From the
Department of Hematology and Oncology,
Division of Clinical Pharmacotherapeutics, Program for Applied Biomedicine,
Graduate School of Biomedical Sciences and
¶Research Institute for Radiation Biology and
Medicine, Hiroshima University, Hiroshima 734-8551, Japan
Received for publication, March 3, 2003 , and in revised form, April 16, 2003.
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ABSTRACT |
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INTRODUCTION |
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IAP is a 50-kDa membrane glycoprotein that has 5 transmembrane-spanning
regions and 1 immunoglobulin (Ig)-like extracellular domain
(15,
16). It was originally
reported to be physically associated with certain integrins including
2
1,
V
3,
and
IIb
3
(1719).
Blockade of IAP with monoclonal antibody inhibits some aspects of integrin
function, and ligation of IAP with activating antibodies induces the
modulation of integrin-dependent cell adhesion. A gene-targeting study showed
that IAP plays a key role in host defense by participation in migration and
activation of leukocytes in response to bacterial infection
(20). Subsequently, it was
reported that IAP is a receptor for the C-terminal cell binding domain of
thrombospondin (TS) (21). TS,
its C-terminal cell binding domain, and a peptide from the C-terminal cell
binding domain, KRFYVVMWKK (4N1K), all stimulate the integrin-dependent
adhesion, spreading, and motility of the cells including endothelial cells,
leukocytes, and smooth muscle cells
(18,
22). On the other hand, IAP is
a ligand for the transmembrane signal-regulatory protein
(23). In this case, IAP likely
plays a role in macrophage function. The role on hematopoiesis
(24) or adhesion of sickle red
blood cell (25) was also
reported.
In platelets, the functional role of IAP was hardly detected using only a
monoclonal antibody against IAP
(26). However, the peptide
4N1K induces platelet aggregation
(27) and spreading on
immobilized fibrinogen or collagen
(19). The ability of the
peptide depends on its interaction with IAP because platelet response was
decreased by treatment with an antifunctional IAP antibody or in platelets
from IAP-deficient mice (17).
There are reports showing that the binding of 4N1K to IAP initiates
intracellular signaling that would result in affinity modulation of
IIb
3
(19). 4N1K induces tyrosine
phosphorylation of several proteins such as Syk and focal adhesion kinase. The
effect of 4N1K is inhibited by pertussis toxin, indicating the participation
of a heterotrimeric Gi protein
(19). This sort of stimulation
of integrin function via G proteins is similar to that caused by agonists
through seven transmembrane spanning receptors, such as ADP, epinephrine, and
thrombin. Recently cloned ADP receptor (P2Y12)
(28) also involves activation
of Gi-containing heterotrimeric GTPases. However, these receptors
are not necessarily present close to
IIb
3.
Therefore, the functional significance of the association of IAP with the
integrin is not clear.
In this report we investigate the mechanism by which IAP modulates the
affinity of IIb
3. We provide evidence that
only the extracellular Ig domain of IAP interacts with
IIb
3 and can change
IIb
3 to a high affinity state without
requirement of intracellular signaling when it binds to TS. These phenomena
might correspond with the dynamic structural change of integrin. The
extracellular event reported here would be a novel mechanism of affinity
modulation of integrin.
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EXPERIMENTAL PROCEDURES |
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Platelet AggregationBlood samples were collected from
consenting healthy volunteer donors in a 1:10 volume of 3.8% trisodium citrate
(w/v). Platelets were isolated from platelet-rich plasma by centrifugation at
800 x g for 10 min in the presence of 0.1 µg/ml
prostaglandin E1 and 1 unit/ml apyrase. The pellet was resuspended and washed
twice in 85 mM sodium citrate, 111 mM dextrose, 71
mM citric acid, pH 7.0, containing prostaglandin E1 and apyrase and
then resuspended at a concentration of 3 x 108 platelets/ml
in a modified Tyrode-HEPES buffer (138 mM NaCl, 0.36 mM
NaH2PO4, 2.9 mM KCl, 12 mM
NaHCO3, 10 mM HEPES, 5 mM glucose, 1
mM MgCl2, and 1 mM CaCl2, pH 7.4).
