From the Department of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania 19104 and the
¶ Department of Medicine, University of California at San
Francisco and the Veterans Administration Medical Center,
San Francisco, California 94121
Received for publication, October 13, 2000, and in revised form, December 27, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanism of signal transduction underlying
the activation of platelets by collagen has been actively investigated
for over 30 years, but the receptors involved remain incompletely understood. Studies of human platelets, which are unresponsive to collagen, mouse knockout models, and platelet biochemical studies support the hypothesis that the recently cloned platelet surface protein GPVI functions as a signaling receptor for collagen. To directly test this hypothesis, we have expressed wild-type and mutant
forms of GPVI in RBL-2H3 cells, which express the Fc Platelet activation is essential for both normal hemostasis and
arterial thrombosis that occurs in the setting of vascular diseases
such as stroke and myocardial infarction. One of the earliest steps in
arterial thrombosis is the adhesion of circulating platelets to areas
of injured vessel wall and the activation of adherent platelets, which
recruits additional platelets to form a hemostatic plug. Activation of
platelets at sites of vascular injury occurs in response to exposed
subendothelial matrix proteins, the most important of which is collagen.
Exposed collagen initiates two essential platelet functions: the
adhesion of circulating platelets to the site of injury and the
activation of platelet signaling, which stimulates thrombus growth.
Platelet adhesion to collagen has been shown to occur both indirectly,
via interaction of platelet GPIb with plasma von Willebrand's factor
bound to exposed collagen (1), and directly, via collagen interaction
with the platelet integrin GPVI is a recently cloned 62-kDa surface protein (6, 7) first
identified by iodination of platelet surface glycoproteins (8). GPVI
was proposed as a signaling receptor for collagen following the
description of individuals with bleeding disorders whose platelets
could not be activated by collagen and lacked GPVI despite having
normal levels of the platelet integrin The recent cloning of human and mouse GPVI (6, 7) reveals that GPVI is
a type I transmembrane protein whose deduced amino acid sequence
identifies it as an Ig domain-containing receptor homologous to the Fc
and killer Ig-like receptors, some of which are known to signal via Fc
R To address the functional role of GPVI, we have stably expressed the
receptor in RBL-2H3 cells, a rat basophilic leukemia cell line that
expresses Fc R Materials--
Type I collagen derived from equine tendons was
obtained from Chronolog (Havertown, PA) and used for all studies
shown. Studies were confirmed with type I collagen derived from bovine
tendon and type III collagen derived from calf skin (Sigma). Convulxin was obtained from Sigma and purified from the venom of the
Crotalus durissus rattlesnake using gel
filtration as previously described (13). CRPs were synthesized as
previously described using cross-linked cysteine residues (21). All
other reagents were obtained from Sigma.
Cloning and Epitope Tagging of GPVI--
A GPVI cDNA was
generated by PCR from human platelet cDNA using primers based on
published 5'- and 3'-untranslated sequences (sense strand primer: 5'-
TCAGGACAGGGCTGAGGAACC-3'; antisense strand primer:
5'-TTGGATACGACCGTGCCTGGG-3'). Three distinct amplified 1.1-kilobase
pair products were sequenced to obtain a consensus sequence that
exhibited several differences from the published cDNA (6) but
agreed with a cDNA sequence deposited directly in
GenBankTM (accession number AB035073). All GPVI receptor
amino acids reported here correspond to the protein predicted by the
open reading frame of this cDNA starting at nucleotide number 13. FLAG-tagged GPVI was generated by replacing the endogenous signal
peptide with that of interleukin-1 and placing the FLAG epitope
(DYKDDDDK) in frame with GPVI at amino acid number 21, the predicted
site of signal peptide cleavage (SignalP VI.I). HA-tagged GPVI was generated in an identical manner. Wild-type and epitope-tagged GPVI
were expressed using the mammalian expression vector pcDNA3.0 (Invitrogen).
Site-directed Mutagenesis of GPVI--
Site-directed mutagenesis
was performed using the QuikChange mutagenesis kit (Stratagene). The
oligonucleotide used for the R272L mutation was 5'-
GCAACCTGGTCCGGATATGCCTCGGGGCTGTG-3'. The oligonucleotide used for the
R295STOP mutation was 5'-GGCAGAGGACTGGCACAGCTAGAGGAAGCGCCTGC-3'. All
mutants were made as epitope-tagged receptors as described above.
Platelet Aggregation Studies--
Blood was collected into
citrate buffer and platelet-rich plasma obtained as previously
described (22). All studies were performed using platelet-rich plasma
at a platelet density of 2 × 108 platelets/ml.
Creation and Screening of RBL-2H3 Cells Stably Expressing
GPVI--
RBL-2H3 cells (ATCC, Manassas, VA) were electroporated with
linearized expression plasmids and selected in G418 (1.0 mg/ml active
concentration; Life Technologies, Inc.) as previously described (23).
