(Received for publication, May 4, 1995; and in revised form, June 5, 1995)
From the
The ligand binding activity of the platelet integrin
The ligand binding activity of many integrins is enhanced by
cell stimulation(1) . A paradigm for this phenomenon is the
interaction of the platelet integrin Like
To
construct a cDNA encoding the extracellular domain of
Figure 1:
Schematic diagram of the subunits of
A cDNA encoding the extracellular domain of
A related strategy was used to construct cDNAs encoding the
extracellular and transmembrane domains of
Figure 2:
Demonstration of
Figure 3:
Photomicrographs of B lymphocytes
expressing
To further characterize
the adherence of lymphocytes expressing
Figure 4:
Characterization of the adherence of
lymphocytes expressing
Next, we compared the
adherence of lymphocytes expressing
Figure 5:
Comparison of the unstimulated and
PMA-stimulated adherence of lymphocytes expressing either
Figure 6:
Comparison of the unstimulated and
PMA-stimulated adherence of lymphocytes expressing either
Figure 7:
Comparison of the unstimulated and
PMA-stimulated adherence of lymphocytes expressing heterodimers
composed of
Intracellular signals initiated by platelet agonists convert
the platelet integrin In addition to stimulating the adherence of lymphocytes expressing
Figure 8:
Alignment of the amino acid sequences of
the cytoplasmic domains of
In contrast to the lack of effect of
exchanging the In our initial
experiments, we expressed double chimeras in which the cytoplasmic and
transmembrane domains of How might the The identity
of the signal transduction pathways that activate integrins in
lymphocytes and platelets are unknown, but our data indicate that
similar pathways involving phorbol ester-sensitive protein kinase C are
present in both cells. On the other hand, it has not been possible to
activate recombinant
IIb
3 is initiated by agonist-generated intraplatelet signals.
We studied this process in vitro by expressing recombinant
IIb
3 in Epstein-Barr virus-immortalized B lymphocytes. We
found that phorbol ester stimulation induced the adhesion of
lymphocytes expressing
IIb
3 to immobilized fibrinogen.
Moreover, replacement of the transmembrane and cytoplasmic domains of
the
and
subunits of
IIb
3 with those of
L
2 significantly increased adherence, whereas replacement of
only the cytoplasmic domains significantly decreased adherence. This
suggests that transmembrane segments are involved in the
agonist-induced modulation of
IIb
3 activity. Similar results
were seen when the
IIb
3 activation-dependent monoclonal
antibody PAC-1 was substituted for immobilized fibrinogen. We also
found that the adherence of lymphocytes expressing
3 with either
of the two
IIb/
L chimeras was similar to that of cells
expressing
IIb
3, whereas the adherence of cells expressing
IIb with either of the two
3/
2 chimeras was
substantially decreased, suggesting that the identity of the
cytoplasmic domain of
3, but not of
IIb, is critical for
IIb
3 function. This report indicates that B lymphocytes
contain signal transduction pathways involving protein kinase C that
can increase the ligand binding activity of
IIb
3 and
demonstrates the utility of these cells as an expression system for the
study of agonist-stimulated
IIb
3 function.
IIb
3 (GPIIb-IIIa) with
fibrinogen and von Willebrand factor(2) . Although
IIb
3 on resting platelets does not bind soluble fibrinogen or
von Willebrand factor, binding readily occurs when platelets are
stimulated by agonists such as thrombin and ADP. The agonist-generated
``inside-out'' signals that convert
IIb
3 from an
inactive to a ligand binding conformation do so by interacting with one
or both cytoplasmic domains of the
IIb
3
heterodimer(3) . The signals are then propagated across the
length of
IIb
3 to expose a ligand binding site located in its
extracellular domain(4) . The nature of the signaling pathways,
their targets on the cytoplasmic domains of
IIb
3, and the
mechanism by which the signals are transmitted across the molecule are
largely unknown. Moreover, it has not been possible to reproduce
agonist-induced
IIb
3 activation in vitro using
recombinant heterodimers because conventional expression systems do not
support agonist-induced
IIb
3 activation(5) .
