(Received for publication, November 26, 1995; and in revised form, January 26, 1996)
From the
The glycoprotein (GP) Ib-IX complex is one of the major platelet
membrane glycoproteins. Its extracellular domain binds von Willebrand
factor at a site of injury, an interaction that leads to activation of
intracellular pathways. Its intracellular domain associates tightly
with the platelet cytoskeleton through actin-binding protein. The goal
of the present study was to investigate the role of the cytoplasmic
domain of the GP Ib-IX complex and its interaction with the
cytoskeleton. Cultured cells were transfected with the cDNAs coding for
GP Ib, GP IX, and full-length or truncated forms of GP
Ib
. Western blots of detergent-insoluble fractions of
Triton X-100-lysed cells showed that deletion of amino acids Trp-570 to
Ser-590 from the cytoplasmic domain of GP Ib
abolished
the interaction of the entire GP Ib-IX complex with the cytoskeleton.
Truncated GP Ib
that was unable to associate with the
cytoskeleton retained its ability to associate with GP
Ib
, to be inserted into the membrane, and to bind von
Willebrand factor. Cells expressing GP Ib
changed their
shape following adhesion to immobilized von Willebrand factor. Cells
expressing truncated GP Ib
also changed their shape
following adhesion but showed a very different morphology as compared
to cells expressing full-length GP Ib
. These results
show that GP Ib-IX-von Willebrand factor interactions lead to
cytoskeletal reorganizations, that the cytoplasmic domain of GP
Ib
regulates these reorganizations, and that the
cytoplasmic domain of GP Ib
is absolutely required for
attachment of the GP Ib-IX complex to the cytoskeleton.
The glycoprotein (GP) ()Ib-IX complex is one of the
major platelet transmembrane
complexes(1, 2, 3, 4) . It consists
of two disulfide-linked subunits, GP Ib
(M
= 145,000) and GP Ib
(M
= 24,000), that are noncovalently
complexed with another glycoprotein, GP IX (M
= 22,000) (5, 6, 7, 8) . This complex binds
von Willebrand factor on exposed vascular subendothelium and is
responsible for mediating the initial interaction of platelets with the
subendothelium at a site of
injury(1, 2, 3, 4) . Several lines
of evidence have shown that the von Willebrand factor-GP Ib-IX
interaction can initiate transmembrane signaling (9, 10, 11, 12) leading to
activation of intracellular pathways, release of platelet granule
contents, and aggregation of platelets.
The cytoplasmic domain of
the GP Ib-IX complex associates with the platelet
cytoskeleton(13, 14, 15) . Based on the
coimmunoprecipitation of GP Ib-IX with actin-binding protein from
detergent lysates, binding of purified GP Ib-IX to purified
actin-binding protein, and association of expressed GP Ib-IX with
actin-binding protein in transfected cells, it has been concluded that
the association of GP Ib-IX with the cytoskeleton is mediated by
actin-binding
protein(16, 17, 18, 19, 20, 21) ,
a homodimer with rod-like 280-kDa subunits that cross-links actin
filaments. Studies with synthetic peptides identified sequences in the
cytoplasmic domain of the GP Ib subunit that can bind
to actin-binding protein in in vitro binding assays (22) but the importance of these sequences in mediating the
interaction of the GP Ib-IX complex with the cytoskeleton in the intact
cell is not known.
The function of several types of adhesion receptors including integrins, selectins, and members of the immunoglobulin-like family of receptors, appears to be affected by the cytoplasmic domain of the receptor(23, 24, 25, 26, 27, 28) . In several cases, a correlation between altered function and altered cytoskeletal association has been noted(23, 27, 28) . Although it is well established that GP Ib-IX associates with the cytoskeleton, the importance of the cytoplasmic domain of this receptor in regulating ligand binding or ligand-induced transmembrane signaling has not been investigated.
