokazu Kashiwagi,
Yoshiaki Tomiyama,
Seiji Tadokoro,
Shigenori Honda,
Masamichi Shiraga,
Hajime Mizutani,
Makoto Handa,
Yoshiyuki Kurata,
Yuji Matsuzawa, and
Sanford J. Shattil
From The Second Department of Internal Medicine, Osaka University
Medical School, and Department of Transfusion, Osaka University
Hospital, Osaka, Japan; the Blood Center, Keio University Hospital,
Tokyo, Japan; and the Department of Vascular Biology and Molecular and
Experimental Medicine, The Scripps Research Institute, La Jolla,
CA.
 |
ABSTRACT |
Inside-out signaling regulates the ligand-binding function of
integrins through changes in receptor affinity and/or avidity. For
example, IIb 3 is in a
low-affinity/avidity state in resting platelets, and activation of the
receptor by platelet agonists enables fibrinogen to bind. In addition,
certain mutations and truncations of the integrin cytoplasmic tails are
associated with a high-affinity/avidity receptor. To further evaluate
the structural basis of integrin activation, stable Chinese hamster
ovary (CHO) cell transfectants were screened for high-affinity/avidity
variants of IIb 3. One clone (AM-1)
expressed constitutively active IIb 3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic anti IIb 3
antibody; and (2) fibrinogen-dependent cell aggregation. Sequence
analysis and mutant expression in 293 cells proved that a single amino
acid substitution in the cysteine-rich, extracellular portion of
3(T562N) was responsible for receptor activation. In
fact, T562N also activated V 3, leading to
spontaneous binding of soluble fibrinogen to 293 cells. In contrast,
neither T562A nor T562Q activated IIb 3,
suggesting that acquisition of asparagine at residue 562 was the
relevant variable. T562N also led to aberrant glycosylation of
3, but this was not responsible for the receptor
activation. The binding of soluble fibrinogen to
IIb 3(T562N) was not sufficient to trigger
tyrosine phosphorylation of pp125FAK, indicating that
additional post-ligand binding events are required to activate this
protein tyrosine kinase during integrin signaling. These studies have
uncovered a novel gain-of-function mutation in a region of
3 intermediate between the ligand-binding region and the
cytoplasmic tail, and they suggest that this region is involved in
integrin structural changes during inside-out signaling.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE 3 SUBFAMILY OF
heterodimeric integrin receptors includes
IIb 3, which is specific for platelets and
critical for adhesive events in hemostasis, and
V 3, which is more widely distributed and
involved in regulation of cell growth, migration, and programmed
death.1-4 The ligand binding function of these integrins is
tightly regulated by two processes often referred to collectively as
inside-out signaling: (1) affinity modulation, which involves
structural changes intrinsic to the heterodimer; and (2) avidity
modulation due to lateral diffusion and clustering of heterodimers into
oligomers.5,6 Ligand binding to and clustering of
3 integrins in turn generate outside-in signals, such as
activation of the protein tyrosine kinases Src and
pp125FAK, which act in concert with signals from growth
factor receptors to regulate many anchorage-dependent cell
functions.6-8
The 3 integrin subunit consists of 762 amino acids
encompassing a large extracellular domain, a single membrane-spanning region, and a 47 amino acid cytoplasmic tail.9 The
extracellular domain is notable for an I-domain like ligand-binding
region (residues 110-294)10 and a cysteine-rich repeat
region (residues 423-622) that contains 31 of the subunit's 56 cysteine residues.9,11 Studies of individuals with variant
Glanzmann thrombasthenia, who bleed due to functional defects of
IIb 3, and of recombinant integrins
expressed in mammalian cells have focused attention on the
ligand-binding region and the cytoplasmic tail as particularly relevant
to the process of inside-out signaling.12-19 In addition, antibodies known as ligand-induced binding site (LIBS), which bind
better to 3 after fibrinogen binds, can in some cases
increase receptor affinity without the need for signals from inside the cells.20,21 Some of these antibodies recognize epitopes
within the cysteine-rich repeats of 3,21,22
raising the possibility that this region may also be involved in
propagating activating signals from inside the cell to the ligand
binding region of the receptor. In this report, we provide the first
direct evidence for this idea by characterizing a single amino acid
substitution in the cysteine-rich repeat region of 3
that results in constitutive activation of both
IIb 3 and
V 3.
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MATERIALS AND METHODS |
Antibodies and plasmids.
