(Received for publication, February 21, 1996; and in revised form, March 13, 1996)
From the
-(GPIIb-IIIa) is the most
abundant integrin expressed on platelets and plays a critical role in
platelet aggregation and normal hemostasis. In response to platelet
stimulation by agonists such as thrombin,
becomes a receptor for the
adhesive proteins fibrinogen, von Willebrand factor, vitronectin, and
fibronectin. Binding of extracellular matrix ligands allows the
integrin to transmit a signal to the inside of the cell, but the exact
mechanisms whereby integrins transduce these signals remain unclear. In
this paper we demonstrate that the
subunit of
was phosphorylated on tyrosine
residues in response to thrombin-induced platelet aggregation. However,
tyrosine phosphorylation was not observed when platelets were
stimulated by thrombin in the presence of an inhibitor of aggregation.
Phosphotyrosine was only detected when platelets were solubilized under
protein-denaturing conditions. A peptide corresponding to residues
740-762 of the
cytoplasmic domain was capable
of binding the signaling proteins SHC and GRB2. GRB2 binding occurred
only when both tyrosine residues (Tyr-747 and Tyr-759) were
phosphorylated. SHC binding also occurred to a peptide
monophosphorylated at Tyr-759. The data suggest that tyrosine
phosphorylation of an integrin
subunit may be important in
initiating outside-in signaling cascades by inducing association of
signaling components directly with the integrin.
Integrins form a widely distributed family of heterodimeric cell surface receptors that play critical roles in cell adhesion events(1) . Integrins function not only to recognize macromolecular ligands but also to transmit signals, a process known as outside-in integrin signaling. These signals can induce a spectrum of cellular responses such as cytoskeleton assembly, gene expression, cell division, and cell motility(2) . How ligand binding to integrins initiates signaling events is an unsolved problem in integrin biology. However, it is clear that the cytoplasmic domains of the integrin subunits play a critical role in the signaling process(3, 4, 5) .
Previous studies have
established that the outside-in integrin signaling through
is critical for platelet function.
The
-dependent aggregation of
platelets causes increased tyrosine phosphorylation of signaling
proteins (e.g. Syk and FAK(6, 7) ), increased
Ca
mobilization(8) , and increased
cytoskeletal organization accompanying changes in cell
shape(9) . Many of these activities also occur when
is expressed and activated in
nucleated cells, and mutations of this receptor have shown that the
cytoplasmic domains are critical for function. Such experiments suggest
a regulatory role for the
cytoplasmic domain in
ligand binding, while the cytoplasmic domain of
is
involved not only in ligand binding to
but also in its signaling
response(4, 5, 10) .
One of the principal
processes by which appears to
mediate the transfer of information from the extracellular to
intracellular milieu is by tyrosine phosphorylation of cytoplasmic
proteins, e.g. FAK, Syk, and cortactin(2) . Ligand
occupancy of
is a prerequisite for
the full range of tyrosine phosphorylation events to
occur(11) , implying an important role for outside-in integrin
signaling in this process.
A primary mechanism by which cell surface
receptors transduce signals is through direct phosphorylation of
receptor subunits. Indeed, the cytoplasmic domain of contains two tyrosine residues. Although previous studies have
determined that the
cytoplasmic domain is
phosphorylated on threonine and serine residues in response to thrombin
stimulation(12) , phosphorylation of the
cytoplasmic tyrosine residues in response to platelet aggregation
has not been documented. Three observations suggest this possibility.
First, tyrosine 747 exists within a motif similar to tyrosines known to
be phosphorylated in the epidermal growth factor and insulin
receptors(13) . Second, tyrosine 747 of
exists in an NPXpY motif (where pY is phosphotyrosine),
known to bind the phosphotyrosine binding (PTB) (
)domain of
signaling proteins(14, 15) . Finally, there is
striking conservation of the tyrosine residues not only within the
,
,
, and
integrin members but also between
species(16) , suggesting a functional role for these residues.
The experiments described here have attempted to determine whether
tyrosine phosphorylation of the cytoplasmic domain of plays a role in signal transduction through
. In this study we have addressed
the issue of the tyrosine phosphorylation state of
after platelet aggregation using an aggressive protein
solubilization protocol. The data show that tyrosine phosphorylation of
the integrin
subunit occurs during
outside-in signaling and suggests
a possible mechanism for integrin signal transduction.
