(Received for publication, May 9, 1995; and in revised form, July 13, 1995)
From the
The platelet membrane is lined with a membrane skeleton that
associates with transmembrane adhesion receptors and is thought to play
a role in regulating the stability of the membrane, distribution and
function of adhesive receptors, and adhesive receptor-induced
transmembrane signaling. When platelets are lysed with Triton X-100,
cytoplasmic actin filaments can be sedimented by centrifugation at low g-forces (15,600 g) but the membrane skeleton
requires 100,000
g. The present study shows that DRP
(dystrophin-related protein) sediments from lysed platelets along with
membrane skeleton proteins. Sedimentation results from association with
the membrane skeleton because DRP was released into the
detergent-soluble fraction when actin filaments were depolymerized.
Interaction of fibrinogen with the integrin
induces platelet aggregation,
transmembrane signaling, and the formation of integrin-rich
cytoskeletal complexes that can be sedimented from detergent lysates at
low g-forces. Like other membrane skeleton proteins, DRP
redistributed from the high-speed pellet to the integrin-rich low-speed
pellet of aggregating platelets. One of the signaling enzymes that is
activated following
-ligand
interactions in a platelet aggregate is calpain; DRP was cleaved by
calpain to generate a
140-kDa fragment that remained associated
with the low-speed detergent-insoluble fraction. These studies show
that DRP is part of the platelet membrane skeleton and indicate that
DRP participates in the cytoskeletal reorganizations resulting from
signal transmission between extracellular adhesive ligand and the
interior of the cell.
Duchenne muscular dystrophy is one of the most common inherited
human diseases. It is caused by a defective gene that codes for a
427-kDa protein,
dystrophin(1, 2, 3, 4, 5) .
The deduced amino acid sequence of dystrophin shows that it consists of
four domains and suggests that it is a cytoskeletal
protein(6) . The major rod-shaped domain contains 24
spectrin-like repeats. This domain is flanked on the amino terminus by
a domain that has a high degree of homology to the actin-binding
domains of spectrin and -actinin, and on the carboxyl terminus by
a cysteine-rich domain that shows some homology to a
Ca
-binding region in
-actinin. The most
carboxyl-terminal end of dystrophin consists of a short domain that has
no homology to any known protein and appears to play a role in linking
the molecule to the plasma membrane(7, 8) . Recent
studies using purified protein or recombinant fragments containing the
putative actin-binding domain (9, 10, 11) have shown that the protein can
bind to actin filaments in vitro, supporting the idea that
this molecule functions as a cytoskeletal protein. The finding that
dystrophin exists in a submembranous location (8, 12, 13) and that the carboxyl-terminal
end of the molecule associates tightly with a complex of membrane
glycoproteins (termed dystroglycan) (14, 15, 16, 17) suggests that
dystrophin is a component of a submembranous cytoskeleton.
Although
there is now considerable information concerning the structure and
interactions of dystrophin, little is known about the way in which the
absence of dystrophin leads to muscle cell necrosis. It is well
established that in the absence of dystrophin, there are increased
concentrations of cytoplasmic Ca and activation of
the Ca
-dependent protease,
calpain(18, 19, 20, 21) , suggesting
that dystrophin may play a role in stabilizing the sarcolemma or in
regulating the activity of Ca
channels(22, 23, 24, 25) . One
of the problems associated with studying the function of dystrophin is
that it is found primarily in muscle and brain tissue. In contrast, a
related protein, dystrophin-related protein (DRP) (
)(26, 27) is present in many different
cell types(28) . DRP is an autosomal gene product that is 80%
homologous to dystrophin(26, 29) . Recent studies have
shown that DRP is present in a submembranous location (30, 31) and associates with the same complex of
transmembrane glycoproteins as does dystrophin(32) . Because
the extracellular domain of dystroglycan can bind laminin (11, 33) and agrin(34, 35) , and DRP
co-localizes with agrin-induced acetylcholine receptor clusters, it has
been suggested that DRP may play a role in the transmission of signals
between the extracellular matrix and intracellular
cytoskeleton(35) . Given the similarities between dystrophin
and DRP, it appears that DRP may serve a similar function to
dystrophin. However, as with dystrophin, direct evidence that DRP is
part of a membrane skeleton in intact cells or that it is involved in
transmembrane signaling is lacking.