PAC1, a ligand-mimetic mouse monoclonal IgM antibody, is specific for
the activated IIb 3 complex.23
Two antibodies to LIBS within 3, LIBS1 and
LIBS6,20,24 were obtained from Dr Mark H. Ginsberg (The
Scripps Research Institute, La Jolla, CA). AP5 (anti- 3
LIBS),22 AP3 (anti- 3; non-function
blocking),25 and AP2
(anti- IIb 3; function
blocking)26 were obtained from Dr Thomas J. Kunicki (The
Scripps Research Institute). PT25-2, an
IIb 3 activating antibody, and its Fab
fraction have been characterized.27 LM142
(anti- V; non-function blocking)28 were
obtained from Dr David A. Cheresh (The Scripps Research Institute). IIb cDNA and 3 cDNA inserted in pCDM8
(resulting in the expression plasmids, CD2b and CD3a)17
were obtained from Dr M.H. Ginsberg. IIb cDNA and
3 cDNA inserted in pcDNA3 ( IIb/pcDNA3,
3/pcDNA3) were obtained from Dr Peter J. Newman (The
Blood Center of Southeastern Wisconsin, Milwaukee, WI).
V cDNA inserted in pcDNA1 was obtained from Dr D.A. Cheresh.
Cell lines.
Chinese hamster ovary (CHO) and 293 cells were maintained as
described.29,30 CD2b and CD3a were cotransfected into a
CHO-AA8 cell line (Clontech, Palo Alto, CA), and stable transfectants expressing IIb 3 were obtained by single
cell sorting by a FACStar flow cytometry (Becton Dickinson, San Jose,
CA). As shown previously, IIb 3
transfectants obtained in this manner do not bind PAC1 or fibrinogen
and are in a low-affinity/avidity state.17,30 Then, one of
these clones (24/12) was stained with PAC1 and subjected to another
round of single-cell sorting to select for rare variants that now bound
PAC1. 192 cells were sorted and PAC1 binding of 48 clones was
reassessed. Only one clone, AM-1, showed constitutive binding of PAC1,
as described below.
Nucleotide sequence analysis of IIb and
3.
Genomic DNA from 24/12 and AM-1 cells was isolated (Qiagen DNA
extraction kit; Qiagen Inc, Chatsworth, CA), and the entire coding
regions of IIb and 3 were amplified using
4 and 3 paired sets of primers, respectively.31,32
Amplified DNA fragments were purified and subjected to direct cycle
sequence analysis using an Applied Biosystems Automated sequencer
(Perkin Elemer-Japan, Chiba, Japan) according to the
manufacturer's directions.
Site-directed mutagenesis of 3 cDNA.
To introduce the T562N mutation in 3 cDNA, two-step
ligation was performed. First, 3/pcDNA3 was cut with
BamHI and Afl II, yielding three fragments, Frag1
(BamHI and Afl II ends: 6 kb), Frag2 (BamHI and
BamHI ends: 1.5 kb), and Frag3 (BamHI and Afl II ends: 0.5 kb). After agarose gel electrophoresis, Frag1 and Frag2
were cut out with Gel extraction kit (Qiagen). The 3
cDNA fragment from AM-1 cells including the mutated site (T562N) was amplified using primers, IIIa3 and IIIa4-AflII, and digested with BamHI and Afl II. The 0.5-kb fragment [Frag3(T562N)]
was cut out and ligated to Frag1 with a ligation kit (Boehringer
Mannheim, Mannheim, Germany). After transformation to
JM109 competent cells (Takara, Shiga, Japan), miniprep DNA was cut with
BamHI and Frag2 was inserted. After transformation, a clone
containing Frag2 in the correct orientation was selected by polymerase
chain reaction (PCR), amplified, and purified with a cesium chloride
gradient. The entire coding region of the plasmid was
sequenced to confirm aquisition of T562N mutation and absence of other
nucleotide change.
To introduce T562A, T562Q, and T564A into 3 cDNA,
PCR-based mutagenesis was performed as described.33 In
brief, first-round PCR was performed using 3/pcDNA3 as a
template with antisense primer containing mutated nucleotide(s) or
sense primer containing mutated nucleotide(s) and IIIa4-AflII. The
sequence of primers used is shown in Table
1. After purification of PCR fragments with a purification kit
(Qiagen), second-round PCR was performed using mixture of first-round
PCR products as template with primers, IIIa3 and IIIa4-AflII. PCR
products were cut with BamHI and Afl II, and the
appropriate fragments were purified and ligated to Frag1 and Frag2. To
make the 3(T562N, T564A) double mutant,
3(T562N)/pcDNA3 was used as a template instead of
3/pcDNA3. All plasmids were sequenced to confirm
authenticity.