Platelet aggregation was measured by the addition of peptides (4N1K or control
peptide) at 37 °C in an aggregometer (Chrono-log, Havertown, PA), with
continuous stirring at 1200 rpm. To inhibit the fibrinogen binding or 4N1K
binding, 100 µg/ml T10 or B6H12 was added 10 min before aggregation
measurements. B6H12 induced direct aggregation of the platelets from some
subjects depending on the polymorphism of Fc receptor, FcRII.
Therefore, the effect of B6H12 was tested using the platelets from
"non-responders." To block the intracellular metabolism, platelets
were incubated with 0.4% sodium azide and 4 mg/ml 2-deoxy-D-glucose
at 37 °C for 1 h. Human fibrinogen was added at a final concentration of
200 µg/ml before the addition of several agonists (100 µM
peptides, 5 µg/ml collagen, or 5 µM ADP).
Flow Cytometric AnalysisThe affinity state of
IIb
3 was determined by flow cytometric
analysis using ligand-mimetic monoclonal antibody PAC1. PAC1 is an IgM
monoclonal antibody that binds only to the activated form of
IIb
3 in the same manner as the
physiological ligand (31).
Platelets were suspended at a concentration of 2 x 107/ml in
a modified Tyrode-HEPES buffer and stimulated with peptides or agonists in the
presence of 20 µg/ml FITC-conjugated PAC1 for 20 min at room temperature
without stirring. Fifty µl of each aliquot was then diluted with 500 µl
of the buffer, and the mixture was directly analyzed by a flow cytometer,
Epics XL (Coulter, Fullerton, CA). Single platelets were gated and analyzed.
In the case of cultured Namalwa and CHO cells, cells were harvested and
resuspended at 1 x 106 cells/ml in a modified Tyrode-HEPES
buffer. Cells were incubated with 100 µM peptides and/or
antibodies (50 µg/ml LIBS6 for stimulation and 100 µg/ml T10 or B6H12
for inhibition) in the presence of 20 µg/ml FITC-PAC1 for 30 min at room
temperature. After washing, cells were stained with 1 µg/ml propidium
iodide, and propidium iodide-negative cells were gated to exclude
permeabilized dead cells.
To estimate the expression of IIb
3 or
IAP, cells were incubated with the first antibody, T10 or B6H12, (10 µg/ml)
in Hanks' balanced salt solution with Ca2+ containing 1%
fetal bovine serum and 0.1% NaN3. After washing, they were
incubated again with FITC-conjugated goat F(ab')2 anti-mouse
immunoglobulins. Each incubation was performed on ice for 1 h. Cells were
finally washed and analyzed in the buffer containing propidium iodide. Because
the expression levels of wild type and mutant forms of IAP in CHO cells were
different, PAC1 binding was standardized by the mean fluorescence intensity of
B6H12 binding to each cell type.
Mutational AnalysisMutant cDNAs of
IIb
3 and IAP were constructed by PCR
according to the strategy previously described
(33). To mutate Ser-752 of
3, PCR was performed using cDNA of wild type
3 as a template. The sense primer contained the
BamHI site at base 1500 within the
3 cDNA, and the
antisense primer contained the Ser-752 to Pro mutation. After gel
purification, the PCR product was used for the second PCR. The second PCR was
performed with the same sense primer and another antisense primer that
overlapped with the first antisense primer and contained the stop codon of
3 cDNA and the HindIII site for cloning. The PCR
fragment was digested by BamHI and HindIII, isolated by gel
electrophoresis, and then used to replace the fragment of the wild type
3 cDNA extending from the BamHI site in the cDNA to
the HindIII site in the multicloning site of the expression
vector.
IAP cDNA in the expression vector was previously described
(26). Wild type IAP in this
study was identical to the so-called form 2, which is the most abundant
isoform (15). Two truncated
mutants were constructed. In the first construct (IAP 291) a stop codon
was introduced just after the 5th transmembrane domain, resulting in deletion
of the C-terminal cytoplasmic tail after Lys-291. In the second (IAP
163) a stop codon was created after the first transmembrane domain,
which resulted in deletion after Lys-163. In both cases PCR was performed with
the sense primer containing the translation initiation codon and the antisense
primer containing the indicated stop codon. In another mutant form (IAP-Tac)
the extracellular domain of IAP was fused with the transmembrane and
cytoplasmic domains of the IL-2 receptor
chain (Tac antigen/CD25).