IIb
3 in platelets, the ligand binding activity of integrins
in leukocytes is enhanced by agonists(1) . For example, the
activity of
L
2 (LFA-1) in lymphocytes can be up-regulated by
cross-linking the T-cell receptor (6) or by stimulating the
cell with a phorbol ester such as phorbol 12-myristate 13-acetate
(PMA)(
)(7) . It has also been reported that agonists
up-regulate the ligand binding activity of lymphocyte
v
3 (8) . Accordingly, we hypothesized that it might also be
possible to generate signals capable of activating
IIb
3 in
lymphocytes and tested this hypothesis by expressing recombinant
IIb
3 in Epstein-Barr virus (EBV)-immortalized B lymphocytes.
In addition, because of the possibility that recognition of
IIb
3 by the signal transduction pathways in lymphocytes might
require the presence of the cytoplasmic and transmembrane domains of
L
2, we also expressed chimeras of
IIb
3 and
L
2 in these cells.
Construction of
Chimeric integrin subunit cDNAs were constructed
in the EBV-based episomal plasmids pREP4 and pREP9 (9) (gifts
of Dr. M. Tykocinski, Case Western Reserve University) by a modified
overlap polymerase chain reaction (PCR) procedure(10) .IIb/
L and
3/
2
Chimeras
IIb and the
transmembrane and cytoplasmic domains of
L (Fig. 1, 2LL), a
full-length
IIb cDNA was first cloned into pREP9 using KpnI and HindIII. An overlap PCR fragment containing
the transmembrane and cytoplasmic domains of
L was then
synthesized using the primers
GCTCCGGGCCTTGGAGGAGCGCCAGATGCTCTACCTCTACGTGCTG and
TGTGAAGCTTCAGACATTCTCTTCCAAGG with cDNA from the EBV-transformed B cell
line GM1500 (11) (a gift of Dr. Y. Tsujimoto, Osaka Medical
College, Japan) as a template. The resulting DNA fragment was used as a
primer along with the
IIb 5`-primer GCGGAATTCACAAGCGGGATCGCAGACAG
to generate a chimeric fragment using
IIb as the template. The PCR
product was sequenced and subcloned into
IIb using BglII
and HindIII.
IIb
3 and of chimeric subunits consisting of the extracellular
domains of
IIb
3 (EX) and the transmembrane (TM) and cytoplasmic (CY) domains of
L
2.
3 and the transmembrane and cytoplasmic domains of
2 (Fig. 1, 3LL) was constructed after a full-length
3 cDNA
was cloned into pREP4 using KpnI and XhoI. An overlap
PCR fragment containing the transmembrane and cytoplasmic domains of
2 was then made using the primers
GTGGTAGAAGAGCCAGAGTGTGTGGCCGGCCCCAACATCGC and
GGTCCTCGAGGGATGTCATTTTATACCCTGAC with Jurkat cell cDNA as a template.
The resulting fragment was used as a primer along with the
3
5`-primer CACGAATTCGAGATTGAGTCAGTGAAAGAGC to generate a chimeric
fragment using
3 as a template. The PCR product was sequenced and
subcloned into
3 in pREP4 using AflII and XhoI.
IIb and
3 and the
cytoplasmic domains of
L and
2, respectively. To construct a
cDNA encoding the extracellular and transmembrane domains of
IIb
and the cytoplasmic domain of
L (Fig. 1, 22L), PCR
mutagenesis was used to introduce a DraI site into the
sequence encoding the amino acids Phe-Lys at the junction between the
transmembrane and cytoplasmic domains of both
IIb (12) and
L(13) . The modified cDNAs were then digested with DraI, and the proximal fragment of
IIb and distal
fragment of
L were ligated and subcloned into pREP4. To construct
a cDNA encoding the extracellular and transmembrane domains of
3
and the cytoplasmic domain of
2 (Fig. 1, 33L), a BclI site was introduced into the sequence encoding for amino
acids Leu-Ile-Thr at the junction between the transmembrane and
cytoplasmic domains of
3 (14) and a BglII site
into the sequence encoding for amino acids Arg-Ile-His of
2(15) . The two portions were digested with their
respective enzymes, ligated at the compatible BclI/BglII ends, and subcloned into pREP9.