Transient transfection.
A human embryonic kidney cell line, 293 (obtained from American Type
Cell Culture, Rockville, MD), was used for transient transfection assays. Transfections were performed using the calcium phosphate method as described.29 Functional assays were
performed 48 hours after transfection.
PAC1 binding and fibrinogen binding assay.
PAC1 binding to cells was assessed as described30 with
minor modification. Cells (5 × 105) were preincubated
in 45-µL aliquots containing Tyrode's buffer supplemented with 2 mmol/L CaCl2 and 2 mmol/L MgCl2 in the presence or absence of 10 µmol/L FK633 (a peptidomimetic
IIb 3 antagonist from Dr Jiro Seki,
Fujisawa Pharmaceutical Co, Osaka, Japan)34 or 10 µg/mL
PT25-2, an IIb 3 activating antibody.
Then, 5 µL of a 1:25 dilution of PAC1 ascites was added to each tube,
and incubations were performed for another 30 minutes at room
temperature. After washing, cells were resuspended in a 1:20 dilution
of fluorescein isothiocyanate (FITC)-conjugated antimouse IgM
(µ-chain specific; Caltag Lab, Burlingame, CA) for 25 minutes on ice.
Then, 1 µL of 1 mg/mL of propidium iodide (PI; Sigma, St Louis,
MO) was added, and 5 minutes later the cells were washed
and resuspended in 500 µL of ice-cold Tyrode's and analyzed on a
FACScan flow cytometer (Becton Dickinson). PAC1 binding (FL1) was
analyzed on the gated subset of single, live cells (PI-negative, FL3).
To compare the PAC1 binding results from one experiment with those from
another, PAC1 binding was expressed as an activation index, defined as (Fx Fi)/(Fm Fi), where Fx is the median fluorescence
intensity of PAC1 binding in the absence of inhibitor, FK633; Fi is the median fluorescence intensity of PAC1 binding in the presence of
inhibitor, FK633; and Fm is median fluorescence intensity of PAC1
binding in the presence of the activating antibody,
PT25-2.30
Fibrinogen was labeled with FITC as described.35 In the
case of IIb 3-stable cell lines, the
binding of FITC-fibrinogen (150 µg/mL) was assessed in the same
manner as described for PAC1. In the case of 293 cells transiently
transfected with V 3, cells were suspended
in calcium-free Tyrode's buffer supplemented with 1 mmol/L
MgCl2 and pretreated for 30 minutes on ice with or without 1 mmol/L RGDW, an inhibitor of fibrinogen binding to
V 3, or 1 mmol/L MnCl2, which
induces a high-affinity state of
V 3.2 LM142 (10 µg/mL), a
non-function blocking anti- V antibody, was added
simultaneously to the tubes to monitor expression of
V 3. After washing, cells were incubated
with 150 µg/mL of FITC-fibrinogen and phycoerythrin (PE)-conjugated
antimouse IgG (Serotec, Oxford, UK) for 25 minutes at room
temperature. Then, after 5 minutes of incubation with PI, cells were
washed and three-color analysis was performed on FACScan. In this case,
fibrinogen binding (FL1) was analyzed on the gated subset of single,
live cells (PI-negative, FL3) that stained positively for
V 3 (FL2).
Cell aggregation.
Fibrinogen-dependent cell aggregation was monitored as
described.36 In brief, 5 × 106/mL cells
were added to wells of a 24-well culture dish precoated with 1 mg/mL of
bovine serum albumin (BSA). Fibrinogen (300 µg/mL) was added with or
without 20 µmol/L FK633 or 20 µg/mL PT25-2 and the dish was rotated
at 100 rpm for 20 minutes on a horizontal rotator (Multi-Mixer;
Lab-Line Instruments Inc, Melrose Park, IL). Aggregate
formation was stopped by adding an equal volume of 0.5% formaldehyde,
and aggregates were visualized and photographed under a phase-contrast
microscope (Olympus, Tokyo, Japan).
Cell surface labeling and immunoprecipitation.