Wild-type GPVI-expressing clones were directly tested for signaling in
response to convulxin (below) and epitope-tagged clones tested for
receptor expression by flow cytometry using M2 anti-FLAG antibody
(Sigma) or anti-HA antibody (Sigma) as primary antibodies and
fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibody.
Intracellular Calcium Studies--
Increases in cytoplasmic
calcium in response to convulxin, CRP, collagen, and thrombin were
measured using the calcium-sensitive dye Fura-2 as previously described
at a final cell concentration of 2 × 106 cells/ml
(24). The buffer used for these studies was RPMI 1640 (Life
Technologies) with HEPES 25 mM and 1 mg/ml BSA.
Functional Rescue of Surface FLAG-Fc R Biochemical Precipitation of GPVI and GPVI Mutants--
CVX was
coupled to CNBr-activated Sepharose 4B according to the manufacturer's
instructions (Amersham Pharmacia Biotech) at a concentration of 200 nM per ml of swollen gel. RBL-2H3 cells (total of
~1 × 107) were lysed for 2 h at 4 °C in
ice-cold lysis buffer (1% (w/v) digitonin (Calbiochem), 0.12% (v/v)
Triton X-100, 150 mM NaCl, 0.01% (w/v) NaN3,
20 mM triethanolamine, pH 7.8, and containing a 1:100 (v/v)
dilution of a mammalian protease inhibitor mixture (Sigma)).
Detergent-insoluble cellular debris was pelleted at 10,000 × gav for 15 min, and 50 µl of CVX-Sepharose
beads were used to immunoprecipitate GPVI from the supernatant in a 2-h
incubation period. Sepharose beads that had been activated by 1 mM HCl and blocked in 0.1 M Tris-HCl, pH 8.0, containing 0.5 M sodium chloride were used as controls for
nonspecific binding. Beads were pelleted by centrifugation and washed
three times in ice-cold washing buffer (50 mM Tris, 150 mM NaCl, pH 8.0, 5 mM CHAPS, containing 1:100 (v/v) protease inhibitors). Finally, beads were heated to 100 °C in
an equal volume of 2× Laemmli sample buffer (1 M Tris-HCl, pH 6.8, 0.2 M DTT, 4% (w/v) SDS, 0.004% bromphenol blue,
20% glycerol), and an aliquot was run on 5-20% (v/v) gradient
SDS-polyacrylamide gels using a standard electrophoresis buffer (25 mM Tris-HCl, 0.25 M glycine, and 0.1% (w/v)
SDS). Gels were transferred (50 mM Tris-HCl, 40 mM glycine, 0.037% (w/v) SDS, 20% (v/v) methanol) to
Hybond-P nitrocellulose membrane (Amersham Pharmacia Biotech), blocked
overnight in blocking buffer (0.1 M Tris-buffered saline, pH 7.4, containing 5% (w/v) nonfat milk, 5% (w/v) BSA, and 0.02% (v/v) Tween 20), and probed with antibodies to the FLAG epitope (Bio-M2
(Sigma)) and the Fc R Measurement of RBL Cell Adhesion--
96-Well polystyrene high
binding plates (Costar 3590; Corning Glass) were coated with 50 µl/well of 20 µg/ml BSA, convulxin, collagen, and fibronectin or 15 µg/ml CRP in PBS with 0.9 mM calcium and 0.4 mM magnesium overnight at 4 °C. Prior to application, the pH of the diluted protein solution was adjusted to pH 7-8. The
plates were blocked with 150 µl of 10 mg/ml BSA for 2 h at room
temperature and washed with PBS with 0.9 mM calcium and 0.4 mM magnesium twice. RBL-2H3 cells were detached from plates
with 5 mM EDTA, washed with PBS or saline solution
containing 3 mM Ca2+ and 1.5 mM
Mg2+, and suspended in the same solution at a cell
concentration of 2 × 106/ml. 2 × 105 cells were applied to each well. After a 1-h incubation
at room temperature, the plates were washed with the same solution
seven times. To read the number of cells bound to each well, the
GPVI-expressing RBL-2H3 Cells Signal in Response to Convulxin and
CRP but Not to Collagen--
RBL-2H3 cells stably expressing wild-type
GPVI (GPVI-RBL) and FLAG-tagged GPVI (FLAG GPVI-RBL) were identified
using adhesion to convulxin and flow cytometry to detect surface FLAG
epitope, respectively. The ability of the putative GPVI ligands
collagen, CRP, and convulxin to initiate calcium signaling in RBL,
GPVI-RBL, and FLAG GPVI-RBL was tested in parallel with
aggregation studies of human platelets
performed using the same reagents on the same day (Fig. 1, Table
I, and data not shown). GPVI-RBL and FLAG GPVI-RBL, but not untransfected RBL cells, responded to convulxin with
a threshold concentration only 1.6-fold greater than that found
necessary to aggregate human platelets (0.3 nM). These
results demonstrate functional GPVI signaling and close concordance
between the two assays for GPVI dose response, suggesting similar GPVI receptor density in the two cell types. In contrast, collagen elicited
no response even at concentrations 500 times greater than that
necessary to aggregate human platelets (100 µg/ml). Similar results
were obtained using three distinct GPVI-RBL clones and three distinct
FLAG-GPVI RBL clones. The data shown are representative of experiments
performed in RPMI with Chronolog Type I collagen, which elicits the
most robust platelet responses in our hands (data not shown). In
addition, no signaling responses were observed using a second source of
type I collagen, type III collagen, or in the presence of higher cation
concentrations (1 mM calcium and 0.5 mM
magnesium; data not shown). Interestingly, CRP was capable of eliciting
a small calcium response but required a concentration 500 times greater
than that necessary to induce platelet aggregation (50 µg/ml). CRP
signaling responses were also only seen with the two GPVI-expressing
RBL-2H3 cell clones that had the greatest sensitivity to convulxin.