One cell-type that has a
membrane skeleton that can be readily isolated and analyzed is the
blood platelet(36) . This skeleton coats the plasma membrane
and associates with membrane glycoproteins. It has been visualized
morphologically(37, 38) , isolated from
detergent-solubilized platelets by centrifugation(37) , and
shown to be composed of short actin filaments, vinculin, spectrin,
actin-binding protein and other unidentified
proteins(37, 38, 39) . Recent work suggests
that the skeleton binds signaling enzymes, and reorganizes following
interaction of the integrin with
its adhesive ligand, fibrinogen, in a platelet
aggregate(40, 41, 42) . In the present study
we have shown that DRP is present in platelets and have used these
cells to demonstrate that DRP is associated with a membrane skeleton,
that it participates in integrin-induced reorganization of the
cytoskeleton, and that it is cleaved by calpain as a consequence of
integrin-ligand interactions. These studies provide direct evidence
that DRP is a component of a membrane skeleton and point to a role of
this protein in mediating the cytoskeletal reorganizations and
transmembrane signaling that occur as a consequence of integrin-ligand
interactions.
Figure 1: Western blots showing the presence of dystrophin-related protein in human platelets. Samples of mouse skeletal muscle or suspensions of human platelets (pls) from a normal control or from a patient with Duchenne's muscular dystrophy (dys) were electrophoresed through SDS gels and transferred to nitrocellulose paper. Blots were incubated with antibodies raised against dystrophin (panel A), dystrophin-related protein (panel B), or actin-binding protein (ABP) (panel C). Antibody-antigen complexes were detected with alkaline phosphatase-conjugated second antibodies.
To assure that the protein detected by the DRP antibody in platelets was authentic DRP, platelet RNA was isolated and used for reverse transcriptase PCR analysis. Primers from the carboxyl-terminal end of DRP (see ``Materials and Methods'') generated the expected 1205-base pair fragment (Fig. 2, lane 1). Similarly, primers from the amino-terminal end of DRP generated the expected band of 815 base pairs (Fig. 2, lane 2). Because platelets contain very little RNA as compared to leukocytes and it is often difficult to isolate platelets completely free of leukocytes, the PCR products could conceivably have arisen from contaminating leukocyte RNA. However, this possibility was eliminated by experiments showing that bands of the appropriate size were not generated when the DRP primers were used under the same amplification conditions on isolated leukocyte RNA (data not shown). Furthermore, primers derived from sequences in the light chain and heavy chain of IgG generated bands of appropriate molecular weight from leukocyte RNA but not from the platelet RNA (data not shown).
Figure 2: Agarose gel electrophoresis showing products from PCR analysis of platelet RNA using primers specific for dystrophin-related protein. Platelet RNA was subjected to reverse transcriptase PCR with primer pairs from the carboxyl-terminal (lane 1) or amino-terminal (lane 2) end of dystrophin-related protein. PCR reaction products were separated on an agarose gel and stained with ethidium bromide. The left-hand lane shows a 1-kilobase pair DNA ladder.
Figure 3:
Western blots showing the sedimentation of
dystrophin-related protein with cytoskeletal fractions from platelet
lysates. Suspensions of platelets (1 10
platelets/ml) were solubilized in an SDS-containing buffer (lane
1) or were lysed by addition of a Triton X-100 lysis buffer (lanes 2-4). Lysates were centrifuged for 4 min at
15,600
g. The resulting pellet was solubilized in
SDS-containing buffer (lane 2) and the Triton X-100
supernatant was centrifuged for a further 2.5 h at 100,000
g. The resulting high-speed pellet (lane 3), and the
high-speed supernatant (lane 4) were solubilized in
SDS-containing buffer. All samples were electrophoresed through
SDS-polyacrylamide gels and transferred to nitrocellulose paper. Blots
were incubated with antibody against DRP. Antibody-antigen complexes
were detected by enhanced
chemiluminescence.