Cells were labeled with sulfo-NHS-biotin (Pierce, Rockford, IL) and
lysed in Triton X-100 buffer (1% Triton X-100, 25 mmol/L Tris-Cl, 100 mmol/L NaCl, pH 7.4, 0.1 mg/mL leupeptin, 4 µg/mL pepstatin A, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L benzamide). Two
hundred micrograms of protein from each sample was immunoprecipitated
with an IIb 3 complex-specific antibody, AP2, as described.32 Immunoprecipitates were resolved in
6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF)
membrane (Immobilon; Millipore, Bedford, MA). After incubation with
peroxidase-conjugated avidin (Vectastain ABC kit; Vector Lab,
Burlingame, CA), immunoreactive bands were visualized by enhanced
chemiluminescence (Renaissance; NEN Life Science, Boston, MA).
Tyrosine phosphorylation of pp125FAK.
Cells were grown in Dulbecco's modified Eagle's medium (DMEM)
containing 0.5% fetal bovine serum for 18 hours and then resuspended to 3 × 106 cells/mL in DMEM. To test the effects of
soluble fibrinogen to IIb 3, suspended
cells were incubated at 37°C for 30 minutes in the presence or
absence of 10 µg/mL of Fab fragments of PT25-2, and then 250 µg/mL
of fibrinogen was added to the cells. After 15 minutes of incubation at
37°C, the cells were washed with phosphate-buffered saline (PBS)
and lysed with Triton lysis buffer supplemented with 1 mmol/L sodium
vanadate. For studies of adherent cells, 1 mL of suspended cells were
seeded onto plastic dishes that had been precoated with 100 µg/mL
fibrinogen or poly-L lysine (Iwaki Glass, Tokyo, Japan). After 30 minutes at 37°C, plates were washed twice with ice-cold PBS. Then
adherent cells were lysed on the plates with Triton lysis buffer
containing sodium vanadate and scraped into microcentrifuge tubes.
Lysates were incubated on ice for 30 minutes and clarified supernatants
were processed for immunoprecipitation.
pp125FAK was immunoprecipitated with 1 µg of rabbit
polyclonal antibody, FAK(C903) (Santa Cruz Biotech, Santa Cruz, CA),
and protein-G sepharose (Pharmacia, Uppsala, Sweden). Precipitates were
separated on 7.5% SDS-PAGE and transferred to a PVDF membrane. Phosphotyrosine was detected with monoclonal antibody, 4G10. To monitor
loading of gel lanes, the blots were stripped (2% SDS, 62.5 mmol/L
Tris, pH 6.7, 100 mmol/L 2-mercaptoethanol for 30 minutes at 70°C)
and reprobed with FAK(C903).
 |
RESULTS |
Establishment of the AM-1 cell line expressing a constitutively
active form of
IIb 3.
IIb 3 expressed in resting platelets or in
a CHO cell model system exists in a low-affinity/avidity state and does
not bind soluble fibrinogen or the ligand-mimetic antibody,
PAC1.6,17 To better understand the structural basis of
IIb 3 activation, a stable CHO cell line
that expresses IIb 3 (24/12) was stained with PAC1 and analyzed by flow cytometry to screen for rare variants that might bind PAC1 constitutively. In so doing, a stable cell line,
AM-1 ( ctivated utant-1), that expressed this
activated phenotype was established. Parental 24/12 cells did not bind
to PAC1 spontaneously, but they did do so as expected in response to
activating antibody, PT25-2 (activation index [AI], 0.04 ± 0.02 [mean ± SD]; n = 5). In contrast, AM-1 cells bound PAC1 even in
the absence of PT25-2 (AI, 0.67 ± 0.09; n = 5) and the binding was
completely inhibited by incubation with an
IIb 3 specific antagonist, FK633
(Fig 1A). The same results were obtained
when another activating antibody (LIBS6) was used to induce ligand binding21 or when another specific antagonist (Integrilin)
was used to block ligand binding37 (data not shown).
Preincubation of AM-1 cells with 0.2% sodium azide and 4 mg/mL
2-deoxy-d-glucose had no effect on the increase in PAC1 binding,
indicating that the high-affinity/avidity state of the
IIb 3 in AM-1 cells was independent of
metabolic energy (data not shown).

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| Fig 1.
Assessment of affinity state of
IIb 3 by PAC1 (A) and fibrinogen (B)
binding. In (A), 24/12 cells (a through c) or AM-1 cells (d through f)
were preincubated with 10 µmol/L FK633 (a and d), 10 µg/mL PT25-2
(c and f), or buffer (b and e) for 30 minutes on ice. Then, 250×
diluted PAC1 ascites were added and incubated for another 30 minutes at
room temperature. After washing, cells were incubated with
FITC-conjugated antimouse IgM for 25 minutes on ice. To exclude dead
cells, PI were added to the cells and incubated for 5 minutes. After
washing, cells were resuspended in buffer and flow cytometric analysis
was performed. In (B), cells were preincubated with 10 µmol/L FK633
(dotted lines), 10 µg/mL PT25-2 (solid lines), or buffer (bold lines)
for 30 minutes on ice. Cells were then incubated with 150 µg/mL of
FITC-labeled fibrinogen for 25 minutes at room temperature and then
with PI for 5 minutes. After washing, flow cytometric analysis was
performed.