Primer sets were prepared in which half of the sequence was identical to the
end of the extracellular domain of IAP, before Asn-142, and the other half was
identical to the beginning of the transmembrane domain of the IL-2 receptor.
Two PCR were performed separately to amplify the extracellular domain of IAP
and the transmembrane and cytoplasmic domains of the IL-2 receptor. The PCR
products were mixed, and the final PCR was performed using the outside
primers.
All constructs were then cloned into the expression vector, pBK-EF, and verified by nucleotide sequencing of the region encoding the PCR products using an automated DNA sequencer (ABI 310, Applied Biosystems, Foster City, CA).
Cells Expressing Recombinant
IIb
3 and
IAPNamalwa and CHO cells were transfected using DMRIE-C and
LipofectAMINE reagents (Invitrogen), respectively, according to the
manufacturer's instructions. Permanent transfectants were selected with G418
as described elsewhere. To establish the cells that stably express
IIb
3 and/or IAP, stable cells were sorted
by reactivity to the antibody T10 and/or B6H12 using a cell sorter (Epics
Elite, Coulter, Fullerton, CA). Cells were cultured again for 57, days
and the positive clones were obtained.
Immunoprecipitation and Immunoblotting AnalysisTo determine
the association of IIb
3 and IAP, platelets
or transfected cells were lysed in an ice-cold lysis buffer (10 mM
Tris, pH 7.4, 100 mM NaCl, 1 mM CaCl2, 0.1%
Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 100 µg/ml
leupeptin). The lysates were chilled on ice for 1 h followed by centrifugation
at 15,000 x g for 10 min. The supernatants were precleared with
protein G-agarose (Immunopure Immobilized Protein G, Pierce), and the
resultant supernatants were incubated for 1 h with protein G-agarose that was
preincubated with antibodies T10, B6H12, or control antibody. Protein
G-agarose was washed with the lysis buffer three times. The samples were then
separated into two aliquots. One was solubilized with SDS sample buffer under
reduced conditions and applied to 7% polyacrylamide electrophoresis gel (PAGE)
for the detection of
IIb
3. The other was
incubated with SDS sample buffer under non-reduced conditions at 60 °C for
30 min and applied to 10% PAGE for IAP. The resolved proteins were then
electrophoretically transferred to polyvinylidene difluoride membranes. The
membranes were blocked with 10% skim milk in TBS buffer (10 mM
Tris, 150 mM NaCl, pH 7.4) and then incubated with
anti-
IIb
3 rabbit polyclonal serum or 1
µg/ml B6H12 for 2 h. After extensive washing with TBS containing 0.1% Tween
20, antibody binding was detected using peroxidase-conjugated anti-rabbit or
anti-mouse IgG and visualized with ECL chemiluminescence reaction reagents
(Amersham Biosciences).
MAP Kinase PhosphorylationTo determine the intracellular signaling events, MAP kinase activation was analyzed in the CHO cells expressing IAPs (34). Cells were incubated with 4N1K or control peptide for 10 min and lysed by the addition of an equal volume of 2x lysis buffer (35) (20 mM Tris, pH 7.4, 40 mM KH2PO4, 10 mM sodium orthovanadate, 40 mM molybdic acid, 80 mM sodium pyrophosphate, 0.2 mM trifluoroperazine, 2 mM EGTA, 20 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 200 µg/ml of leupeptin, and 2% Triton X-100). After removal of the insoluble fraction by centrifugation, cell lysates were subjected to immunoblotting with anti-phospho-MAPK antibody. Immunoblotting was performed as described above, except that blocking was with 2% bovine serum albumin. To reprobe with anti-MAPK antibody to detect the total MAPK levels, membranes were incubated in stripping buffer (62.5 mM Tris, pH 6.7, 100 mM 2-mercaptoethanol, 2% SDS) at 70 °C for 1 h, washed, and then used for the second immunoblotting.
Recombinant Soluble Form of IAP and Pull-down AssayAn
expression vector that sequentially introduces a FLAG epitope, His tag, and a
stop codon at 3'-end of cDNA in pBK-EF was kindly provided by Dr. K.