While membrane skeleton proteins
sediment in the high-speed detergent-insoluble fraction because they
are associated with the detergent-insoluble membrane skeleton
fragments, other proteins can sediment at these g-forces
because they are inherently insoluble in Triton X-100. To distinguish
between these possibilities, a lysis buffer that induces
depolymerization of actin filaments (39, 51) was used
(the buffer contained DNase I and free Ca) and the
effect of this on the solubility of DRP determined. Analysis of the
high-speed detergent-insoluble pellets confirmed that the amount of
filamentous actin was decreased in lysates containing free
Ca
and DNase I (Fig. 4, compare the first and
second lanes of panel A). Depolymerization of actin filaments
in the detergent lysates was accompanied by decreased sedimentation of
several proteins known to be associated with the membrane skeleton in
unstimulated platelets(42, 51) : glycoprotein
Ib
(Fig. 4, panel B),
(Fig. 4, panel C),
and pp60
( Fig. 4panel D).
Depolymerization of actin was also accompanied by decreased
sedimentation of DRP (Fig. 4, panel E).
Figure 4:
Effect of actin depolymerization on the
recovery of DRP and membrane skeleton proteins from the high-speed
detergent-insoluble fraction of platelets. Suspensions of platelets (1
10
platelets/ml) were lysed by addition of an equal
volume of a Triton X-100 lysis buffer that contained 10 mM EGTA to chelate Ca
present in the Tyrode's
buffer and platelet extracts (lanes 1), or the same buffer
lacking EGTA and containing 2 mg/ml DNase I (lanes 2). Lysates
were incubated at 4 °C for 1 h and then centrifuged for 2.5 h at
100,000
g. The resulting pellets were solubilized in
SDS-containing buffer, samples were electrophoresed through
SDS-polyacrylamide gels, and transferred to nitrocellulose paper. Panel A represents a Coomassie Brilliant Blue-stained gel.
Blots were incubated with antibodies against glycoprotein (GP)
Ib
(panel B),
(panel C), pp60
(panel
D), or dystrophin-related protein (DRP) (panel E).
Antibody-antigen complexes were detected by enhanced
chemiluminescence.
Figure 5:
Western blots showing that
dystrophin-related protein redistributes along with membrane skeletal
proteins to the low-speed detergent-insoluble fraction of aggregating
platelets. Suspensions of platelets (1 10
platelets/ml) were incubated with thrombin for the indicated
times. Suspensions were either agitated occasionally (left-hand
panels) or stirred (middle- and right-hand
panels). The samples shown in the right-hand panels had
been preincubated with 0.5 mM RGDS for 5 min prior to thrombin
addition. Incubations were terminated by addition of Triton X-100 lysis
buffer. Lysates were centrifuged for 4 min at 15,600
g to obtain the low-speed detergent-insoluble pellet. All samples
were electrophoresed through SDS-polyacrylamide gels and transferred to
nitrocellulose paper. Blots were incubated with antibodies against
(A), talin (B),
pp60
(C), or DRP (D) as
shown. Antibody-antigen complexes were detected by enhanced
chemiluminescence.
Platelets from patients
with Glanzmann's thrombasthenia are deficient in
(43) . Thus, although they
undergo other activation-induced events(42) , they do not
undergo
-induced transmembrane
signaling. Therefore, the integrindependent redistribution of membrane
skeleton proteins to the low-speed detergent-insoluble fraction does
not occur when these platelets are activated(42) . In the
present study, we observed that although DRP redistributed to the
low-speed detergent-insoluble pellet of normal platelets (Fig. 6A), it did not redistribute to the low-speed
detergent-insoluble fraction of thrombasthenic platelets that were
activated in the same way (Fig. 6B). This finding is
consistent with the idea that DRP is part of a submembranous skeleton
that is incorporated into integrin-rich cytoskeletal complexes as a
result of integrin-induced transmembrane signaling in platelets.