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To establish whether AM-1 cells also bind a soluble physiological
ligand in a constitutive fashion, binding of FITC-labeled fibrinogen
was examined by flow cytometry. Consistent with the PAC1 results,
fibrinogen bound to AM-1 in the absence of PT25-2 and the binding was
completely blocked by FK633 (Fig 1B). To determine the functional
relevance of this spontaneous fibrinogen binding, the ability of AM-1
cells to aggregate was assessed. The parental 24/12 cells exhibited
fibrinogen-dependent aggregation only after addition of the activating
antibody. In contrast, AM-1 cells exhibited fibrinogen-dependent and
FK633-inhibitable aggregation even in the absence of the activating
antibody (Fig 2). AP2, a function blocking
anti- IIb 3 antibody,26 also
inhibited the fibrinogen-dependent aggregation of AM-1 cells, and this
aggregation was divalent cations dependent, because the aggregation was
not observed when cells were resuspended in divalent cation free
Tyrodes buffer with 2 mmol/L EDTA (data not shown). These results
indicate that IIb 3 in AM-1 cells is in a
constitutively activated state, fully capable of supporting spontaneous
cell aggregation in the presence of fibrinogen.

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| Fig 2.
Spontaneous, fibrinogen-dependent aggregation of AM-1
cells. Cells were resuspended in Tyrode's buffer in the presence of
300 µg/mL of fibrinogen and with or without 20 µmol/L FK633 or 20 µg/mL PT25-2. Cells were then rotated for 20 minutes on a horizontal
rotator. Aggregate formation was stopped by adding equal amount of
0.5% formaldehyde and incubating for 30 minutes. Shown are photographs
representative of three independent experiments.
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IIb 3
in AM-1 cells is in a high-affinity state.
Integrins can become activated to bind ligands through conformational
changes in the heterodimer (affinity modulation) and/or receptor
clustering (avidity modulation).5-7 To determine whether the activation of IIb 3 in AM-1 cells was
associated with conformational changes in the receptor, the binding of
various anti-LIBS antibodies (LIBS1, LIBS6, and AP5) was examined. It
has been shown that LIBS1, LIBS6, and AP5 preferentially recognize
epitopes on 3 after ligand-binding, and the epitope of
LIBS6 and AP5 has been defined within the cysteine-rich repeat region
and the N-terminal region, respectively.20,22 FK633 (5 µmol/L) was used to induce LIBS expression, because preliminary studies showed that it could induce full expression of LIBS epitopes in
platelets and CHO cell lines (Kashiwagi et al, unpublished observation). In parental 24/12 cells the LIBS antibodies
bound to IIb 3 only after cell incubation
with FK633. In contrast, LIBS6 and AP5 bound fully to AM-1 cells in the
absence of FK633 and LIBS1 showed a slight increase in binding
(Fig 3). Thus, some but not all LIBS
epitopes are constitutively exposed on
IIb 3 in AM-1 cells, even in the absence
of added ligand.

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| Fig 3.
LIBS expression of 24/12 (the left row) and AM-1 cells
(the right row). Cells were preincubated with 5 µmol/L FK633 (solid
lines) or buffer (bold lines) for 30 minutes on ice, and then 5 µg/mL
of LIBS1 (upper row), LIBS6 (middle row), or AP5 (lower row) was added.
After 30 minutes of incubation, cells were washed and then incubated
with FITC-conjugated antimouse IgG for 30 minutes. After washing, flow
cytometric analysis was performed. Dotted lines indicate MOPC21, a
control mouse IgG antibody, binding.
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Molecular analysis of
IIb 3 in AM-1
cells.