In the present study, we expressed full-length and
truncated forms of the GP Ib-IX complex in cultured cells. The results
show that either all or part of the amino acid sequence Trp-570 to
Ser-590 in the cytoplasmic domain of GP Ib is necessary
for mediating association of the entire GP Ib-IX complex with the
cytoskeleton. Although GP Ib-IX complex lacking this cytoplasmic region
of GP Ib
did not associate with the cytoskeleton, it
was incorporated into a complex with the other subunits and was
inserted into the membrane of cultured cells. The truncation of the
cytoplasmic domain that ablated the interaction of the receptor with
the cytoskeleton had no detectable effect on the binding of adhesive
ligand. However, cells expressing truncated receptor showed a very
different morphology following adhesion to immobilized von Willebrand
factor than did cells expressing full-length receptor. These results
show that the cytoplasmic domain of GP Ib
is absolutely
required for attachment of the GP Ib-IX complex to the cytoskeleton and
suggest that the cytoplasmic domain of GP Ib
plays an
important role in regulating von Willebrand factor-induced
transmembrane signaling.
For Scatchard analysis, von Willebrand factor was labeled with I by the lactoperoxidase method and various
concentrations incubated with transfected cells as described previously (30) . Samples were rocked at room temperature for 1 h,
aliquots sedimented through sucrose cushions, and the amount of
I-labeled von Willebrand factor associated with the
sedimented cells measured. Preliminary experiments used excess
unlabeled von Willebrand factor to determine nonspecific binding. In
subsequent experiments nonspecific binding was determined by measuring
I-labeled von Willebrand factor that bound to cells which
had not been transfected with GP Ib-IX and subtracting the counts from
those of the corresponding transfected samples. The two methods of
determining nonspecific binding gave comparable results; the
experiments in the present study were performed using the latter method
for determining nonspecific binding.
Figure 1:
Map of GP
Ib mutations. Stop codons were introduced into the
cytoplasmic domain of GP Ib
cDNA by single base pair
changes at Gln-545 (C to T at base pair 1723), Ser-559 (C to A at base
pair 1766), Trp-570 (G to A at base pair 1800), Gln-591 (C to T at base
pair 1861), and Tyr-605 (C to A at base pair 1905) to generate the
m545, m559, m570, m591, and m605 constructs, each coding for a
different truncation of GP Ib
. The solid bars in the lower portion of the figure represent synthetic peptides
previously assayed in in vitro binding
studies(22) .
Figure 2:
Expression of truncated forms of GP Ib-IX
on the surface of transfected CHO cells. CHO cells (5
10
/sample) transfected with GP Ib-IX constructs encoding
either full-length GP Ib
(panel A) or
truncated forms (panels B-F) were labeled with 5 µg/ml
FITC-conjugated monoclonal antibody AN51 (against GP
Ib
) and analyzed by flow cytometry. Solid lines represent the samples indicated, while broken lines represent a negative control (CHO cells transfected only with GP
Ib
and GP IX constructs).
Figure 3:
Western blot showing the differential
recovery of truncated forms of GP Ib in the high speed
pellets from lysates of transfected CHO cells. CHO cells from single
confluent 90-mm dishes (5
10
cells/dish) were
harvested, suspended in lysis buffer containing 1% Triton X-100, and
ultracentrifuged. The Triton X-100-insoluble pellet (p) and
resulting supernatant (s) for each sample were solubilized in
an SDS-containing buffer with
-mercaptoethanol (reducing
conditions), electrophoresed through SDS-polyacrylamide gels, and
transferred to nitrocellulose paper. Blots were incubated with a
monoclonal antibody directed against the extracellular domain of GP
Ib
. CHO, nontransfected cells;
IX, cells transfected with constructs for GP
Ib
, GP IX, and native GP Ib
;
``m'' cell types, cells transfected with constructs
for GP Ib
, GP IX, and the various truncations of GP
Ib
. Platelets (1
10
/ml) solubilized
in SDS-containing buffer were included for reference. The GP
Ib
-reactive band with a slightly lower molecular weight
than GP Ib
that is indicated with an arrow is
assumed to be GP Ib
that had been cleaved at a
protease-sensitive site in the extracellular amino-terminal region of
the protein (see (34) ).