Figure 6:
Western blots showing that
dystrophin-related protein does not redistribute to the low-speed
pellets from lysates of platelets of patients with Glanzmann's
thrombasthenia. Suspensions of platelets (1 10
platelets/ml) were agitated with thrombin for the indicated
times. Incubations were terminated by addition of Triton X-100 lysis
buffer. Lysates were centrifuged for 4 min at 15,600
g to obtain the low-speed detergent-insoluble pellets. The Triton
X-100 supernatants were centrifuged for a further 2.5 h at 100,000
g to obtain the high-speed detergent-insoluble pellets
and the detergent-soluble fractions. All samples were electrophoresed
through SDS-polyacrylamide gels, transferred to nitrocellulose paper,
and blotted with antibodies against DRP. Antibody-antigen complexes
were detected by enhanced
chemiluminescence.
Figure 7:
Western blots showing the cleavage of
dystrophin-related protein in thrombin-activated platelets. Suspensions
of platelets (1 10
platelets/ml) were preincubated
alone (lanes 1 and 2), or with 0.5 mM RGDS (lane 3) for 5 min. Suspensions were then stirred in the
absence (lane 1) or presence of thrombin (lanes 2 and 3) for 30 min. Incubations were terminated by addition of an
SDS-containing buffer. Samples were electrophoresed through
SDS-polyacrylamide gels and transferred to nitrocellulose paper. Blots
were incubated with antibody against dystrophin-related protein.
Antibody-antigen complexes were detected by enhanced
chemiluminescence.
The
finding that cleavage of DRP could be inhibited with RGDS and did not
occur in thrombasthenic platelets is consistent with cleavage being
induced by calpain. To directly test this, platelets were incubated
with agonist in the presence of the membrane permeable inhibitors of
calpain, MDL (44) and EST(45) . The concentration of
MDL used was such that it partially inhibited the activity of calpain,
as shown by the partial inhibition of the appearance of the degradation
products of actin-binding protein (Fig. 8, panel A)
(actin-binding protein is cleaved to generate fragments of 200 and
100 kDa; the
100-kDa fragment is then cleaved further to
generate a fragment of
91 kDa(50) ). The concentrations of
EST used were such that they almost completely prevented degradation of
actin-binding protein in thrombin-treated platelets (Fig. 8, panel A). Similarly, MDL partially inhibited degradation of
DRP while EST completely prevented any detectable degradation (Fig. 8, panel B). The amount of calpain activation in
thrombin-activated platelets can be quite variable. The experiment
presented in Fig. 8is one in which considerable activation of
calpain occurred, as indicated by the extensive cleavage of
actin-binding protein (panel A). In this experiment, there was
also considerable cleavage of DRP as shown by the almost complete loss
of intact DRP (panel B); when cleavage of DRP was extensive,
the fragment of
140 kDa was no longer detected (Fig. 8, panel B). When calpain activity was partially inhibited (with
MDL) the
140-kDa fragment was detected again (panel B).
Thus, the
140-kDa fragment presumably represents an intermediate
cleavage product of DRP that contains the epitope in the
carboxyl-terminal domain against which the antibody was raised; further
cleavage of the
140-kDa fragment results in loss of this epitope.
Like DRP, the
140-kDa hydrolytic fragment of the protein was
present in the low-speed detergent-insoluble fraction from aggregating
platelets (see Fig. 5and Fig. 6).