The cytoplasmic domains of contain two tyrosines, both on
(Tyr-747 and
Tyr-759 (13) ). In preliminary experiments we failed to detect
tyrosine phosphorylation of
in either activated,
non-aggregated platelets, in agreement with a previous
report(12) , or activated, aggregated platelets. In these
experiments,
was isolated by
immunoprecipitation following lysis of platelets in mild detergents
containing vanadate. Reasoning that dephosphorylation of
phosphotyrosine may have occurred during detergent solubilization
(particularly as tyrosine phosphatases, such as PTP1B, are abundant in
platelets(23) ), we examined the tyrosine phosphorylation state
of
from control and thrombin-aggregated platelets
using an aggressive strategy for protein solubilization. Using the
experimental design detailed in procedures an 8-fold increase (n = 6) in tyrosine phosphorylation of
was
observed in aggregated as compared with control platelets (see Fig. 1B). Increased tyrosine phosphorylation of other
proteins was also observed, most notably FAK and Syk as previously
documented(6, 7) . These increases were either
abrogated (e.g.
and FAK) or blunted (e.g. Syk) by the addition of integrin antagonist peptide (Fig. 1B), demonstrating that phosphorylation occurred
primarily in response to aggregation and was not due to
thrombin-induced platelet stimulation. Platelets also express the
integrin but at very low levels,
approximately 0.1-0.2% of the amount of
(24) . To determine whether
this integrin contributed to the observed phosphorylation of
, the experiments were also performed in the presence
of LM609, an antibody that blocks the ligand binding activity of
. However, even at 20 µg/ml
LM609, where binding to platelets could be observed by
fluorescence-activated cell sorter, the same enhancement in the level
of
tyrosine phosphorylation was observed as occurred
in its absence (data not shown).
Figure 1:
Aggregation-induced tyrosine
phosphorylation of in platelets. Nonreduced-reduced
two-dimensional gel electrophoresis was carried out on lysates from
platelets either resting, thrombin-aggregated, or treated with thrombin
in the presence of a cyclic RGD peptide antagonist to block
aggregation. A, Coomassie Blue staining of a gel to visualize
the proteins present and to demonstrate the migration patterns and
loading integrity of the proteins. The position of the
protein is indicated by the arrows. B,
anti-phosphotyrosine (Anti-ptyr) immunoblot of a duplicate
gel, which was transferred to nitrocellulose.
protein
is again indicated by arrows and was confirmed by stripping
and reprobing the immunoblot with the anti-
monoclonal
antibody C3a.19.5. C, the spots obtained with the
anti-
antibody lined up exactly with the spots on the
anti-phosphotyrosine blot marked with the arrows. Molecular
mass standards are indicated to the left. Densitometry was
performed using ImageQuant software on a Molecular Dynamics
densitometer, and the results were normalized for the amount of
present. An average 8-fold increase in the tyrosine
phosphorylation of
from aggregated as compared with
control platelets was observed (n =
6).
Phosphorylation of cytoplasmic
tyrosine residues has been shown to be important in recruiting
signaling molecules to a number of cell surface receptors. Because
was dephosphorylated in non-reducing detergent,
synthetic peptides corresponding to residues 740-762 of
were utilized to identify signaling proteins that can
become associated with the phosphorylated cytoplasmic domain of
. The peptide containing two phosphorylated tyrosine
residues (Tyr(P)-747 and Tyr(P)-759) was found to bind both GRB2 and
SHC from platelet lysates (Fig. 2). GRB2 was not bound by
peptides in which the two tyrosines were either not phosphorylated or
in which only one was phosphorylated. Thus, GRB2 binding required
phosphorylation of both tyrosines. Our data also showed that SHC was
not bound by peptides in which tyrosines were not phosphorylated or
when only Tyr-747 was phosphorylated. However, SHC was bound by the
Tyr(P)-759 peptide (Fig. 2B), indicating that
phosphorylation of only the more carboxyl tyrosine of
was sufficient for interacting with this signaling protein. We
failed to detect the association of other signaling molecules such as
FAK, Syk, and phosphatidylinositol 3-kinase with any of the
peptides (data not shown).
Figure 2:
Binding
of the signaling proteins SHC and GRB2 to peptides encompassing the
tyrosine-phosphorylated cytoplasmic region. A, single-letter amino acid sequence of peptides used in these
studies. B and C, immunoblots of proteins
precipitated by the indicated peptides. 0.5-1 mg of 1% Nonidet
P-40 lysates from resting platelets were incubated with peptides
followed by avidin-agarose precipitation. The resulting immunoblots
were probed with anti-SHC (B) or anti-GRB2 (C)
antibodies.