Expression of
pREP4 and pREP9 containing cDNAs encoding
wild-type IIb
3 and Chimeras of
IIb
3 and
L
2 in Human B
Lymphocytes
IIb and
3 or the
and
subunit chimeras
described above were cotransfected into 7.5
10
GM1500 B lymphocytes by electroporation (Gene Pulser, Bio-Rad,
250 V and 960 millifarads). Stable cotransfectants were selected by
growth in RPMI media containing 20% fetal calf serum and both G418 (750
µg/ml) and hygromycin (250 µg/ml). The presence of
IIb
3 and the chimeras of
IIb
3 and
L
2 on
the lymphocyte surface was detected by flow cytometry and by
immunoprecipitation. For flow cytometry, cells were stained with either
fluoroscein-conjugated anti-murine IgG alone or with the
IIb
3-specific murine monoclonal antibody (mAb) A2A9 (16) followed by fluorosceinconjugated anti-murine IgG. Flow
cytometry was performed using a FACScan flow cytometer (Becton
Dickinson) as previously described(17) . For
immunoprecipitation, the lymphocyte surface was labeled with
I using lactoperoxidase(17) .
IIb
3 and
the chimeric integrins were then immunoprecipitated using the
3-specific mAb SSA6 (17) and visualized following
SDS-polyacrylamide gel electrophoresis and autoradiography as
previously described(18) .
Measurement of Lymphocyte Adherence to Immobilized
Fibrinogen
The function of IIb
3 and the chimeras
of
IIb
3 and
L
2 in B lymphocytes was tested by
measuring the adherence of PMA (Sigma)-stimulated transfected cells to
immobilized fibrinogen. In some experiments, bovine serum albumin or
the mAb PAC-1 (19) was substituted for fibrinogen. For these
assays, the wells of Immulon 2 flat bottom microtiter plates (Dynatech
Laboratories) were coated with either 50 µg/ml purified human
fibrinogen in 50 mM NaHCO
buffer, pH 8.0,
containing 150 mM NaCl, or 40 µg/ml PAC-1 in the same
buffer. Unoccupied protein binding sites on the wells were then blocked
with 5 mg/ml bovine serum albumin dissolved in the same buffer. 1.5
10
lymphocytes, metabolically labeled overnight
with [
S]methionine, were suspended in 100 µl
of 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.5 mM CaCl
, 0.1% glucose, and 1%
bovine serum albumin and added to the protein-coated wells. Following
an incubation at 37 °C without agitation, the plates were
vigorously washed four times with the lymphocyte suspension buffer, and
adherent cells were dissolved using 2% SDS. The SDS solutions were then
counted for
S in a liquid scintillation counter.
Expression of
Heterodimers composed of IIb
3 and Chimeras of
IIb
3 and
L
2 in B
Lymphocytes
IIb
3 or
chimeras of
IIb
3 and
L
2 (Fig. 1) were stably
expressed in EBV-immortalized human GM1500 B lymphocytes using the
EBV-based plasmids pREP9 and pREP4. As shown in Fig. 2A, the
and
subunits of these
heterodimers were immunoprecipitated from detergent extracts of
surface-labeled cells using the anti-
3 mAb SSA6, demonstrating
that the expressed heterodimers had been transported to the lymphocyte
surface. Moreover, the presence of the
IIb heavy chain
(
IIb
) in the immunoprecipitates indicates that the
heterodimers had undergone correct intracellular processing in these
cells(12) . Faint bands corresponding to
3 and the heavy
chain of the vitronectin receptor,
V, were present in
immunoprecipitates from mock-transfected cells, consistent with the
previous reports that B lymphocytes constitutively express low levels
of
V
3(8) . As shown in Fig. 2B, flow
cytometry using the
IIb
3 heterodimer-specific mAb A2A9
revealed that comparable levels of
IIb
3 and the chimeras were
expressed on the lymphocyte surface.
IIb
3 and
chimeras of
IIb
3 and
L
2 on the surface of
transfected lymphocytes by immunoprecipitation and flow cytometry. A,
IIb
3 and the 2LL/3LL and 22L/33L chimeras were
immunoprecipitated using the
3-specific mAb SSA6 from Triton X-100
extracts of transfected lymphocytes whose surface had been labeled with
I as described under ``Experimental
Procedures.''