Fukudome (Saga Medical College, Saga City, Japan)
(36). To make recombinant
soluble IAP (rsIAP), the extracellular domain of IAP until Asn-142 was
PCR-amplified, digested, and ligated to this vector. Human 293 cells were
transfected and selected with G418. High producing clones were screened in
culture dishes by agarose diffusion immunoassay for secreted protein as
previously described (37).
Immunoassay was performed with B6H12 and the anti-FLAG antibody, M2. The cells
producing the highest amount of rsIAP were grown until semi-confluent, and the
culture medium was changed to serum-free medium for 293 cells, SFMII
(Invitrogen). After 5 days of culture, the medium was collected, and the rsIAP
was purified by a His-Trap chelating column (Amersham Biosciences) according
to the manufacturer's instructions. The purification procedure was repeated
twice, and the protein concentration was determined by BCA protein assay
(Pierce). The effects of rsIAP on the affinity state of
IIb
3 in CHO cells were analyzed by flow
cytometer.
To analyze the association of IIb
3 and
rsIAP in vitro, pull down assay was performed. Purified human
IIb
3
(32) and rsIAP (10 µg each)
were mixed in a buffer containing 50 mM Tris, pH 7.4, 150
mM NaCl, 1 mM CaCl2, 0.5 mM
MgCl2, 1% CHAPS, and then 100 µg peptide was added in a total
volume of 100 µl for 3 h. Protein G-agarose, which was preincubated with 5
µg of antibodies Tab or M2, was added and further incubated for 1 h. The
protein G-agarose was subsequently washed three times with the above buffer.
Bound proteins were released in the SDS sample buffer and then determined by
immunoblotting.
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RESULTS |
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The activation state of IIb
3 was
estimated by the binding of ligand mimetic antibody, PAC1
(Fig. 2). 4N1K induced PAC1
binding to platelets, whereas control peptide did not. The binding was
completely inhibited by T10 and also partially inhibited by B6H12. In the
energy-depleted platelets, 4N1K still induced PAC1 binding, although the
binding induced by ADP was completely blocked. B6H12 largely inhibited the
binding in this case. The effects were similar to that of activating antibody,
LIBS6. These results suggested that there are pathways in which 4N1K induces
the activation of
IIb
3 via IAP, and some
pathways do not require the intracellular signaling. In the following study,
we used the expression system in the heterologous cultured cells since 4N1K
did not induce the agglutination of these cells.
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4N1K Activates
IIb
3 Independently
with Intracellular Signaling EventsTo determine the mechanism
underlying the activation of
IIb
3 induced
by 4N1K, an expression study was performed in a human B-lymphocytic cell line,
Namalwa cells. These cells expressed a high amount of intrinsic IAP
(Fig. 3A). Wild type
cDNA of
IIb
3 was transfected into the
cells, and stable clones were selected. In the transfected cells, the
association of
IIb
3 and IAP was detected
because
IIb
3 and IAP was
co-immunoprecipitated by T10 and B6H12, as observed in platelets
(Fig. 3B). The
activation of
IIb
3 was analyzed with these
cells. 4N1K induced PAC1 binding although control peptide did not. PAC1
binding was almost completely blocked by T10 and B6H12, indicating that
activation of
IIb
3 was thoroughly mediated
by IAP in these cells (Fig. 4).
Treatment with sodium azide and 2-deoxy-D-glucose did not abolish
the binding of PAC1 (not shown). We prepared mutant cDNA of
3
(Ser-752 to Pro) and its transfectants. Platelets containing
IIb
3 with this mutation fail to bind
fibrinogen or aggregate upon agonist stimulation probably because
intracellular signaling cannot be transduced to the cytoplasmic domains
(39). The mutant
3 formed a complex with
IIb and associated
with IAP in the transfected cells, as did wild type
3, which
was demonstrated by immunoprecipitation assay
(Fig. 3B). 4N1K
induced PAC1 binding to the mutant
IIb
3 as
well as to the wild type. The binding was also likely mediated by IAP because
of B6H12 inhibition (Fig. 4).
The same results were observed in the three independent stable transfectants.
The results further support the theory that the activation of
IIb
3 can be induced by 4N1K independently
with intracellular signaling events.