To determine whether the
COOH-terminal region of the cytoplasmic domain of GP Ib was necessary for association of the other components of the GP
Ib-IX complex with the cytoskeletal fraction, detergent-insoluble and
-soluble fractions from CHO cells expressing GP Ib-IX that contained
either full-length GP Ib
or the truncated m559 form
(lacking 52 amino acids) were analyzed on Western blots using
polyclonal antibodies that recognizes all three subunits of the GP
Ib-IX complex. Fig. 4shows that release of GP Ib
from the detergent-insoluble fraction was accompanied by release
of the other two components of the GP Ib-IX complex. Although these
results do not exclude the possibility that GP Ib
or GP
IX may play a role in mediating the interaction of the GP Ib-IX complex
with the cytoskeleton in the intact cell, they show that the
cytoplasmic domain of GP Ib
is absolutely required for
this interaction.
Figure 4:
Western blot showing the release of all
three subunits of GP Ib-IX from the high speed pellet of CHO cells
transfected with the truncated m559 form of GP Ib.
Transfected CHO cells from single confluent 90-mm dishes (5
10
cells/dish) were harvested, suspended in lysis buffer
containing 1% Triton X-100, and ultracentrifuged. The Triton
X-100-insoluble pellet (p) and resulting supernatant (s) for each sample were solubilized in an SDS-containing
buffer with
-mercaptoethanol, electrophoresed through an
SDS-polyacrylamide gel, and transferred to nitrocellulose paper. The
blot was incubated with a polyclonal antibody that recognizes all three
subunits of the GP Ib-IX complex. The band indicated with an arrow is assumed to be GP Ib
that had been cleaved at a
protease-sensitive site in the extracellular amino-terminal region of
the protein(34) .
To determine whether the truncation of GP
Ib that prevented its association with the cytoskeleton
had an effect on its incorporation into the GP Ib-IX complex,
detergent-insoluble and -soluble fractions from cells expressing
full-length or truncated forms of GP Ib
were analyzed
on SDS gels in the presence and absence of reducing agent; like
full-length GP Ib
in the cytoskeletal fractions,
truncated forms of GP Ib
in the detergent-soluble
fractions migrated higher in the absence of reducing agent than in the
presence (data not shown) indicating that the truncated form of GP
Ib
that was unable to associate with the cytoskeleton
was still disulfide-linked to the
-subunit of GP Ib. Since GP IX
associates with GP Ib
, not GP
Ib
(35) , we assume that it also remained a part
of the glycoprotein complex when the GP Ib
cytoplasmic
domain was truncated. FACS analysis with SZ1, a complex specific
antibody(5) , provided further evidence that truncated GP Ib-X
was present in the membrane in the form of a complex (data not shown).
Figure 5:
Botrocetin-dependent binding of von
Willebrand factor to truncated GP Ib-IX on the surface of transfected
CHO cells. CHO cells (5 10
/sample) transfected with
constructs for native GP Ib-IX (panel A) or the truncated m559
form of GP Ib-IX (panel B) were incubated with 0.5 µg/ml
purified von Willebrand factor in the presence (solid line) or
absence (dashed line) of 5 µg/ml botrocetin for 1 h at 4
°C. Following subsequent incubations with polyclonal antibody
against von Willebrand factor (1:1000) and (5 µg/ml)
FITC-conjugated anti-rabbit IgG, samples were analyzed by flow
cytometry.