IIb
corresponds to the heavy chain of
IIb;
corresponds to the
subunit of the heterodimer. B, lymphocytes transfected with the plasmids pREP 4 and pREP 9
(mock-transfected cells) and lymphocytes expressing
IIb
3 or
the 2LL/3LL and 22L/33L chimeras were stained with either
fluoroscein-conjugated anti-murine IgG alone (No A2A9) or with the
IIb
3 heterodimer-specific mAb A2A9 followed by
fluoroscein-conjugated anti-murine IgG (A2A9) and analyzed by flow
cytometry as described under ``Experimental Procedures.'' The
histograms of mock-transfected cells stained under both sets of
conditions overlap, indicating no detectable A2A9 binding to these
cells.
Adherence of B Lymphocytes Expressing
The ability of IIb
3
to Immobilized Fibrinogen
IIb
3 in
B lymphocytes to interact with ligands was tested by measuring the
adherence of phorbol ester-stimulated lymphocytes to the wells of
microtiter plates coated with purified fibrinogen. Because the ligand
binding activity of the endogenous lymphocyte integrin
L
2
requires the presence of Mg
(20) , whereas the
interaction of
IIb
3 with fibrinogen occurs in the presence of
either Mg
or Ca
(21) ,
L
2-mediated lymphocyte function was prevented by performing
the assays in a Mg
-free buffer containing 0.5
mM Ca
. As shown by the photomicrographs in Fig. 3, few unstimulated lymphocytes expressing
IIb
3
attached to immobilized fibrinogen over a 30-min period. However,
stimulation of these cells with 50 nM PMA resulted in a marked
increase in the number of adherent cells over the same time period.
Adherence was inhibited by the presence of 1 mM EDTA,
indicating that it was calcium-dependent. Because flow cytometry before
and after PMA stimulation revealed no differences in the extent of
IIb
3 expression (data not shown), it is likely that the
increased adherence induced by PMA resulted from an increase in the
avidity of
IIb
3 for fibrinogen.
IIb
3 adherent to immobilized fibrinogen. The
adherence of unstimulated lymphocytes (No PMA), lymphocytes stimulated
with 50 nM PMA (PMA), and lymphocytes stimulated with 50
nM PMA in the presence of 1 mM EDTA (PMA + EDTA)
to fibrinogen immobilized on microtiter plates was examined as
described under ``Experimental Procedures.'' The plates were
then washed four times and examined by light
microscopy.
IIb
3 to fibrinogen,
cells were metabolically labeled with
[
S]methionine and the number of adherent cells
quantitated under a variety of conditions. Fig. 4A shows the time course of lymphocyte adherence. In the absence of
PMA stimulation, adherence was minimal throughout a 60-min period of
incubation. In contrast, PMA stimulation resulted in a significant
increase in adherence within 5 min (p = 0.014), and
adherence was maximal at 30 min. To confirm that adherence was mediated
by the extracellular domain of
IIb
3, lymphocytes were
preincubated with 50 µg/ml A2A9, a concentration of antibody
sufficient to prevent fibrinogen binding to platelet
IIb
3(16) . In the presence of A2A9, PMA-stimulated
adherence was reduced to the level observed when albumin was
substituted for fibrinogen or when the assay was performed in the
presence of 1 mM EDTA (Fig. 4B). Moreover, an
identical concentration of the
3-specific mAb SSA6, an antibody
that has no effect on fibrinogen binding to platelet
IIb
3(17) , had no effect on lymphocyte adherence.
Fibrinogen binding to
IIb
3 is also inhibited by peptides
containing the amino acid sequence RGDS (22) . RGDS, at a
concentration of 5 mM, completely inhibited PMA-stimulated
adherence, whereas the same concentration of the control peptide RGES
had only a minimal effect (Fig. 4B). The IC
of RGDS in this assay was
400 µM (Fig. 4C),
20-fold greater than its IC
for inhibiting thrombin-stimulated platelet adhesion to
immobilized fibrinogen(23) , suggesting that the avidity of
lymphocytes expressing
IIb
3 for immobilized fibrinogen is
substantially greater than that of platelets.