These data demonstrate that GPVI is a functional receptor but that GPVI
expression alone is insufficient to reproduce the collagen and CRP
signaling responses observed in platelets.
Adhesion of RBL and GPVI-RBL to the Putative GPVI Ligands
Convulxin, Collagen, and CRP--
To determine whether expression of
GPVI is sufficient to mediate adhesion but not signaling to collagen,
static adhesion assays were performed with wild-type and
GPVI-expressing RBL cells (GPVI RBL and FLAG-GPVI RBL generated
identical results; data not shown). Expression of GPVI conferred strong
adhesion to convulxin and moderate adhesion to CRP, but no adhesion to
collagen was detected (Fig. 2). GPVI
R272L- and GPVI R295STOP-expressing RBL clones, which express ~5-fold
and 10-50-fold more surface GPVI than FLAG-GPVI, respectively (Fig.
3), also failed to bind collagen despite
binding both CRP and CVX. Performing the assay using normal saline with 1 mM calcium and 0.5 mM magnesium yielded
identical results (data not shown). In addition, no adhesion of
GPVI-expressing or wild-type RBL cells was observed to bovine type I
collagen or to bovine type III collagen (data not shown). Wild-type RBL
cells adhered only to fibronectin (Fig. 2). Unlike fibronectin binding,
GPVI-mediated adhesion to CRP and convulxin was not disrupted by 5 mM EDTA (Fig. 2B), consistent with a
nonintegrin-mediated mechanism of adhesion. Thus, adhesion assays of
GPVI-expressing RBL cells are consistent with signaling assays and show
strong GPVI interaction with convulxin, weaker GPVI interaction with
CRP, and no GPVI interaction with collagen.
The GPVI Transmembrane Arginine and Intracellular C-tail Are Both
Necessary for GPVI Signaling--
The signaling roles of the GPVI
transmembrane (TM) domain arginine (R272) and intracellular C-tail were
tested by generating RBL cell lines expressing FLAG-tagged receptors in
which the TM arginine is replaced by leucine (R272L-RBL) and the C-tail
is truncated shortly following the TM domain (R295STOP-RBL). Both mutant GPVI receptors were expressed on the surface of RBL cells at
levels equal to or greater than clones expressing wild-type GPVI (Fig.
3B). Consistent with the results of analogous mutations in
related receptors, R272L-RBL did not signal in response to convulxin,
confirming a necessary role for the GPVI TM domain arginine for signal
transduction. Surprisingly, unlike similar C-tail truncation mutants of
the Fc R Loss of the GPVI Transmembrane Domain Arginine and C-tail Results
in Loss of Coupling to Fc R Recent studies of human platelets that are unresponsive to
collagen (10), mouse knockouts (27), and platelet signaling (reviewed
in Ref. 14) have generated the hypothesis that the platelet surface
protein GPVI mediates collagen signaling and does so through its
interactions with the immunoreceptor signaling adaptor Fc R Expression of wild-type and FLAG-GPVI conferred robust calcium
signaling to the snake venom protein convulxin at a threshold concentration equivalent to that necessary to activate human platelets but no detectable response to collagen at a concentration more than 500 times greater than that necessary to activate human platelets (Fig. 1).
Unlike collagen, convulxin has been demonstrated to directly bind GPVI
(13, 29) and was used by Clemetson et al. (6) to purify the
GPVI protein from platelets. Thus, GPVI is a functional receptor in
RBL-2H3 cells and signaling in RBL-2H3 cells closely resembles that in
human platelets, but GPVI alone is not sufficient for collagen signaling.
To address GPVI-ligand interaction independent of signal transduction,
we tested the adhesion of GPVI-RBL to putative GPVI ligands. GPVI
expression conferred strong binding to convulxin and weaker binding to
CRP but no detectable binding to collagen. Thus, the adhesion to
immobilized proteins conferred by GPVI expression parallels the
signaling responses observed to soluble agonists. These results are in
contrast to those recently reported by Jandrot-Perrus et al.