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Activation of
IIb
3 by 4N1K Does Not
Require the Transmembrane and Cytoplasmic Domains of IAPWe next
examined the effects of deletion mutants of IAP on the activation of
IIb
3. For this purpose, we used CHO cells
since in our previous study
(26) intrinsic IAP of CHO
cells did not affect the function of human
IIb
3, although CHO cells may express
hamster IAP. In addition, 4N1K did not induce the activation of
IIb
3 in CHO cells, indicating that 4N1K
peptide derived from human TS fails to bind hamster IAP or fails to affect the
IIb
3 even if the peptide is bound to it.
Wild type IAP and two truncated mutants were prepared. However, a mutant with
a single transmembrane domain (IAP
163) was not effectively expressed
on the cell surface. Therefore, another mutant form was created in which the
extracellular domain of IAP was fused with the transmembrane and cytoplasmic
domains of an irrelevant transmembrane protein, IL-2 receptor (Tac antigen).
Three constructs (Fig.
5A: wild type IAP, IAP
291, and IAP-Tac) were
co-transfected into CHO cells with the cDNA of
IIb
3. The established clones expressed
comparable amounts of
IIb
3
(Fig. 5B). All IAP
constructs were recognized by antibody B6H12 and detected by immunoblotting at
an expected molecular size (Fig.
5C). The association of
IIb
3 with the wild type or mutant form IAP
(IAP
291) was detected by immunoprecipitation. However, the association
with IAP-Tac was hardly detected (Fig.
5D).
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4N1K induced PAC1 binding to all the cells expressing wild type IAP, IAP
291, and IAP-Tac, whereas the binding was not observed with control
peptide (Fig. 6A). T10
and B6H12 inhibited the binding, as observed in Namalwa cells (not shown).
Because the expression level of the three IAP forms varied
(Fig. 5B), PAC1
binding was standardized by each surface expression level. Namely, mean
fluorescence intensity of PAC1 binding in the presence of 4N1K was divided by
mean fluorescence intensity of B6H12 binding and calculated as an activation
index. The activation index was
0.8 with IAP
291 and 0.5 with
IAP-Tac, whereas the index with wild type was 1.0. We tested at least three
independent clones expressing each IAP construct and obtained the same
results.
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To confirm that the construct with only the extracellular domain of IAP did
not cause the intracellular signaling, we analyzed the phosphorylation of MAP
kinase. It was reported that the binding of 4N1K to IAP caused
Gi-mediated strong inhibition of MAP kinases, a unique phenomenon
among IAP-mediated signal pathways
(34). 4N1K caused
down-regulation of MAP kinase phosphorylation (mainly p44) in the CHO cells
expressing the wild type and the mutant IAP (IAP 291)
(Fig. 6B), although
total MAP kinase levels were not changed (not shown). In contrast, 4N1K did
not influence the phosphorylation in CHO cells expressing IAP-Tac, indicating
that intracellular signaling was not induced by the mutant form with only the
extracellular domain of IAP. We concluded that the alteration of
IIb
3 to the active form induced by 4N1K was
possible by only the extracellular domain of IAP.
4N1K-bound Extracellular Domain of IAP Can Associate with
IIb
3 and Activate
ItWe further analyzed the effects of rsIAP. Purified rsIAP was
recognized by B6H12 and detected as a single band at
30 kDa in
immunoblotting (Fig.
7A). Using CHO cells that expressed a high amount of
IIb
3 alone, the affinity state of
IIb
3 was analyzed. To our surprise, the
addition of srIAP together with 4N1K caused PAC1 binding to
IIb
3 on the surface of CHO cells
(Fig. 7B). With the
control peptide or with only srIAP or 4N1K, PAC1 binding was not induced.
Although the physical association of rsIAP and
IIb
3 was hardly detected by
immunoprecipitation using the CHO cells (not shown), the data suggested that
the rsIAP functionally interacted with
IIb
3
and caused its activation in the presence of 4N1K.
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To detect the association of srIAP with
IIb
3, we finally tried a pull-down assay
using purified proteins in the relatively mild detergent, 1% CHAPS
(Fig. 8). After the two
proteins were mixed,
IIb
3 was precipitated
by anti-
IIb antibody, Tab, and srIAP was precipitated by M2,
which recognized the FLAG epitope introduced at the C-terminal end of srIAP.