IIb
3 to immobilized fibrinogen. The
adherence of lymphocytes metabolically labeled with
[
S]methionine and expressing
IIb
3 was
quantitated by dissolving adherent cells in 2% SDS and measuring the
amount of
S in the SDS solution as described under
``Experimental Procedures.'' Each measurement of cell
adherence was made in quadruplicate. A, time course of
unstimulated and PMA-stimulated lymphocyte adherence. B,
effect of various inhibitors on PMA-stimulated lymphocyte adherence.
PMA-stimulated lymphocyte adherence to immobilized fibrinogen or bovine
serum albumin was quantitated as described above. The consequences of
preincubating the cells with 50 µg/ml mAb A2A9, 1 mM EDTA,
50 µg/ml mAb SSA6, or 5 mM of the tetrapeptides
Arg-Gly-Asp-Ser (RGDS) or Arg-Gly-Glu-Ser (RGES) on adherence to
fibrinogen were then determined. The data were normalized to 100%
adherence in the absence of inhibitors to facilitate comparison. C, effect of RGDS concentration on PMA-stimulated adherence.
100% represents adherence in the absence of
RGDS.
IIb
3 to that of cells
expressing the 2LL/3LL and 22L/33L chimeras (Fig. 5). In the
absence of PMA stimulation, 0.3% of mock-transfected cells, 3.8% of
cells expressing
IIb
3, 7.3% of cells expressing 2LL/3LL, and
4.5% of cells expressing 22L/33L were adherent to fibrinogen. Following
PMA stimulation, the adherence of the mock-transfected cells did not
change, while the adherence of cells expressing
IIb
3,
2LL/3LL, and 22L/33L increased to 26.2, 37.4, and 17.9%, respectively.
Adherence of both the unstimulated and PMA-stimulated cells expressing
IIb
3, 2LL/3LL, and 22L/33L was reduced to that of the
mock-transfected cells by the addition of 1 mM EDTA or the mAb
A2A9 (data not shown). Moreover, the differences in adherence of the
PMA-stimulated cells expressing
IIb
3 versus 2LL/3LL
and 2LL/3LL versus 22L/33L were statistically significant at p < 0.02 and p < 0.0005, respectively (t test for unpaired samples). Thus, these data indicate that in B
lymphocytes, the ligand binding activity of
IIb
3 is
unaffected by replacing its cytoplasmic domains with those of
L
2. Furthermore, the difference in adherence of cells
expressing 2LL/3LL versus 22L/33L suggests that transmembrane
segments are involved in agonist-induced modulation of
IIb
3
activity.
IIb
3 or the
IIb
3-
L
2 chimeras to
immobilized fibrinogen. Mock-transfected lymphocytes and lymphocytes
expressing
IIb
3 or the 2LL/3LL and 22L/33L chimeras were
metabolically labeled with [
S]methionine and
incubated with immobilized fibrinogen as described in Fig. 3and Fig. 4and under ``Experimental Procedures.'' Each
measurement of cell adherence was performed in quadruplicate. The data
are the mean and standard error of 4-8 separate
experiments.
Adherence of B Lymphocytes Expressing
Platelet agonists, including PMA, activate
IIb
3
and
IIb
3-
L
2 Chimeras to Immobilized
PAC-1
IIb
3 by inducing a conformational change in the molecule that
exposes its ligand binding site(24) . Similarly, PMA enhances
lymphocyte adherence to ICAM-1 by converting
L
2 from a low
affinity to a high affinity state(7, 25) .
Nevertheless, it has been suggested that PMA-stimulated adhesion is a
post-receptor event(26) , perhaps the result of a
cytoskeleton-mediated rearrangement of integrins on the cell surface (27) . To address the mechanism of PMA-stimulated lymphocyte
adherence to fibrinogen, we repeated the lymphocyte adherence assay,
substituting the mAb PAC-1 for immobilized fibrinogen (Fig. 6).