To
determine whether the binding affinity of von Willebrand factor to the
GP Ib-IX receptor was affected by truncation of the receptor's
cytoplasmic domain, melanoma cells transfected with GP Ib-IX containing
either full-length GP Ib (Fig. 6, panel
A) or m545 (Fig. 6, panel B) were incubated with
I-labeled von Willebrand factor and the binding of von
Willebrand factor measured. Nonspecific binding was determined by
measuring binding of
I-labeled von Willebrand factor to
non-GP Ib-IX-expressing cells. Preliminary experiments using excess
unlabeled von Willebrand factor to determine nonspecific binding gave
comparable results (data not shown). The specific binding could be
inhibited by a monoclonal antibody directed against the von Willebrand
factor binding site on GP Ib
(data not shown). Binding
of von Willebrand factor to cells expressing both full-length GP
Ib
or m545 was saturable (Fig. 6, upper
panels). While the binding capacity for cells expressing m545 was
slightly higher than that of cells expressing normal GP
Ib
, this was due to a higher number of receptors on the
surface of the m545 cells. The Scatchard analysis of the binding curves
detected a single class of binding sites (Fig. 6, A and B, lower panels). The affinity of von Willebrand factor for
receptor was virtually identical for normal GP Ib
and
m545. These results demonstrate that even when the cytoplasmic domain
of GP Ib
is truncated and the GP Ib-IX complex is not
associated with the cytoskeleton, the complex is still capable of
binding ligand with the same affinity as full-length
cytoskeleton-associated receptor.
Figure 6:
Scatchard analysis of the binding of I-labeled von Willebrand factor to transfected melanoma
cells. Melanoma cells expressing GP Ib-IX containing either full-length
GP Ib
(A) or m545 (B) were incubated
for 1 h in the presence of 5 µg/ml botrocetin and various
concentrations of
I-labeled von Willebrand factor (vWF). Cells were layered on sucrose cushions, spun at 15,000
g for 4 min, and counted in a
-scintillation
counter following aspiration of sucrose. Each data point represents the
mean of duplicate determinations. Scatchard analysis of the binding
curves was performed using the Microsoft Excel computer
program.
Figure 7:
Fluorescence microscopy showing
differential spreading of transfected CHO cells on von Willebrand
factor-coated slides. CHO cells transfected with constructs for GP
Ib, GP IX, and either GP Ib
(panels
A and B) or m559 (panels C and D) were
seeded on von Willebrand factor-coated chamber slides (10,000 cells per
well) in the absence (panels A and C) or presence (panels B and D) of botrocetin. After 2 h, cells were
fixed, lysed, and incubated with a polyclonal antibody against GP Ib-IX
followed by FITC-labeled anti-rabbit IgG. Bar, 25
µm.
The GP Ib-IX complex is one of the major platelet membrane
receptors and is responsible for mediating the initial interaction of
platelets with the subendothelium at a site of
injury(1, 2, 3, 4) . Following
interaction of GP Ib-IX with its adhesive ligand, von Willebrand
factor, transmembrane signaling is initiated and intracellular pathways
leading to the secretion of granule contents and aggregation are
induced(9, 10, 11, 12) . The way in
which binding of von Willebrand factor to the GP Ib-IX complex induces
intracellular changes is not known. In the present study, we have
investigated the importance of the cytoplasmic domain of GP
Ib in regulating the function of the complex. We show
that truncation of the carboxyl-terminal half of the cytoplasmic domain
of GP Ib
does not affect incorporation of GP
Ib
into the GP Ib-IX complex, insertion of the complex
into the membrane, or binding of von Willebrand factor to the
extracellular domain of the complex. However, truncation of this
cytoplasmic domain results in a GP Ib-IX complex that cannot associate
with the cytoskeleton in transfected cells. Moreover, cells expressing
complex with truncated GP Ib
cytoplasmic domain showed
a very different shape following adhesion to immobilized von Willebrand
factor than did cells expressing full-length GP Ib
.
Previous work has demonstrated that the link between GP Ib-IX and
the platelet cytoskeleton is actin-binding
protein(16, 17, 18, 19, 20, 21) .