(7), who detected a small amount of collagen binding in a monocytic
cell line (U937) stably expressing human or mouse GPVI. This
discrepancy could reflect a difference in GPVI receptor density between
the stable cell lines used or differences in methodology. In our hands,
however, even clones expressing very high levels of GPVI such as the
R272L clones (whose extracellular domains are wild type) confer
adhesion to both CRP and convulxin but not collagen (data not shown and
Fig. 3).
Is GPVI a bona fide collagen receptor, and, if
so, why is GPVI expression insufficient to confer collagen signaling?
Inadequate receptor density on RBL-2H3 cells is not a likely
explanation, since the dose-response to CVX is similar in platelets and
in GPVI-expressing RBL cells (Table I). One potential explanation for
these results is that GPVI does mediate collagen signaling but that
another coreceptor is required. This coreceptor might facilitate direct
GPVI-collagen binding, or GPVI might mediate collagen signaling
indirectly by linking a ligand-binding coreceptor to the signaling
adaptor Fc R Several lines of evidence support the model that collagen is a GPVI
ligand but that GPVI absolutely requires a coreceptor such as
Studies of Fc R The human GPVI C-tail is ~50 amino acids long and can be divided into
basic, proline-rich, and serine/threonine-rich domains (Fig.
6). The mouse GPVI C-tail is shorter and
lacks the serine/threonine-rich region (data not shown, and see Ref.
7). Truncation of the GPVI C-tail does not interfere with receptor
expression but results in complete loss of signaling in RBL cells and
loss of Fc R receptor
-chain (Fc R
), the putative signaling co-receptor for GPVI in platelets, but lack GPVI itself. Expression of GPVI in RBL-2H3 cells
confers strong adhesive and signaling responses to convulxin (a snake
venom protein that directly binds GPVI) and weak responsiveness to
collagen-related peptide but no responsiveness to collagen. To
elucidate the mechanism of GPVI intracellular signaling, mutations were
introduced in the receptor's transmembrane domain and C-terminal tail.
Unlike reported studies of other Fc R
partners, these studies reveal
that both the GPVI transmembrane arginine and intracellular C-tail are
necessary for coupling to Fc R
and for signal transduction. To our
knowledge, these studies are the first to demonstrate a direct
signaling role for GPVI and the first to directly test the role of GPVI
as a collagen receptor. Our results suggest that GPVI may be necessary
but not sufficient for collagen signaling and that a distinct
ligand-binding collagen receptor such as the
2
1 integrin is likely to
play a necessary role for collagen signaling as well as adhesion in platelets.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 (2). In
contrast, although the activation of platelets by collagen has been
observed for over 30 years (3), the receptors and signaling pathways
that mediate platelet activation by collagen are only beginning to be
fully understood. Indirect evidence suggests that both
2
1 and GPIb can initiate signaling when
bound to collagen (1, 4, 5). However, this signaling does not appear to be sufficient to account for the magnitude of the platelet response to collagen.
2
1 (9, 10). Significant evidence suggests that GPVI signaling is
sufficient to activate platelets and that GPVI may mediate collagen
signaling in platelets. Platelets are activated by cross-linked anti-GPVI antibodies (11) and by convulxin
(CVX),1 a multimeric snake
venom protein isolated from a South American rattlesnake which is
capable of desensitizing platelets specifically to collagen and which
binds specifically to GPVI (12, 13). In addition, collagen signaling
and GPVI signaling in platelets both employ the immunoreceptor
signaling pathway (14) and require Fc R
(15). These data have led to
a model of collagen activation of platelets in which adhesive roles are
played by the integrin
2
1 as well as GPIb
and signaling roles are played by GPVI-Fc R
,
2
1, as well as perhaps GPIb (14). This
model has not been adequately tested, however, because of the absence
of systems in which the contribution of each receptor can be studied in isolation.
(16). Consistent with its putative role as an Fc R
partner,
GPVI has a charged arginine residue in its transmembrane domain that
may mediate interaction with the Fc R
transmembrane domain in a
manner analogous to that of the known Fc R
partners Fc
RI
and PIR-A (16-18). Direct functional evidence demonstrating that GPVI
is a collagen receptor and that GPVI signaling is mediated by Fc R
,
however, are lacking. Transient expression of GPVI has been
demonstrated to confer a slight calcium signal to collagen in the DAMI
megakaryocytic cell line (6), but these cells express endogenous GPVI
(data not shown and Ref. 6),
2
1 (19), and
perhaps other collagen receptors; it is therefore not clear if
that response is mediated directly by GPVI or if GPVI expression is
sufficient to enhance signaling by
2
1 or
other identified (e.g. p65 (20)) or unidentified collagen receptors.