As a test of the physiological
importance of GRB2 or SHC binding to phosphorylated ,
a peptide containing a naturally occurring mutation of
(S752P) was used. This single point mutation was identified in a
patient with Glanzmann's thrombasthenia(25) . This
autosomal dominant bleeding disorder is caused by platelets that fail
to aggregate (26) and has been shown to prevent outside-in
signaling through
(27) . The doubly
tyrosine-phosphorylated mutant peptide was unable to precipitate GRB2
and showed a marked decrease in its ability to precipitate SHC (Fig. 2), suggesting that the Ser to Pro mutation in these
patients alters protein structure such that interactions with signaling
proteins cannot occur.
It was not possible to determine whether SHC
or GRB2 bound to phosphorylated during platelet
aggregation, due to the rapid dephosphorylation of
in
detergent lysis conditions required for immunoprecipitation
experiments. However, as shown in Fig. 3, both proteins were
tyrosine-phosphorylated in response to platelet aggregation and
furthermore appeared to associate with several unidentified
phosphoproteins (e.g. p120 and p170) in an
aggregation-dependent manner.
Figure 3: SHC and GRB2 are tyrosine-phosphorylated in response to thrombin and co-immunoprecipitate other tyrosine-phosphorylated proteins. A, anti-phosphotyrosine (Anti-ptyr) immunoblot of the indicated immunoprecipitations (I.P), or total lysate, from control(-) and thrombin-aggregated (+) platelets. 3 mg of 1% Nonidet P-40 platelet lysates were immunoprecipitated with 10 µg of the indicated antibody (Ab) plus protein A-Sepharose. Immunoblots were first probed with a mix of PY-20 and 4G10 anti-phosphotyrosine antibodies (A) and then stripped and reprobed with either anti-SHC (B) or anti-GRB2 (C) antibodies to show loading integrity between control and thrombin-aggregated samples. Molecular mass standards are indicated to the left of each blot. The migrations of particular proteins are indicated by the arrows to the right of the blots. Asterisk in A and B indicates the position of Ig heavy chain.
The experiments above indicated that
was a substrate for a tyrosine kinase(s). A number of
tyrosine kinases are present in platelets and indeed several have been
implicated in integrin signaling, including Src, Fyn, and
Syk(6, 11) . To determine whether any of these kinases
could be involved in
tyrosine
phosphorylation, Src family tyrosine kinases p60
,
p59
, and p56
as well as Syk were tested for
their activities in phosphorylating full-length
and a
peptide consisting of the cytoplasmic domain. Of these kinases p60
and Syk showed the greatest ability to
phosphorylate
(Fig. 4A). p59
showed modest activity, whereas the partially purified
p56
showed less activity. However, the p56
also showed a decreased ability to phosphorylate the control
substrate enolase (Fig. 4A). That p59
and
p56
could phosphorylate
was confirmed
in experiments using kinases immunoprecipitated from platelet lysates
(data not shown). In contrast, the SH2-containing tyrosine kinase
p93
, which has been implicated in signaling via cytokine
receptors(28) , showed little ability to phosphorylate
. Although increased kinase activity was observed with
the
immune complex from thrombin-aggregated as
compared with resting platelets (Fig. 4B), we were
unable to detect direct association of Src, Fyn, Lyn, or Syk with
using co-immunoprecipitation and
immunoblotting techniques (data not shown).
Figure 4:
In vitro phosphorylation of
and association of tyrosine kinase activity with
upon platelet aggregation. A, in vitro kinase assay demonstrating the ability of the indicated tyrosine
kinases to phosphorylate either the
subunit of
affinity-purified
(no
phosphorylation of
was observed), a peptide
consisting of residues 715-762 of
, or enolase.
4 units of p60
and p59
,
12 units of p56
, 50 ng of
p93
, and baculovirus-expressed Syk were used. Asterisk indicates the band corresponding to p93
autophosphorylation. The p56
appeared
to be less active than the other Src kinases as shown by its reduced
ability to phosphorylate the exogenous substrate enolase, as well as
. B, kinase activity co-immunoprecipitating
with
from control and thrombin-aggregated platelets.