Furthermore, in vitro binding studies utilizing synthetic
peptides derived from the sequence of the cytoplasmic domains of GP
Ib and GP Ib
showed that sequences in
the cytoplasmic domain of the GP Ib
subunit could bind
to purified actin-binding protein(22) . In particular, a
hydrophilic amino acid sequence from Thr-536 to Phe-568 in GP
Ib
was most effective, while the adjacent Trp-570 to
Ala-588 sequence bound to a lesser extent. Because of potential
problems involving the binding of synthetic peptides to purified
proteins in in vitro assays, it was necessary to identify
sequences that were involved in mediating the interaction with the
cytoskeleton in an intact cell. Based on our previous results with the
synthetic peptides, we chose to focus on GP Ib
as the
subunit most likely to be involved in mediating the interaction. The
present study shows that GP Ib
missing 20 amino acids
(truncated at Gln-591) or less is still able to associate with the
cytoskeleton while GP Ib
missing 41 or more
COOH-terminal amino acids (truncated at Trp-570) loses its ability to
do so. Thus, it appears that all or part of the Trp-570 to Ser-590
sequence in the cytoplasmic domain of GP Ib
is
essential to maintaining the cytoskeletal association in an intact
cell, either by interacting with submembranous actin-binding protein or
perhaps by binding and subsequently conferring stability or a favorable
secondary structure to the adjacent Thr-536 to Phe-568 region, making
it also amenable to interaction with actin-binding protein.
Truncation of the cytoplasmic domain of a number of different cell
adhesion molecules has been shown to affect the ligand binding
properties of the receptors. In several cases, a correlation between
altered function and an inability to associate with the cytoskeleton
has been suggested. For example, truncation of the cytoplasmic domain
of the integrin subunit eliminated its incorporation
into focal contacts and eliminated its ability to promote adhesion of
cultured cells(23) . Deletions in the carboxyl half of the
cytoplasmic domain of E-cadherin caused it to lose its ability both to
associate with the cytoskeleton and to promote cell-cell adhesion,
while mutations in other regions of the cytoplasmic domain had no
effect on either property(27, 28) . In other studies,
the ability of another cadherin, L-CAM, to mediate cell aggregation was
abolished (41) following treatment of transfected cells with
cytochalasin D, an agent that disrupts cytoskeletal microfilaments. In
the case of GP Ib-IX, it has been suggested that association of the GP
Ib-IX complex with actin-binding protein in the platelet membrane
skeleton might be responsible for maintaining the uniform distribution
of the receptor in the platelet membrane and that this distribution
might in turn be important in regulating the binding of von Willebrand
factor multimers (38, 42) . However, in the present
study using transfected cells we found no evidence that association of
the complex with the cytoskeleton regulates its ability to bind
adhesive ligand. The number of binding sites per cell was slightly
different on the cells in which the cytoplasmic domain of GP
Ib
was truncated but the difference could be accounted
for entirely by a difference in the number of receptors in the
membrane; the affinity of the interaction was the same whether the
cytoplasmic domain of GP Ib
was truncated or not. Thus,
in transfected cells GP Ib-IX complex containing a truncated form of GP
Ib
that could not associate with the cytoskeleton was
able to bind von Willebrand factor just as well as GP Ib-IX containing
full-length subunit. These findings suggest a difference between GP
Ib-IX and other adhesion receptors. However, the distribution of GP
Ib-IX could be maintained by the cytoskeleton differently in cultured
cells than it is in platelets. Furthermore, in order to induce binding
of soluble von Willebrand factor to GP Ib-IX in cultured cells,
modulators such as ristocetin or botrocetin (21, 30, 39, 40) were used. It is
conceivable that if it were possible to determine the effect of the
cytoplasmic domain mutations in a more physiological system (e.g. shear-induced binding of von Willebrand factor to platelets or
platelets adhering to von Willebrand factor in the extracellular
matrix) a different result would be obtained. Future studies using
transgenic mice in which the receptor is truncated may help to resolve
these issues. The identification of the subunit that mediates the
interaction of the entire GP Ib-IX complex with the cytoskeleton and of
truncations that ablate the interaction while still allowing the
complex to be inserted into the membrane should prove useful in
allowing the design of such experiments.