and reproduces the platelet collagen responses of
intracellular calcium mobilization and degranulation but does not
express endogenous GPVI or
2
1. Our
studies reveal that GPVI cross-linking by the GPVI-specific ligand
convulxin initiates intracellular signaling but that GPVI alone is
incapable of mediating a signaling response to collagen. A small
signaling response is elicited by collagen-related peptides (CRPs),
however, and static adhesion studies support interaction with convulxin and CRP but not collagen. Finally, site-directed mutagenesis of the
GPVI transmembrane domain and intracellular C-tail demonstrates that
both the GPVI transmembrane arginine and the receptor C-tail are
necessary for Fc R
interaction and intracellular signaling. These
results provide insight into GPVI signal transduction and suggest that
GPVI-signaling in response to collagen requires coreceptors for
both ligand binding and intracellular signal transduction.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in HEK-293T Cells to
Measure GPVI-Fc R
Interaction--
A stable HEK-293T cell line that
stably expressed an N-terminally FLAG-tagged Fc R
in a manner
identical to that previously described for DAP-12 (25) was a kind gift
of Steve Spusta (University of California at San Francisco). These
cells expressed FLAG-Fc R
at a level such that no cell surface
FLAG-Fc R
was detectable by fluorescence-activated cell sorting
despite readily detectable intracellular FLAG-Fc R
by Western blot
analysis (data not shown). HA-tagged wild-type and mutated GPVI
receptors were transiently expressed in FLAG-Fc R
-expressing
HEK-293T cells (Fugene6 transfection reagent; Roche Molecular
Biochemicals). Surface expression of GPVI was followed with anti-HA
antibody, and surface expression of Fc R
was followed with anti-FLAG
antibody using flow cytometry as previously described for DAP-12 (25).
Rescue of surface expression of FLAG-Fc R
was quantitated by
comparing surface FLAG expression in GPVI-transfected versus
mock-transfected 293T-FLAG-Fc R
cells as previously described
(25).
(Upstate Biotechnology, Inc., Lake Placid, NY)
diluted 1:3000 (v/v) and 1:1500 (v/v), respectively, in blocking
buffer. These antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (Sigma) diluted 1:10,000 (v/v) in 0.1 M Tris-buffered saline, pH 7.4, and membranes
were developed using the ECL method (ECL-Plus; Amersham Pharmacia Biotech).
-hexosaminidase activity assay was used as previously described
(26). Briefly, 20 µl of 0.5% Triton X-100 in PBS were added to each
well to lyse the bound cells. 80 µl of 1 mM substrate
(p-nitrophenol
N-acetyl-
-D-glucosaminide; Sigma catalog no.
N9376) in 0.05 M citrate buffer, pH 4.5, were subsequently
added. After 1 h of incubation at 37 °C, 100 µl of 0.1 M sodium carbonate, 0.1 M sodium bicarbonate
were added per well. The A405 was
measured with an Emax precision microplate reader (Molecular Devices,
Inc., Sunnyvale, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
[in a new window]
Fig. 1.
GPVI signaling in RBL-2H3 cells. Wild
type (a) and RBL cells stably expressing GPVI
(b-e) were exposed to collagen (100 µg/ml), convulxin (1 nM), CRP (50 µg/ml), or thrombin (10 nM), and
calcium signaling was measured using the calcium-sensitive dye Fura-2.
a, wild-type RBL cells have no response to convulxin or CRP
but signal in response to thrombin. b, RBL cells expressing
GPVI signal in response to the GPVI ligand CVX. c,
GPVI-expressing RBL cells do not respond to collagen. d and
e, GPVI-expressing RBL cells signal weakly in response to
CRP. Results are representative of eight experiments performed with
three distinct GPVI-expressing lines of RBL cells.
Comparison of signaling by putative GPVI ligands in GPVI-expressing RBL
cells and in platelets
View larger version (11K):
[in a new window]
Fig. 2.
Adhesion of GPVI-expressing RBL cells to
putative GPVI ligands. Microtiter plates were coated with type I
collagen, CRP, CVX, fibronectin, or BSA, and adhesion of wild-type and
GPVI-expressing RBL cells was measured at A405
using a colorimetric substrate of the endogenous RBL enzyme
hexosaminidase. a, adhesion of wild type RBL cells
(open bars) and GPVI-expressing RBL cells
(black bars) in the PBS with 0.9 mM
calcium and 0.4 mM magnesium. b, adhesion of
wild type RBL cells (open bars) and
GPVI-expressing RBL cells (black bars) in the
presence of EDTA (5 mM). The results shown are the mean and
S.D. of quadruplicate samples from a single experiment. Each experiment
is representative of 3-5 similar experiments performed with distinct
GPVI-expressing clones. Note the strong adhesion of GPVI-expressing
cells to CVX, intermediate adhesion to CRP, and lack of detectable
adhesion to collagen.
View larger version (26K):
[in a new window]
Fig. 3.