To begin to analyze the conformational change of
IIb 3 in AM-1 cells,
IIb 3 was immunoprecipitated with AP2. In
contrast to 3 in the parental cells, 3 in
AM-1 cells migrated more slowly and as a broad band under both reduced
and nonreduced conditions, suggesting that mutation(s) and/or change of
glycosylation state existed in 3 of AM-1 cells
(Fig 4). Next, the sequence of the entire
coding region of IIb and 3 cDNA
integrated in genomic DNA of AM-1 cells was determined. Amplification
of genomic DNA using primer pairs that located in far separated exons
excluded the possibility to amplify intrinsic hamster
IIb and 3 DNA. A consecutive dinucleotide
change in 3 cDNA integrated in AM-1 cells
(1732A A ) was
discovered, leading to a single putative amino acid substitution, T562N
(Fig 5A). No other sequence abnormalities were detected. This mutation is in the third of four cysteine-rich repeats in 3, and it would establish a new putative
N-glycosylation site at the mutated residue
(562 RT RT: NXT/S is a
consensus sequence of N-glycosylation). Amino acid alignment around the
mutated site shows that amino acid residues in C560-C567, that may make
a small loop in the third repeat of the cysteine-rich repeat
region,11 are completely identical in Xenopus, chicken,
rodent, and human 3. By contrast, they are poorly
conserved between human 3 and other human integrins (Fig 5B).

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| Fig 4.
Immunoprecipitation of IIb 3
from 24/12 and AM-1 cells. Cell surface was labeled with
sulfo-NHS-LC-biotin and lysed with 1% Triton X-100 lysis buffer.
Lysates from 24/12 cells (lanes 1 and 3) and AM-1 cells (lanes 2 and 4)
were incubated with anti- IIb 3 antibody,
AP2, and immunoprecipitates were separated on 6% SDS-PAGE under
reducing (lanes 1 and 2) or nonreducing conditions (lanes 3 and 4).
After transfer, the membrane was incubated with peroxidase-conjugated
avidin and developed with chemiluminescence.
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| Fig 5.
Sequence analysis of 3 cDNA from 24/12 or
AM-1 cells. (A) The results of sequencing using an antisense primer are
shown. The same results were obtained using a sense primer for
sequencing (data not shown). The mutated nucleotides and the changed
amino acid are in bold. (B) Amino acid alignment around the mutated
site is shown. T562 in 3 and corresponding amino acids
in other integrins are in bold. NRT (underlined) is a consensus
sequence for N-glycosylation. Amino acid sequences were
obtained from Wippler et al,44 Mimura et al,53
and Ransom et al.54
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Substitution of asparagine for threonine at position 562 of
3 causes a high-affinity state of
IIb 3.
To prove that this single amino acid change was responsible for the
constitutive activation of IIb 3, we
introduced this mutation into wild-type 3 and
transiently transfected it into 293 cells. Indeed, in the absence of
activating antibody PT25-2, PAC1 bound spontaneously to cells
transfected with IIb 3(T562N) (AI, 0.80 ± 0.10; n = 5), but not to cells transfected with wild-type IIb 3 (AI, 0.06 ± 0.02; n = 5), indicating that the T562N mutation is responsible for constitutive
activation of the receptor (Fig 6A). When a
D119Y mutation in 3 that abrogates fibrinogen binding to
IIb 312 was introduced into
3(T562N), PAC1 failed to bind to
IIb 3(D119Y, T562N) either in the absence
or presence of PT25-2 (data not shown).


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| Fig 6.
Assessment of the activation state of wild-type and
mutant IIb 3 in transiently transfected
293 cells. (A) Wild-type IIb cDNA was transfected with
wild-type 3 cDNA (WT) (a through c) or mutant
3 cDNAs (T562N [d through f], T562A [g through i],
T562Q [j through l]) to 293 cells, and PAC1 binding was determined.
Plots in the upper row (a, d, g, and j) represent nonspecific
PAC1 binding determined in the presence of FK633. Plots
in the lower row (c, f, i, and l) represent maximal PAC1
binding in the presence of PT25-2. Plots in the middle row (b, e, h,
and k) represent PAC1 binding in the absence of the
antagonist and the activating antibody. (B) Wild-type
IIb 3,
IIb 3(T564A), or
IIb 3(T562N,T564A) transfected cells were
incubated with PAC1 and biotinylated-AP3, a non-function blocking
anti- 3 antibody, followed by incubation with
FITC-conjugated antimouse IgM and PE-conjugated streptoavidin, and
analyzed by flow cytometry. The overlay histogram represents PAC1
binding to cells expressing high levels of
IIb 3 determined by AP3 (denoted by the
rectangle in the dot blots) [wild-type
IIb 3, dotted line;
IIb 3(T564A), solid line;
IIb 3(T562N,T564A), bold line]. (C)
Wild-type and mutant IIb 3 were
surface-labeled with biotin, and immunoprecipitation was performed with
AP2. Immnoprecipitates were electrophoresed on 6% polyacrylamide gel
under reducing conditions. After transfer, membrane was incubated with
peroxidase-conjugated avidin and developed with chemiluminescence.