PAC-1 is an IgM mAb that only interacts with
IIb
3 in its
activated conformation(19) . In the absence of PMA stimulation,
4.4% of cells expressing
IIb
3, 4.7% of cells expressing
2LL/3LL, and 4.4% of cells expressing 22L/33L were adherent to
immobilized PAC-1. Following PMA stimulation, the adherence of cells
expressing
IIb
3, 2LL/3LL, and 22L/33L increased to 29.8,
35.1, and 24.7%, respectively. Adherence of each cell type declined to
0.5, 0.7, and 0.2, respectively, in the presence of 1 mM EDTA
(data not shown), and there was essentially no adherence of the cells
to immobilized non-immune IgM, regardless of whether they were
unstimulated or stimulated with PMA. Although the differences in the
magnitude of adherence of the PMA-stimulated cells expressing
IIb
3 versus 2LL/3LL and 2LL/3LL versus 22L/33L were smaller than when fibrinogen was the ligand, the
differences remain statistically significant at p < 0.001
and p < 0.0001, respectively. Thus, these data support a
conclusion that the PMA-stimulated adherence we observed resulted from
a conformational change in the extracellular domain of
IIb
3.
Whether a cytoskeletal rearrangement was also involved in this process
remains to be determined.
IIb
3 or the 2LL/3LL and 22L/33L chimeras to immobilized PAC-1
or non-immune IgM. The wells of microtiter plates were coated with
either purified PAC-1 or non-immune murine IgM (Sigma), each at a
concentration of 40 µg/ml. The adherence of unstimulated
lymphocytes and lymphocytes stimulated with 50 nM PMA was
measured as described in Fig. 5. Each measurement of cell
adherence was performed in quadruplicate. The data are the mean and
standard error of 2-8 separate
experiments.
Adherence of B Lymphocytes Expressing either
The preceding experiments indicated that
IIb
or
3 and a Complementary Chimeric Subunit to Immobilized
Fibrinogen
IIb
3 activation in lymphocytes was most efficient when the
transmembrane and cytoplasmic domains of the heterodimer were derived
from the same integrin. To study the role of individual transmembrane
and cytoplasmic domains in this process, we measured the adherence of
lymphocytes expressing
IIb with either 3LL or 33L and
3 with
either 2LL or 22L to immobilized fibrinogen (Fig. 7). In the
absence of PMA stimulation, 6.5% of lymphocytes expressing 2LL/
3
and 7.6% of lymphocytes expressing 22L/
3 were adherent to
fibrinogen. Following PMA stimulation, adherence increased to 28.6 and
33.9%, respectively. In contrast, only 0.9 and 1.5% of cells expressing
IIb/3LL or
IIb/33L were adherent in the absence of PMA
stimulation, and adherence increased to only 10.5 and 13.3% following
PMA stimulation. Moreover, because GM1500 cells express a low level of
endogenous
3 (Fig. 2), it is possible that part of the
adherence of cells expressing
IIb/3LL or
IIb/33L was actually
mediated by
IIb
3. Thus, the measured adherence may overstate
the function of the
IIb/3LL and
IIb/33L chimeras.
Accordingly, these experiments indicate that the cytoplasmic domain of
3 is specifically involved in
IIb
3 activation in
lymphocytes. They also suggest that compatibility of the
IIb
3
cytoplasmic domains is important in this process.
3 and the 2LL and 22L chimeras or
IIb and the 3LL
and 33L chimeras to immobilized fibrinogen. The adherence of
unstimulated lymphocytes and lymphocytes stimulated with 50 nM PMA was measured as described in Fig. 5. Each measurement
of cell adherence was performed in quadruplicate. The data are the mean
and standard error of the mean of 2-10 separate
experiments.
IIb
3 from a functionally inactive to a
functionally active conformation, enabling it to interact with ligands
such as fibrinogen(2) . The signaling pathways responsible for
inducing this conformational change may be unique to platelets because
it has not been possible to use cellular agonists to activate the
endogenous
IIb
3 expressed by megakaryocyte-like cell lines or
recombinant
IIb
3 expressed in heterologous cells(5) .