Expression and function of GPVI transmembrane
and C-tail mutants in RBL-2H3 cells. FLAG-tagged wild-type
(WT), R272L, and R295STOP GPVI receptors were stably
expressed in RBL cells and tested for their signaling responses to the
GPVI ligand CVX using the calcium-sensitive dye Fura-2. a,
schematic diagrams of the wild-type and mutant receptors expressed are
shown to highlight the sites of mutation and the proposed point of
interaction with the signaling adaptor Fc R . EC, receptor
extracellular domain; TM, receptor transmembrane domain;
IC, receptor intracellular domain; R, the GPVI
transmembrane domain arginine; L, the leucine substituted
for R272 in the R272L mutant; D, the Fc R
transmembrane
domain aspartate; Y, Fc R
ITAM tyrosine residues.
b, receptor surface expression measured with anti-FLAG
antibody in control RBL cells (thin lines) and
GPVI-expressing RBL cells (thick lines).
c, calcium signaling of GPVI-expressing RBL cells in
response to CVX (10 nM) and thrombin (10 nM).
These results are representative of identical experiments performed
with 3-5 distinct clones for each receptor type.
partners Fc
RI and Fc
RIII (see Fig. 6), RBL cells
expressing the GPVI C-tail truncation mutant R295STOP also failed to
signal to convulxin, demonstrating an unexpected necessary role for the
GPVI C-tail (Fig. 3C).
--
To determine why the R272L and
R295STOP mutants of GPVI no longer supported signaling by CVX, we
compared the ability of wild-type and mutant GPVI receptors to interact
with Fc R
with a functional assay in HEK-293T cells stably
expressing FLAG Fc R
and by direct biochemical means using
coprecipitation. As for HEK-293T cells engineered to express low levels
of the homologous immunoreceptor signaling adaptor DAP-12(25), FLAG-Fc
R
is not expressed on the cell surface of these cells in the absence
of a coexpressed Fc R
partner (Fig.
4A). The ability of an
expressed receptor to rescue surface expression of FLAG-Fc R
therefore measures functional association with Fc R
. Wild-type GPVI
expression rescued 10 times more surface FLAG-Fc R
than
mock-transfected cells (Fig. 4A). In contrast, GPVI R272L
expression failed to rescue any FLAG-Fc R
, consistent with a
complete loss of association with Fc R
(Fig. 4A). GPVI
R295STOP expression also failed to rescue surface FLAG-Fc R
,
indicating a lack of Fc R
interaction (Fig. 4A). Surface
staining for the HA epitope confirmed that wild-type and mutant GPVI
receptors were expressed at equivalent levels (Fig. 4B) and
demonstrates that, as for the related Fc R
partner PIR
(18), GPVI
expression in HEK-293T cells does not require Fc R
interaction. Loss
of Fc R
interaction in GPVIR272L and GPVIR295 STOP was confirmed
biochemically using convulxin to precipitate GPVI and subsequently
assaying for associated Fc R
by immunoblotting (Fig.
5). Convulxin precipitation of wild-type
GPVI, but neither mutant receptor resulted in coprecipitation of Fc
R
(Fig. 5). Interestingly, immunoblotting of GPVI-R295STOP protein
with anti-FLAG antibody reveals the presence of mature protein at the
predicted molecular mass of ~57 kDa (5 kDa smaller than the
wild-type and R272L receptors) and the presence of a large amount of
protein at 36-38 kDa, the predicted size for unglycosylated,
incompletely processed protein. It is possible that this lower
molecular mass species represents protein that cannot reach the cell
surface because it cannot couple to Fc R
partners and is therefore
targeted for degradation. The fact that the abundantly expressed R272L mutant escapes this fate supports the role of the transmembrane arginine in targeting unpartnered receptors for degradation. These results show that the GPVI transmembrane arginine is necessary but not
sufficient for functional association with the receptor's signaling
coreceptor Fc R
and that loss of signaling following truncation of
the GPVI C-tail is due to loss of Fc R
interaction rather than loss
of an unidentified, distinct signaling function.
View larger version (17K):
[in a new window]
Fig. 4.
Wild-type and mutant GPVI receptor
interaction with Fc R . The interaction
between wild type and mutant GPVI receptors and the signaling
coreceptor Fc R
was measured using surface rescue of FLAG-Fc R
in
a HEK-293T cell line that stably expresses a low level of FLAG-tagged
Fc R
as described under "Experimental Procedures" a,
surface FLAG-Fc R
expression in cells transfected with HA-tagged
wild-type GPVI (WT), HA-tagged GPVI R272L (R272L), and
HA-tagged GPVI R295STOP (R295STOP) is shown relative to
mock-transfected cells (control). These results are the mean and S.D.
of three experiments. b, expression of wild-type GPVI
(blue line), GPVI R272L (red
line), and GPVI R295STOP (green line)
receptors was measured using anti-HA antibody and is shown relative to
non-GPVI-transfected cells (black line).
View larger version (44K):
[in a new window]
Fig. 5.