When 4N1K was present, a significant amount of srIAP was coprecipitated with
IIb
3 by Tab, whereas without the peptide or
with the control peptide, only a faint band of srIAP was detected. Conversely,
IIb
3 was detected in the precipitates by M2
only when 4N1K was added. These results indicated that the binding of 4N1K to
the extracellular Ig domain of IAP facilitated the physical association with
IIb
3.
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DISCUSSION |
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Tulasne et al.
(38) recently reported that
4N1K induces platelet agglutination in addition to aggregation, and the
agglutination is independent of IIb
3. Our
data corresponded in part with this since
anti-
IIb
3 inhibitory antibody only
partially inhibited platelet aggregation by 4N1K. They further concluded that
platelet aggregation or agglutination was not mediated by IAP because the
ability of 4N1K to stimulate aggregation was not altered in the platelets from
IAP-deficient mice. However, this is a still controversial. In previous
studies, Chung et al. conclude that the effect of 4N1K or TS on
platelet aggregation was absolutely dependent on IAP because the platelets
from IAP-deficient mice did not form aggregates in response to 4N1K or TS on
the collagen surface (17) and
also showed little spreading of the B6H12-treated platelets on immobilized
fibrinogen (19). The disparity
in findings may have resulted from different measurement conditions and is
probably due to difficulty in separating the platelet agglutination and
aggregation.
Our results supported the presence of IAP-mediated platelet aggregation
since B6H12 partially inhibited it. The data suggested that there are at least
three components by which platelets form aggregates in response to 4N1K. The
first part is aggregation, which requires inside-out signaling. This was shown
by the decrease in aggregation or intensity of PAC1 binding in the
energy-depleted platelets compared with non-treated platelets. The second is
agglutination, which was shown by the residual response of the energy-depleted
platelets in the presence of antibodies to
IIb
3 and IAP. The third is aggregation,
which was represented by the deceased part by the antibodies in the
energy-depleted platelets. The last aggregation does not require intracellular
signaling but is mediated by
IIb
3 and
IAP.
In previous studies using cultured cells, the effects of 4N1K were clearly
demonstrated probably because cells other than platelets did not show
agglutination. 4N1K can modulate the function of
V
3 on endothelial or melanoma cells and
2
1 on smooth muscle cells
(15,
34). IAP-expressing cells
spread much more rapidly on vitronectin in the presence of 4N1K
(18). The attachment and
migration of smooth muscle cells on matrices through
2
1 was dramatically stimulated by 4N1K, and
this increased migration was blocked by antibodies recognizing either
2
1 or IAP
(22). In smooth muscle cells
from IAP-deficient mice, 4N1K did not stimulate migration
(34). All these experiments
represent clear proof that the 4N1K peptide acts through IAP to augment
integrin functions. In this study, 4N1K induced PAC1 binding to
IIb
3 in Namalwa and CHO cells, and the
effects were dependent on IAP since PAC1 binding was completely inhibited by
B6H12 in these cells.
It has been reported that the activation of Gi-containing
heterotrimeric GTPases is involved in intracellular signaling induced by IAP
(15,
16). 4N1K caused an immediate
and dramatic fall in cAMP levels within the cells, including platelets. The
inhibition of phosphorylation of MAP kinase was also likely mediated by
Gi protein (34).
Another study showed that the Fc receptor -chain was associated with
signaling in 4N1K-induced platelet aggregation
(38). Our data also suggested
the presence of these signaling pathways in addition to the direct activating
mechanism of
IIb
3. Indeed, platelet
aggregation induced by 4N1K was partially inhibited by energy depletion. By
this treatment, Gi-coupled or Fc
chain-associated signaling
was completely abolished; this was monitored by the absence of ADP or
collagen-induced response since it is known that the major platelet ADP
receptor, P2Y12, is coupled with Gi
(28) and collagen receptor
GPVI is coupled with the Fc
chain
(40). The binding of PAC1 to
the energy-depleted platelets was detected but decreased compared with that of
non-treated platelets. 4N1K induced the activation of the mutant
IIb
3 (Ser-752 to Pro) but to a lower extent
than the activation of wild type
IIb
3 in
Namalwa cells. Activation of
IIb
3 was
induced with the truncated form of only the extracellular Ig domain of IAP
(IAP-Tac) in CHO cells, but when PAC1 binding was standardized by its surface
expression, the PAC1 intensity was approximately half compared with that with
wild type IAP. One possible explanation for this is a lower efficiency of the
association with
IIb
3 since the association
between IAP-Tac and
IIb
3 was hardly
detected by immunoprecipitation. However, it might account for the absence of
intracellular signaling.