Figure 8:
Western blots showing the calpain-induced
cleavage of actin-binding protein (ABP) and dystrophin-related
protein in thrombin-activated platelets. Suspensions of platelets (1
10
platelets/ml) were preincubated at 37 °C
with carrier (no addition), or with one of the membrane-permeable
inhibitors of calpain, MDL (25 mM; see (45) ) and EST
(100 µg/ml; see (45) ), for 20 min. Suspensions were then
stirred with thrombin for the indicated times. Incubations were
terminated by addition of an SDS-containing buffer. Samples were
electrophoresed through SDS-polyacrylamide gels and transferred to
nitrocellulose paper. Blots were incubated with antibody against
actin-binding protein (panel A) or dystrophin-related protein (panel B). Antibody-antigen complexes were detected by
enhanced chemiluminescence. ABP; 200, 100, and 91 indicate
calpain-induced actin-binding protein fragments of
200,
100,
and
91 kDa (50) respectively; 140 indicates a
fragment of DRP of
140 kDa.
Although the molecular cause of muscular dystrophy is now
known, little information is available about the way in which the
absence of functional dystrophin leads to muscle necrosis. One of the
problems in elucidating the function of dystrophin is that it is
present predominantly in muscle and brain. In contrast, a related
protein, DRP, is present in many non-muscle cells. In the present
study, we show that DRP exists in platelets. In this cell, the membrane
is lined by a skeleton that is readily isolated from detergent-lysed
platelets by centrifugation (37) and has been visualized by
electron microscopy(37, 38) . Several lines of
evidence show that DRP is a component of the platelet membrane
skeleton. First, it was recovered along with the membrane skeleton in
the high-speed fraction from detergent-lysed platelets. Second, like
other membrane skeleton proteins, it was released from the
detergent-insoluble material when actin filaments were depolymerized.
Third, DRP redistributed, along with other membrane-skeleton proteins,
to the low-speed detergent-insoluble fraction from aggegating
platelets. Finally, like other membrane-skeleton
proteins(40, 42) , the redistribution from the high-
to the low-speed pellet in aggregating platelets was dependent on
binding of adhesive ligand to and
did not occur in platelets that lacked this integrin. The observations
that dystrophin binds to actin in
vitro(9, 10, 11) , that dystrophin and
DRP associate with membrane
glycoproteins(14, 15, 16, 17, 32) ,
and that dystrophin and DRP exist in a submembranous
location(8, 12, 13, 30, 31) ,
have provided circumstantial evidence that these proteins are part of a
submembranous cytoskeletal structure. The present study provides direct
evidence that DRP is indeed a component of a membrane skeleton in an
intact cell.
The finding that DRP is a component of the platelet
membrane skeleton suggests a number of potential functions for this
protein. For example, the skeleton coats the entire plasma membrane and
it is thought that it may regulate the function and distribution of
membrane glycoproteins (37, 52) , and also stabilize
the membrane, preventing microvesicles from being shed(55) . It
also binds signaling molecules and appears to be involved in
transmembrane signaling following integrin-ligand
interactions(42) . In other cells, binding of extracellular
ligand to dystroglycan has been implicated in inducing changes in the
organization of a membrane skeleton(34, 35) . It is
not known whether dystroglycan is present in platelets; the
reorganizations of the DRP-containing membrane skeleton detected in the
present study were initiated by interaction of extracellular ligand
with the integrin . In platelets,
at least two adhesive receptors are associated with the membrane
skeleton (glycoprotein Ib-IX and
) (42, 51) and transmembrane signaling is induced as a
consequence of ligand binding to both of
them(42, 56, 57, 58, 59) .
Thus, it appears possible that DRP is a component of a skeletal
structure that reorganizes in response to interaction of associated
glycoproteins with their extracellular adhesive ligands. Additional
work will be needed to investigate this possibility and to identify the
membrane glycoprotein with which DRP associates in platelets.
Based
on the fact that the integrin-rich cytoskeletal complexes in platelets
associate with cytoplasmic actin (42) and that they contain a
number of the proteins present in focal contacts of cultured cells (e.g. talin and vinculin(42) ), we have suggested that
they may be analagous to focal contacts in adherent
cells(36, 42) . Interestingly, there have been
previous indications that dystrophin may be present in integrin-rich
domains in other cells(60) . As in focal contacts, a number of
signaling molecules appear to associate with the integrin-cytoskeletal
complexes in platelets (e.g. pp60,
pp125
, phosphoinositide 3-kinase, and
calpain(41, 42, 57, 58, 61, 62, 63, 64) ).