Coprecipitation of Fc R
with wild-type and mutant GPVI receptors expressed in RBL
cells. The interaction of Fc R
with wild-type and mutant GPVI
receptors in RBL cells was determined biochemically by precipitation of
GPVI receptors with convulxin-coated beads. CVX-precipitated protein
was probed for GPVI using anti-FLAG antibody (upper
panel) and for Fc R
using anti-Fc R
antibody
(lower panel). RBL, untransfected RBL
cells; RBL-GPVI, RBL cells stably expressing wild-type GPVI;
RBL-GPVIR272L, RBL cells stably expressing GPVIR272L;
RBL-GPVIR295STOP, RBL cells stably expressing GPVI R295STOP;
+, precipitation with convulxin-coated beads;
, control
precipitations with BSA-coated beads. The GPVI-expressing RBL cell
lines used were the same as those analyzed for surface expression and
signaling in Fig. 3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. We have
expressed GPVI in RBL-2H3 cells and studied GPVI signaling in a
heterologous system to directly and formally address this hypothesis.
RBL-2H3 cells express endogenous Fc R
and are a model cell line for
studying Fc
RI receptor signaling (28). Like platelets, the
signaling end points achieved by Fc
RI receptor cross-linking and Fc
R
signaling in RBL-2H3 cells include mobilization of intracellular
calcium and degranulation. Unlike the megakaryocytic cell lines DAMI,
HEL, and MEG-01, however, RBL-2H3 cells express neither endogenous GPVI
nor the integrin receptor for collagen
2
1
(data not shown). Thus, RBL-2H3 cells express the appropriate signaling
machinery to study GPVI signaling without the ambiguity of endogenous
collagen receptor expression.
. Of the reported platelet collagen receptors, including
the integrin
2
1 (2), glycoprotein IV (30), and p65 (20), the most likely candidate is the
2
1 integrin, whose high affinity for
collagen may bring collagen to the platelet surface in an apparent
concentration and/or configuration necessary for GPVI binding and
signal transduction, although the precise role of
2
1 remains controversial (31).
Alternatively, GPVI may not be involved in collagen signaling, and an
as yet unrecognized Fc R
partner may be the true collagen receptor.
2
1 for productive collagen interaction.
CRPs, which structurally closely resemble collagen but are more potent
activators of platelets (21), initiate a small amount of intracellular signaling in GPVI-expressing but not wild-type RBL-2H3 cells (Fig. 1).
The CRP signaling response in GPVI-expressing RBL-2H3 cells, however,
requires 500 times the concentration necessary to activate platelets,
suggesting that the lack of observed signaling to the related ligand
collagen may reflect an extremely low affinity rather than a complete
lack of direct interaction. A necessary role for
2
1 as a coreceptor for collagen signal
transduction through GPVI is also supported by the description of an
individual with reduced levels of
2
1
whose platelets failed to aggregate in response to collagen (32), but
the lack of genetic and molecular characterization of this individual
precludes exclusion of associated defects in the platelet expression of
GPVI, Fc R
, or other unidentified platelet receptors. Finally,
platelets from an individual lacking GPVI also demonstrated a loss of
Fc R
expression despite normal Fc R
expression in other cell
types (33), suggesting that GPVI may be the only Fc R
partner
expressed in platelets and therefore must play a role in collagen
signaling. Lack of genetic and molecular characterization of this
individual, however, limits interpretation of this observation, and a
megakaryocyte-specific block in Fc R
expression cannot be excluded.
Thus, the preponderance of data support a complex model of collagen
signal transduction at the platelet surface with necessary roles played
by no fewer than four transmembrane proteins, GPVI,
2
1, and Fc R
.
-deficient mouse platelets have revealed that, like
Fc R
partners expressed in immune cells, GPVI is not expressed in
the absence of Fc R
(27). Amino acid analysis of human and mouse
GPVI reveals the presence of an arginine in the receptor transmembrane
domain in a position identical to that of related Fc R
partners such
as the Fc
receptor and the NK receptors PIR
and NKp46 (6). As
found for the Fc
and PIR
receptors (16, 18), mutation of this
arginine does not interfere with receptor expression but results in a
complete loss of receptor signaling (Fig. 3) and loss of interaction
with the Fc R
(Figs. 4 and 5). Thus, the GPVI transmembrane arginine
is required for Fc R
interaction, and Fc R
interaction is
required for GPVI signaling.