The structural requirement for physical and functional association was
examined only for IAP association with V
3.
Lindberg et al. (41)
demonstrate that the Ig domain of IAP was required for functional cooperation
with
V
3. Cells expressing only Ig domain
attached to the plasma membrane with a glycan phosphoinositol anchor or a CD7
single transmembrane segment facilitated the ligand binding function of
V
3. They also showed that these constructs
were hardly immunoprecipitated with
V
3 or
only a small minority of total
V
3 was
coprecipitated. Our data support the importance of Ig domain also in the
association with
IIb
3. Using a similar
construct of IAP-Tac and the recombinant soluble Ig domain of IAP, we further
demonstrated direct evidence that upon binding to TS, the Ig domain can
physically associate with the extracellular domain of
IIb
3 with a higher affinity and then change
the
IIb
3 to the active form. It was
suggested that the physical association requires a higher affinity interaction
between IAP and integrins than functional association since the association
between IAP-Tac and
IIb
3 was not detected
by immunoprecipitation, which was in agreement with the above report. The
multiple membrane-spanning domain of IAP may stabilize the physical
association with integrins
(42).
Rebres et al. (43)
report the presence of a long range disulfide bond between the extracellular
and membrane-spanning domains of IAP (Cys-33 and Cys-263). They demonstrated
that IAP with the mutation of the cysteines showed impaired signal
transduction and reduction of binding to another IAP ligand, signal-regulatory
protein. But loss of the disulfide bond did not affect the association with
V
3. Therefore, this disulfide bond is
likely to be important for signaling events rather than interaction with
integrins. The construct IAP-Tac or rsIAP used in our study theoretically did
not contain this disulfide bond. However, consistent with their report, these
recombinant forms were sufficient for interaction with
IIb
3.
Cells can regulate the integrin-mediated response by changing their
affinity for ligands. Rapid changes in affinity have been widely documented
among a number of integrins including not only 3 but also
1,
2, and
7 classes
(2). The regulation of integrin
function or inside-out signaling is believed to be mediated by the cytoplasmic
domains. The interaction between integrin
and
subunit
cytoplasmic tails or the linkage with cytoplasmic proteins can regulate the
affinity of the extracellular domain by an allosteric mechanism
(8). This may involve
transmission of long range dynamic conformational rearrangements, namely from
a bent conformation to a high affinity extended structure
(13). Our data suggested that
one of the signals that can induce such conformational change may be
transduced at the extracellular domains of
IIb
3 by the Ig domain of IAP, which may not
require the long range conformational rearrangement from its cytoplasmic tail.
Ligand mimetic peptides or Mn2+ can directly induce a
conformational change in integrin extracellular domains
(13). Several activating
monoclonal antibodies, including LIBS6, may also change the extracellular
conformation. The mechanism of
IIb
3
activation by TS-bound IAP Ig domain might resemble that by such antibodies.
The activating antibodies usually recognize the
subunit of the
integrin. Some mutations within the cysteine-rich region of the
subunit
can confer the naturally active form of
IIb
3 integrin
(44). Thus, it should be
determined which subunit of
IIb
3 integrin
would be a binding site for TS-bound IAP. In addition, further study is needed
to determine whether the phenomenon observed here could be widely demonstrated
among other integrins.
Nonetheless, the extracellular event demonstrated in this study would facilitate the dynamic change in the conformation of the integrin extracellular domain and represents a novel mechanism of affinity modulation of integrins.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Hematology and Oncology,
Division of Clinical Pharmacotherapeutics, Program for Applied Biomedicine,
Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi,
Minami-Ku, Hiroshima 734-8551, Japan. Tel.: 81-82-257-5298; Fax:
81-82-257-5299; E-mail:
fujimot{at}hiroshima-u.ac.jp.
1 The abbreviations used are: IAP, integrin-associated protein; srIAP,
soluble recombinant form of IAP; TS, thrombospondin; FITC, fluorescein
isothiocyanate; MAP, mitogen-activated protein; CHO, Chinese hamster ovary;
IL-2, interleukin 2; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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