At least in some cases, the recruitment of signaling molecules to the
integrin-rich cytoskeletal complexes appears to be involved in
activation of the enzymes(41, 64) . The specific
protein-protein interactions that mediate the recruitment of the
signaling enzymes to the integrin-cytoskeletal complexes are not known.
A number of the proteins present in these cytoskeletal complexes
contain the SH2 and SH3 domains that have been implicated in
protein-protein interactions. Interestingly, an additional motif
present in a number of signaling molecules has recently been identified
and shown to be present in DRP (65, 66) . It will be
of interest to determine whether DRP plays a simple structural role in
the integrin-cytoskeletal complexes or whether it is also involved in
binding and regulating signaling molecules.
One enzyme that is
recruited to focal contacts in cultured cells (67) and is
incorporated into the detergent-insoluble integrin-rich cytoskeletal
fraction in aggregating platelets (64) is calpain. This
protease is selectively activated at sites where the integrin clusters
with cytoskeletal proteins(64) . Thus, we have suggested that
recruitment of the protease to the ``focal-contact-like''
structures in platelets is the first step in activation of this
protease(64) . The finding in the present study that DRP is
part of the integrin-rich detergent-insoluble cytoskeletal fraction is
of interest because one of the characteristics of muscle from patients
with Duchenne's muscular dystrophy is activation of calpain and
subsequent degradation of muscle proteins. In muscle, it is thought
that the absence of dystrophin may result in decreased membrane
stability and thus, increased Ca concentrations and
calpain activation. An alternative idea is that dystrophin normally
serves to directly regulate Ca
fluxes(21, 24, 25) . In platelets,
integrins have been implicated in the regulation of Ca
fluxes (68, 69) and calpain
activation(54) . An increased understanding of the role of the
integrin-cytoskeletal complexes and of DRP in regulating calpain
activation in platelets may shed light on the way in which the absence
of dystrophin leads to increased calpain activation in muscle.
The
fact that DRP is cleaved by calpain suggests that it plays an active
role in inducing the cytoskeletal remodeling that is induced by
integrin-ligand interactions. Previous studies have shown that
dystrophin is a substrate for calpain in vitro(70, 71) but it has not been known whether it is
cleaved by this protease in an intact cell. The major DRP fragment
detected in the present study was one of 140 kDa that reacted with
an antibody against the carboxyl-terminal end of the molecule. While
this end of the molecule contains the binding site for dystroglycan, it
does not contain the binding site for actin; despite this, the fragment
remained associated with the cytoskeleton in aggregating cells. Future
work will be needed to determine whether additional proteolytic
fragments remain associated with the cytoskeleton and to identify
cytoskeletal and membrane proteins that mediate the interaction of DRP
and its calpain-induced fragment with the integrin-rich cytoskeletal
fraction.
In summary, it is becoming increasingly apparent that the membrane skeleton in platelets binds signaling molecules and is involved in transmitting signals from extracellular adhesive proteins to the interior of the cell. The present study shows that DRP is part of this structure. The finding that integrin-induced transmission of signals from extracellular ligand to the interior results in cleavage of DRP by calpain, suggests that DRP may play an important role in mediating integrin-induced cytoskeletal remodeling and transmembrane signaling. Because the platelet membrane skeleton can be readily obtained from detergent-solubilized platelets, signaling events can be rapidly induced, and DRP-containing integrin-rich cytoskeletal complexes can be isolated, the platelet may provide a useful model in which to characterize the interactions of DRP and to identify the function of this protein. The high degree of homology between dystrophin and DRP suggests that these proteins may serve the same function. Thus, studies on the platelet could lead to an increased understanding of the way in which the absence of dystrophin in patients with Duchenne's muscular dystrophy leads to cell necrosis.