Nevertheless, the ability of cells to modulate integrin function is a
general phenomenon (1) . Consequently, we postulated that
lymphocytes, as cells of hematopoietic origin, might contain signal
transduction pathways capable of activating
IIb
3 and
expressed this integrin in EBV-immortalized B lymphocytes. Using an
adherence assay similar to that used by others to study the activity of
L
2 in lymphocytes (7) , we found that the adherence
of lymphocytes expressing
IIb
3 to immobilized fibrinogen
increased nearly 7-fold when they were incubated with the phorbol ester
PMA. Moreover, like platelets, the interaction of the transfected
lymphocytes with fibrinogen was inhibited by calcium chelation, by the
mAb A2A9, and by the peptide
RGDS(16, 21, 22) . Nevertheless, it has been
observed that unstimulated platelets can adhere to fibrinogen but by a
process that is independent of platelet metabolic
activity(23, 28) . Similarly, we found that
4% of
transfected lymphocytes adhere to fibrinogen in the absence of PMA
stimulation. Thus, it is possible that the PMA-stimulated adherence we
observed simply represents an enhanced interaction of inactive
IIb
3 with fibrinogen. To address this possibility, we
measured lymphocyte adherence to the activation-dependent
anti-
IIb
3 mAb PAC-1 (19) and found that adherence to
PAC-1 correlated well with adherence to fibrinogen. Accordingly, these
data suggest that the
IIb
3 in a fraction of the unstimulated
lymphocytes is present in an activated state and that the increase in
adherence we observed following PMA stimulation resulted from an
increase in the number of lymphocytes expressing active
IIb
3.
IIb
3 to fibrinogen, we found that PMA stimulated the
adherence of lymphocytes expressing chimeras of
IIb
3 and
L
2 to the same substrate. This result allowed us to examine
the contribution of individual cytoplasmic domains to
IIb
3
function. Despite substantial differences in the sequences of the
cytoplasmic domains of
IIb and
L (Fig. 8), we found
that PMA-stimulated adherence mediated by heterodimers composed of
3 and either the 22L or 2LL
subunit chimeras was equivalent
to adherence mediated by
IIb
3. This result is consistent with
previous studies of the function of the integrin
2
1 in which
it was found that replacing the cytoplasmic domain of
2 with the
cytoplasmic domains of either
4 or
5 did not affect
constitutive or PMA-stimulated
2
1 activity in RD or K562
cells(29) . However, truncation of the
2 cytoplasmic
domain distal to its GFFKR sequence (Fig. 8) eliminated both
constitutive and PMA-stimulated
2
1 activity, demonstrating
that the presence of an
subunit cytoplasmic domain is necessary
for
2
1 function. On the other hand, it has been reported that
truncation of the cytoplasmic domain of
L distal to GFFKR has no
effect on
L
2 function in COS cells(7) , and
truncation of the cytoplasmic domain of
IIb proximal to GFFKR or
replacing the cytoplasmic domain with the cytoplasmic domain of
5
results in constitutive
IIb
3 activity in Chinese hamster
ovary cells(5) . Nevertheless, our results indicate that like
2, the exact identity of the cytoplasmic domain of
IIb is not
critical for
IIb
3 function, perhaps because the GFFKR
sequence was preserved.
IIb
3 and
L
2. The :
indicates the position of identical amino acids in the alignment. The numbers represent the carboxyl-terminal amino acid of each
sequence(12, 13, 14, 15) . The
positions of the membrane-proximal sequences postulated to be involved
in integrin activation are underlined.
IIb and
L cytoplasmic domains on lymphocyte
adherence to fibrinogen, we found that both unstimulated and
PMA-stimulated adherence was significantly diminished when the
cytoplasmic domain of
3 was replaced by that of
2. This
result is consistent with the virtual elimination of constitutive and
PMA-stimulated lymphocyte adherence to ICAM-1 following deletion of the
2 cytoplasmic domain (7) and suggests that specific amino
acids in the cytoplasmic domain of
3 are involved in modulating
IIb
3 function. Mutagenesis of the
2 cytoplasmic domain
indicated that the carboxyl-terminal amino acids 757-761 (ATTTV)
and Phe
are required for
L
2 function (Fig. 8)(30) . The corresponding amino acids in the
3 cytoplasmic domain are ATSTF (residues 750-754) and
Tyr
(14) . Moreover, a naturally occurring
mutation in
3, Ser
Pro, prevents platelet
aggregation(31) , confirming the importance of this residue for
IIb
3 activity. However, conversion of
2 Thr
Ser or
2 Phe
Tyr, such that this
portion of the
2 cytoplasmic domain resembles that of
3, has
no effect on
L
2 function(30) . Consequently, it is
unlikely that differences in the carboxyl-terminal regions of the
2 and
3 cytoplasmic domains can account for the results of
our experiments. It has also been reported that amino acids located in
the membrane-proximal region of the
1 cytoplasmic domain,
corresponding to the amino acid sequence DRKEFAKFEEE in human
3 (Fig. 8), are involved in localizing
1-containing integrins
in focal adhesions (32) . The secondary structure of this
1 sequence was predicted to be an
-helix(32) . Thus,
it is conceivable that a similar helix in
3 interacts with as yet
unidentified signaling molecules, with the cytoskeleton, or with a
complementary sequence in the
subunit cytoplasmic domain to
up-regulate
IIb
3 function. The stretch of amino acids in
2 analogous to
3, DLREYRRFEKE, contains notable differences,
perhaps accounting for the diminished ability of the
2 cytoplasmic
domain to support
IIb
3 function.