Anti-
(E8) or control antibody was used to precipitate
protein from 0.5 mg of 1% Brij 96 platelet lysates. The resulting
immunoprecipitates were subjected to an in vitro kinase assay,
and phosphorylated proteins were visualized by
autoradiography.
Platelet aggregation, a process mediated by
, is known to initiate an
outside-in signal through
, as
evidenced by increased phosphorylation of proteins such as Syk and FAK
(Refs. 6 and 7 and Fig. 1). The present data demonstrate for the
first time that the
integrin subunit of
is tyrosine-phosphorylated in
response to aggregation. We also found that SHC and GRB2, two proteins
known to participate in the Ras signaling pathway(29) , which
is active in platelets(30) , could associate with
phosphorylated
cytoplasmic domain peptides. SHC and
GRB2 were also phosphorylated during platelet aggregation. These data
suggest that the initial event triggered by outside-in signaling via
the
integrin on platelets may in
fact be the tyrosine phosphorylation of
and that the
phosphorylated cytoplasmic domain has the capability of assembling
signaling complexes, which also become tyrosine-phosphorylated.
Platelets express two integrins containing the subunit:
and
.
is abundant on platelets (50,000 molecules/platelet; 1% of total
protein mass(31) ) and has been shown to mediate platelet
aggregation(32) . In contrast,
is expressed at very low levels (50-100 molecules/platelet,
0.1-0.2% of the levels of
(24) ), and it is unclear
what role, if any, this integrin plays in platelet function. The
presence of LM609, an antibody that blocks the ligand binding activity
of
(19) , had no effect on
the amount of
tyrosine phosphorylation observed,
indicating that the
was phosphorylated as the
integrin.
participates in both outside-in
and inside-out signal transduction(32) . Inside-out signaling
results in
having enhanced
affinity for fibrinogen and von Willebrand factor following platelet
stimulation by agonists such as thrombin and ADP. Although the
mechanism of inside-out signaling is not known, it most likely is
mediated by membrane perturbation that affects a large number of
receptors or is induced by a direct modification of all receptors, as
the affinity state of all receptors is capable of being up-regulated.
Outside-in signaling results when platelet stimulation occurs upon
integrin ligation to a substrate that allows
clustering, e.g. during
platelet aggregation. Given the abundance of
on platelets and the known
transient nature of tyrosine phosphorylation of other cell surface
signaling receptors (e.g. Ig-
and Ig-
of the B cell
receptor complex(33, 34) ), it is not unexpected that
tyrosine phosphorylation would occur on a small
number of receptors and be transient and that dephosphorylation
mechanisms would be essential and robust. This perhaps explains why
tyrosine phosphorylation has not been observed previously for this
integrin. In other cell systems, such as B lymphocytes, it has been
possible to increase the amount of detectable B cell receptor tyrosine
phosphorylation by performing stimulations at 0-4
°C(33) . Since this temperature does not allow for platelet
aggregation, these conditions cannot be utilized to characterize
tyrosine phosphorylations during aggregation-induced
signaling.
The
integrin is not expected to have
any intrinsic tyrosine kinase activity and, so far, we have been unable
to detect the direct association of a tyrosine kinase(s) with this
integrin. However, our in vitro data suggest that possible
candidates include members of the Src family of tyrosine kinases or
possibly Syk. Support for the role of these kinases in the
phosphorylation of
in vivo has been provided by Dorahy et al.(35) , who have
demonstrated that Src and Lyn can be cross-linked to
in intact platelets by chemical cross-linking agents.
Although
this is the first report of outside-in signaling inducing the tyrosine
phosphorylation of , it should be noted that tyrosine
phosphorylation of the
integrin subunit has been
observed in response to integrin ligation(36) .
is novel among integrin subunits. Its cytoplasmic domain is 1,018
amino acids in length, bears no sequence homology to
or other integrin
subunits, and contains an immune receptor
tyrosine-based activation motif (ITAM)(37) . ITAMs are defined
amino acid motifs containing two tyrosine residues as well as other
critical amino acids. It is well documented that phosphorylated ITAMs
play essential roles in signaling via T cell, B cell, and Fc receptor
complexes (reviewed in (38) ).
The two tyrosines in
are separated by an 11-amino acid peptide sequence.