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To determine the mechanism of activation by the T562N mutation, we
introduced T562A and T562Q mutations into 3. The T562Q mutant was constructed because asparagine and glutamine have the same
amide in their side chain. PAC1 did not bind to cells transfected with
either IIb 3(T562A) or
IIb 3(T562Q) in the absence of PT25-2 (AI,
0.08 ± 0.03 and 0.07 ± 0.06, respectively; n = 3; Fig 6A), indicating that it was the acquisition of asparagine at residue 562 that was important for spontaneous receptor activation. Next, we
introduced the T564A mutation into 3 to disrupt the
aberrant consensus sequence of N-glycosylation at residue 562. Immunoprecipitation of this form of 3 showed that
3(T564A) and 3(T562N,T564A) migrated on
SDS gels similarly to the migration of 3(WT) (Fig 6C).
Because the T564A mutation led to a mild reduction in the expression of IIb 3, we monitored
IIb 3 expression by a non-function
blocking anti- 3 antibody, AP3, and analyzed only cells
expressing high levels of IIb 3 for PAC1
binding. Although IIb 3(T564A) showed a
slight increase in the activation index (0.28 ± 0.03; n = 3), the
activation of IIb 3(T562N, T564A) was even
greater (AI, 0.46 ± 0.01; n = 3), and this difference was
statistically significant (P < .001; Fig 6B). These results
suggest that the glycosylation at N562 may not be essential for the
constitutive activation of IIb 3 in AM-1
cells. A lack of relationship between the aberrant N-glycosylation of
3(T562N) and IIb 3
activation was also suggested by the observation that 24 hours of
incubation of the cells with 1 to 5 µg/mL of tunicamycin, a specific
inhibitor of N-glycosylation,38 had no effect on the
activation state of IIb 3 in AM-1 cells (AI, 0.67 ± 0.09, 0.67 ± 0.04, and 0.66 ± 0.02; 0, 1, and 5 µg/mL of tunicamycin, respectively; n = 3). On the other hand, the
activation state of wild-type IIb 3 in
24/12 cells was slightly increased by tunicamycin treatment in a
concentration-dependent manner (AI, 0.04 ± 0.02, 0.13 ± 0.04, and 0.18 ± 0.01; 0, 1, and 5 µg/mL of tunicamycin,
respectively; n = 3).
The T562N mutation also leads to activation of
V 3.
To determine whether the activating mutation in 3 would
affect the affinity state of the related integrin
V 3, this integrin was transiently
expressed in 293 cells. In this case, FITC-labeled fibrinogen was used
to determine the activation state of V 3 and a non-function blocking anti- V antibody, LM142, was
used to monitor V 3 expression. When
V was transfected with wild-type 3,
fibrinogen bound only if integrin affinity was upregulated by the
addition of manganese. In contrast, fibrinogen binding to cells
expressing V 3(T562N) could be detected
even in the absence of manganese (Fig 7).
These results indicate that the T562N mutation is capable of increasing
the activation state of both IIb 3 and
V 3.

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| Fig 7.
Soluble fibrinogen binding to
V 3 and
V 3(T562N) transfected cells. Cells were
preincubated with 1 mmol/L RGDW, 1 mmol/L manganese, or buffer with 10 µg/mL of anti- V antibody, LM142. After 30 minutes of
incubation, cells were washed and then incubated with 150 µg/mL
FITC-conjugated fibrinogen and PE-conjugated antimouse IgG for 30 minutes and analyzed by flow cytometry. (A) Dot blots represent
FITC-fibrinogen (horizontal) and LM142 (vertical) binding in the
absence of RGDW and manganese. (B) Fibrinogen binding to cells
expressing high levels of V 3 (denoted by
the rectangle in the dot blots) was analyzed on the histograms. (a
through c) Wild-type V 3 transfected
cells; (d through f) V 3(T562N)
transfected cells. (a and d) With RGDW; (b and e) with buffer; (c and
f) with manganese.
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Tyrosine phosphorylation of pp125FAK in AM-1 cells.