To investigate the
possibility that the cytoplasmic domain of GP Ib-IX regulated events
that occurred following von Willebrand factor binding to the complex,
we allowed transfected cells to settle onto von Willebrand
factor-coated slides. Nontransfected cells showed little adhesion,
those that adhered remained round. Similarly, in the absence of
botrocetin transfected cells showed little adhesion; those that did
adhere did not spread. In contrast, when botrocetin was present, cells
that expressed GP Ib-IX adhered and spread. Even when the GP Ib-IX
complex was not associated with the cytoskeleton, the cells spread
showing that neither the interaction with the cytoskeleton nor the
interaction of the carboxyl-terminal half of the cytoplasmic domain of
GP Ib with other intracellular proteins is needed to
transmit the signals that allow this ligand-induced change in cell
behavior. However, the morphology of the spreading cells was very
different between the two transfected cell types. Cells containing
full-length GP Ib
extended a few broad pseudopods.
Cells expressing truncated GP Ib
extended numerous
finger-like projections around the entire cell. Because non-transfected
cells did not spread, the cellular morphologies observed were the
specific result of GP Ib-IX-von Willebrand factor interactions. These
results indicate that the reorganization of the cytoskeleton resulting
from ligand-receptor interactions was regulated in an altered way in
the cells in which the cytoplasmic domain of the receptor was truncated
compared to those in which it was not.
In platelets, the major
spreading that occurs following adhesion of platelets to exposed
extracellular matrix is thought to occur as a consequence of
-induced transmembrane
signaling(43, 44, 45) .
Ligand-
interactions have been
shown to induce cytoskeletal reorganizations (46, 47) and the incorporation of signaling molecules
into the newly formed integrin-cytoskeletal complexes has been studied
in
detail(47, 48, 49, 50, 51, 52) .
In contrast, dramatic spreading of platelets does not appear to be
induced as a direct consequence of GP Ib-IX-matrix interactions (43, 44, 45) and little consideration has
been given to the possibility that GP Ib-IX-von Willebrand factor
interactions induce cytoskeletal reorganizations. However, a recent
publication (12) suggests that signaling molecules are induced
to associate with the cytoskeleton following GP Ib-IX-ligand
interactions. The present study indicates that cytoskeletal
reorganizations are also induced; ligand-receptor interactions resulted
in a compact cell morphology and the formation of only a few broad
protrusions. In platelets, GP Ib-IX-von Willebrand factor interactions
are known to induce the stable attachment of platelets to the
extracellular matrix(43, 44, 45) ; the
cytoskeletal reorganizations detected in the present study may
stabilize the GP Ib-IX-induced adhesion of platelets to the
extracellular matrix while subsequent
-induced signaling results in
spreading of the platelets over the surface.
The GP Ib-IX-induced
cytoskeletal reorganizations occurred even in the absence of the
COOH-terminal 52 amino acids of GP Ib. However, in the
absence of these amino acids a very different cytoskeletal
reorganization was induced. Because the cells exhibited a much more
irregular morphology with many finger-like projections around the cells
in the absence of GP Ib's COOH-terminal domain, we propose that
this cytoplasmic domain has a restraining effect on von Willebrand
factor-induced cytoskeletal reorganizations, regulating both the
location and extent of the reorganizations. Because truncation of the
cytoplasmic domain also prevented association of the GP Ib-IX complex
with the cytoskeleton, it is possible that it is the association of the
complex with the cytoskeleton that allows it to directly exert an
influence on the ligand-induced reorganization of the cytoskeleton.
Previously, we have shown that in platelets the
-subunit of the GP
Ib-IX complex is phosphorylated on serine 166 (53) and have
provided evidence that this phosphorylation is involved in inhibiting
the polymerization of actin within platelets(54) . Thus, it is
conceivable that the
-subunit of GP Ib-IX can regulate the extent
and location of actin polymerization and that it is unable to do this
if the glycoprotein complex is not associated with the cytoskeleton.
Another possibility is that the cytoplasmic domain of GP Ib
associates with molecules that are involved in the transmission
of signals across the GP Ib-IX complex and in the absence of this
association a regulatory pathway is lost. One candidate is 14.3.3, a
protein that has recently been found to co-isolate with GP Ib-IX from
platelet lysates (55) and to associate with the COOH-terminal
region of GP Ib
. (
)Further experiments will
be needed to investigate these possibilities.