This spacing is similar to that of the two tyrosines found in the ITAM
domains of immune receptor complexes. Although the tyrosines of the
integrin subunit do not fulfil the ITAM sequence
requirement of obligatory leucine or isoleucine residues +3 from
the tyrosine residues, it becomes of interest to compare the
interactions of the two phosphorylated receptor types. The tyrosine
residues of ITAM domains are phosphorylated upon receptor ligation,
which allows for the recruitment of cytoplasmic proteins. In
particular, the direct binding to the tandem SH2 domains of the
tyrosine kinases ZAP-70 and Syk has been
documented(21, 39) . While we failed to detect the
association of Syk with doubly phosphorylated
peptides, we did detect the association of two intracellular
signaling proteins, GRB2 and SHC. SHC has been shown to associate with
phosphorylated ITAMs from both the T cell and B cell receptor
complexes(40, 41) . However, neither tyrosine in
exists in a GRB2 SH2 binding motif(42) .
Also, GRB2 only contains a single SH2 domain while phosphorylations of
both tyrosines were required for binding. Thus, it seems probable that
binding of GRB2 is bridged by another protein, possibly one containing
tandem phosphotyrosine binding domains. SHC can bind to
phosphotyrosines through SH2 domain interactions as well as to
NPXpY (where pY is phosphotyrosine) motifs via a PTB domain (14, 15) . Our data showed that SHC did not bind to
monophosphorylated Tyr-747 peptide, which contains an NPXY
motif. However, it did bind to the monophosphorylated Tyr-759 peptide (Fig. 2). The sequence surrounding Tyr-759 does not appear to be
in a motif recognized by either the SH2 (42) or PTB domain (14, 15) of SHC. Although this may indicate that
binding of SHC, too, is bridged by another protein, perhaps in a
signaling complex distinct from that involving GRB2, we cannot rule out
a direct association between SHC and
. This suggestion
derives from recent data showing that the proline may not be required
in the motif recognized by proteins containing PTB domains (43) and that a hydrophobic residue in the -5 position
promotes PTB(44) . This position on
, residue
754, is a phenylalanine. Interestingly, we found that GRB2, as well as
SHC, was tyrosine-phosphorylated in thrombin-aggregated platelets. To
our knowledge, this is the first example of tyrosine phosphorylation of
GRB2 during a signaling response. Since tyrosine phosphorylation does
not appear to be necessary for GRB2 association with the Ras exchange
factor SOS(45) , tyrosine phosphorylation of GRB2 may be
involved in an as yet undescribed function of this adaptor protein.
The doubly phosphorylated peptide containing the
S752P point mutation failed to bind to GRB2 and showed markedly reduced
binding with SHC.
bearing the
S752P mutation is known to be defective in outside-in signaling as
Chinese hamster ovary cells expressing this receptor show reduced
ability to spread on immobilized fibrinogen, form focal adhesions, and
retract fibrin clots, compared with cells expressing wild type
(27) . Our data suggest
that this defect may be due to the inability, or reduced ability, of
the S752P
cytoplasmic domain to recruit signaling
proteins even if the tyrosines are phosphorylated.
endonexin, a protein of unknown function that was identified by
its binding to the
cytoplasmic region in the yeast
two-hybrid system, also has reduced ability to bind the
S752P cytoplasmic domain(46) .
These data provide a
mechanism by which can mediate
outside-in signaling. This mechanism surprisingly shares many of the
overall features of growth factor and immune receptor-mediated
signaling; receptor cross-linking leads to tyrosine phosphorylation of
the receptor cytoplasmic domains, followed by the induced binding of
signaling complexes to the phosphorylated receptor. The tyrosines in
do exist in sequences bearing striking homology to
several other integrin
subunits (e.g. 740-762, the
carboxyl terminus, is 50% identical to
, 36% to
, and 54% to
(14) ). This
suggests that tyrosine phosphorylation may also be of importance in
outside-in signaling via these integrins. As yet, these homologous
subunits have not been shown to be tyrosine-phosphorylated in
response to integrin signaling, but
is constitutively
tyrosine-phosphorylated in v-Src-transformed cells(47) .
Although phosphorylation of
prevents the
incorporation of
in focal adhesion
sites(48) , it is not clear whether phosphorylated
contributes to cellular signaling
processes. Taken together our data make it attractive to hypothesize
that the tyrosine phosphorylation of integrin
subunits is a
general method for initiating outside-in signaling, allowing the
phosphorylated
subunits to recruit phosphotyrosine-binding
cytoplasmic signaling molecules to the membrane, thereby activating
intracellular signaling pathways.