IIb and
3 were replaced by the
corresponding domains of
L and
2. We found that the adherence
of lymphocytes expressing 2LL/3LL to fibrinogen was increased compared
to lymphocytes expressing
IIb
3, perhaps reflecting a
preference of the lymphocyte signaling mechanism for
2 integrins.
Conversely, we found that replacing the transmembrane domains of
2LL/3LL with those of
IIb
3 significantly reduced adherence,
implying that transmembrane domains play a role in
IIb
3
activity. This inference is underscored by the complete conservation of
the transmembrane domains of human, rat, and mouse
IIb and
3(33, 34) and the nearly complete conservation
of the transmembrane domains of human and rat
L and
2(35, 36) . Furthermore, the ability of
heterodimers composed of 2LL and
3 to respond to PMA as well as
those composed of 22L and
3 suggests that
IIb can tolerate
other acceptable sequences in its transmembrane region. Similar results
have been reported for the insulin (37) and epidermal growth
factor (EGF) receptors (38) but not for the platelet-derived
growth factor (39) and nerve growth factor
receptors(40) , implying that there are differences in the way
various transmembrane segments contribute to receptor activity. We also
found little difference in lymphocyte adherence mediated by
heterodimers composed of
IIb and 33L or 3LL. However, the absence
of a
3 cytoplasmic domain in this circumstance likely obscures the
contribution of the
3 transmembrane domain to
IIb
3
function.
IIb and
3 transmembrane domains
contribute to
IIb
3 function? One possibility is that the
transmembrane and cytoplasmic domains exist as a conformational unit
such that the composition of the transmembrane domain influences the
conformation of the cytoplasmic domain. As a precedent for this
possibility, it was found that the ability of an EGF receptor-nerve
growth factor receptor chimera to transduce EGF signals was lost when
the transmembrane domain of the nerve growth factor receptor was
replaced by that of the EGF receptor(40) . A second possibility
is based on analogy to many cytokine and growth factor receptors where
transmembrane signaling requires homo- or heterodimerization of
receptor subunits(41, 42) . Examination of solubilized
IIb
3 by electron microscopy reveals that the tails of
IIb and
3 containing their transmembrane segments are
flexible and interact with each other in
30% of images (43) . In view of the fluid nature of biological
membranes(44) , it is likely that the transmembrane segments of
IIb and
3 would interact in unstimulated platelets unless
constrained by factors such as cytoskeletal associations or unfavorable
interactions between its cytoplasmic domains(41, 45) .
By relieving these constraints, agonist-generated signals could allow
the transmembrane domains of
IIb and
3 to associate, thereby
transmitting the signals to the extracellular domain.
2 or
3 integrins using agonists like PMA
in fibroblast-like cells(5, 7, 46) ,
suggesting that there are differences between the repertoire of signal
transduction pathways in cells of fibroblast origin and in cells of
hematopoietic origin where activation of
2 or
3 integrins
normally occurs. Previous studies of
IIb
3 activation have
depended on the ability of mutations to induce constitutive
IIb
3 activity(5, 46) . Besides restricting
such studies to activating mutations, the relationship of the changes
in
IIb
3 induced by the mutations to the changes induced by
cellular agonists remains problematic. Our results indicate that the
ligand binding activity of
IIb
3 and of
IIb
3-
L
2 chimeras can be modulated by phorbol esters
in human B lymphocytes. Accordingly, this lymphocyte system establishes
a model that permits study of both agonist-initiated signal
transduction pathways and the agonist-stimulated function of intact or
mutant integrins.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.