Ligand binding and clustering of integrins stimulate outside-in
signaling, manifested by responses that include protein tyrosine phosphorylation and cytoskeletal reorganization.6-8 Focal
adhesion kinase (FAK), a 125-kD cytoplasmic tyrosine kinase, is a
component of focal adhesions and is a well-established component of
integrin signaling pathways.39,40 Consequently, the
tyrosine phosphorylation state of pp125FAK in AM-1 cells
was studied. pp125FAK was not tyrosine-phosphorylated in
AM-1 cells or in control 24/12 cells maintained in suspension for 15 minutes, either in the presence or absence of fibrinogen. Furthermore,
neither cell type exhibited pp125FAK phosphorylation when
adherent to plates coated with poly-L-lysine. On the other hand, both
showed pp125FAK phosphorylation in response to cell
adhesion to fibrinogen (Fig 8). These
results indicate that receptor activation by the T562N mutation or the
mere binding of soluble fibrinogen to the activated receptor is not
sufficient to cause activation of pp125FAK; nonetheless,
this mutant receptor is fully capable of mediating this outside-in
signaling response upon cell adhesion.

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| Fig 8.
pp125FAK phosphorylation. (A) 24/12 cells
(lanes 1 through 3) or AM-1 cells (lanes 4 through 6) were maintained
in suspension for 15 minutes with no addition (S; lanes 1 and 4), with
the addition of 250 µg/mL of fibrinogen (S, Fg; lanes 2 and 5), and
with addition of fibrinogen and 10 µg/mL of Fab fragments of PT25-2
(S, Fg+PT; lanes 3 and 6). (B) 24/12 cells (lanes 1 through 3) or
AM-1 cells (lanes 4 through 6) were allowed to become adherent to
immobilized fibrinogen (Fg; lanes 3 and 6) or poly-L lysine (PL; lanes
2 and 5) or were maintained in suspension (S; lanes 1 and 4) for 30 minutes. The cells were lysed and pp125FAK was
immunoprecipitated with an anti-FAK polyclonal antibody.
Phosphotyrosine was detected with 4G10 (upper panel), and blots were
reprobed with anti-FAK to assess gel loading (lower panel).
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DISCUSSION |
In this report, we analyzed a cell line in which the ligand-binding
function of integrin IIb 3 was
constitutively activated, in contrast to the usual, default
low-affinity/avidity state of this integrin in platelets and
transfected tissue culture cells.6,17 We found the
following: (1) A single amino acid change in the extracellular
cysteine-rich repeat region of 3, T562N, is responsible for this constitutive activation. (2) The presence of the asparagine as
opposed to the loss of the threonine appears responsible for this
phenotype. (3) Although the T562N mutation leads to aberrant glycosylation, it is unlikely that this posttranslational modification actually causes the activated integrin phenotype. (4) The T562N mutation is capable of activating V 3 as
well as IIb 3. (5) Activation of
IIb 3 by T562N or binding of soluble
fibrinogen to the mutant is not sufficient to trigger tyrosine
phosphorylation of pp125FAK. Ligand binding as the result
of the T562N mutation was similar to that induced through the more
physiological process of inside-out signaling, because (A) it was
completely blocked by synthetic IIb 3-specific antagonists, (B) it was
abrogated by a mutation in 3(D119Y) associated with a
variant form of thrombasthenia and clinical bleeding,12 and
(C) it mediated fibrinogen-dependent cell aggregation.
Three types of activating mutations in
IIb 3 have been described previously. (1)
Bajt et al41 showed that swapping the ligand-binding site
(residues 129-133) of 3 with the corresponding sequence
in 1 led to a gain of function in
IIb 3. (2) The proximal regions of
cytoplasmic tails of IIb and 3 are highly
conserved, and deletions or amino acid changes that may break the
interaction between them lead to activation of
integrins.13,17,18 (3) Liu et al42 recently
reported that disruption of the C5-C435 disulfide bond in
3 resulted in an increase in affinity of
IIb 3. In this context, it has been shown
that mild reducing agents, such as dithiothreitol, can increase the
ligand-binding function of
IIb 3.43 In addition, it has
been shown that some LIBS antibodies that bind to epitopes within the
cysteine-rich repeats of 3, such as LIBS2, LIBS3, and
LIBS6, lead to activation of IIb 3 without
ligand-binding.20-22 Wippler et al44 also
demonstrated that recombinant IIb 3
lacking the cysteine-rich repeats of 3 showed
high-affinity binding to fibrinogen. Furthermore, sequence alignment of
3 integrins indicate that about 90% of noncysteine residues in the cysteine-rich repeats are conserved between rodents and
human 3, whereas noncysteine residues in the region are
poorly conserved among 1, 2, and
3 integrins (~15%).45 These results suggest that the cysteine-rich repeats may have an important role for
regulation of 3 integrin functions.
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