interaction despite the presence of the transmembrane
arginine (Figs. 3-5). Thus, both the transmembrane arginine and the
receptor C-tail are necessary, but neither alone is sufficient for
intracellular signaling. Interestingly, similar truncation mutants
(within 5-10 amino acids of the TM domain) have been studied with two
other Fc R
partners, the Fc
RI (34) and the Fc
RIII (35)
receptors, with no loss of Fc R
interaction or signaling. It is
intriguing to note that while all of these receptors couple
functionally to the Fc R
, the transmembrane domains of Fc
RI and
Fc
RIII are homologous to each other but demonstrate little homology
to those of GPVI or Fc
receptor and lack the signature arginine residue of that subfamily of Fc R
partners. In addition, Fc
RI
and Fc
RIII share a chromosomal locus on human chromosome 1 (36),
while GPVI and the related receptors discussed share a locus on
chromosome 19, consistent with the existence of distinct ancestral
receptors from which two receptor families may have evolved. Our
results suggest that GPVI couples to the signaling adaptor Fc R
in a
manner distinct from that of the previously studied Fc R
partners
Fc
RI and Fc
RIII. Precisely how the GPVI C-tail facilitates Fc
R
interaction and whether GPVI-related receptors also require their
C-tails for Fc R
coupling remains uncertain and awaits further
mutational analysis.
View larger version (23K):
[in a new window]
Fig. 6.
Amino acid alignment of the transmembrane and
intracellular domains of GPVI and other Fc R
receptor partners. Alignment of the deduced amino acid
sequences of human GPVI (hGPVI), mouse GPVI
(mGPVI), Fc
, PIR
, Fc
RI, and Fc
RIII receptors
was performed using the ClustalW program (Macvector). Amino acid
identities shared among the GPVI, Fc
, and PIR
receptors are
shaded. Amino acid identities shared between the Fc
RI
and Fc
RIII receptors are boxed. hGPVI R272 is in
boldface type. *, the site of C-tail truncation
for GPVI R295STOP; **, the site of C-tail truncation for an Fc
RI
receptor mutant; ***, the site of C-tail truncation for an Fc
RIII
receptor mutant; TM, transmembrane domain; basic,
portion of GPVI C-tail containing a significant number of basic amino
acids; proline, portion of GPVI C-tail containing a cluster
of proline residues; S/T, portion of human GPVI C-tail with
a significant number of serine and threonine residues.
These studies provide the first functional analysis of GPVI as a
signaling receptor and raise several important questions regarding the
role of GPVI in vivo. The inability of GPVI to respond directly to collagen may suggest the evolution of a receptor adapted to
operate in a highly specialized cellular environment in cooperation with other collagen receptors such as the integrin
2
1 (a hypothesis supported by the
megakaryocytic-specific pattern of expression of the receptor's
mRNA (data not shown, and see Ref. 27), but the possibility that
GPVI does not mediate collagen signaling cannot yet be definitively
excluded. Our results extend the proposed model of platelet collagen
signaling to one requiring no fewer than four receptor subunits and
establish a heterologous system in which to further dissect this
signaling pathway. Identification of the receptors involved in
collagen activation of platelets and the molecular basis for this
response may provide novel targets for anti-platelet therapies,
which act at a critical point in thrombogenesis, the activation of
newly adherent platelets at sites of atherosclerotic rupture.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the helpful suggestions of Dr. Reuben Siraganian for generation of RBL-2H3 cell clones and the thoughtful comments of Drs. Skip Brass and Gary Koretzky regarding the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the American Heart Association and the W. W. Smith Charitable Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: University
of Pennsylvania, 421 Curie Blvd., BRB II/III Rm. 952, Philadelphia, PA
19104-6100; Tel.: 215-898-9007; Fax: 215-573-2094; E-mail: markkahn@mail.med.upenn.edu.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M009344200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
CVX, convulxin;
Fc
R, Fc
-chain;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PBS, phosphate-buffered saline;
TM, transmembrane;
HA, hemagglutinin;
BSA, bovine serum albumin;
CRP, collagen-related peptide.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Savage, B., Almus-Jacobs, F., and Ruggeri, Z. M. (1998) Cell 94, 657-666[Medline] [Order article via Infotrieve] |
2. | Santoro, S. A. (1986) Cell 46, 913-920[Medline] [Order article via Infotrieve] |
3. | Wilner, G. D., Nossel, H. L., and LeRoy, E. C. (1968) J. Clin. Invest. 47, 2616-2621[Medline] [Order article via Infotrieve] |
4. |
Du, X.,
Harris, S. J.,
Tetaz, T. J.,
Ginsberg, M. H.,
and Berndt, M. C.
(1994)
J. Biol. Chem.
269,
18287-18290 |
5. |
Keely, P. J.,
and Parise, L. V.
(1996)
J. Biol. Chem.
271,
26668-26676 |
6. |
Clemetson, J. M.,
Polgar, J.,
Magnenat, E.,
Wells, T. N.,
and Clemetson, K. J.
(1999)
J. Biol. Chem.
274,
29019-29024 |
7. |
Jandrot-Perrus, M.,
Busfield, S.,
Lagrue, A. H.,
Xiong, X.,
Debili, N.,
Chickering, T.,
Couedic, J. P.,
Goodearl, A.,
Dussault, B.,
Fraser, C.,
Vainchenker, W.,
and Villeval, J. L.
(2000)
Blood
96,
1798-1807 |
8. | Clemetson, K. J., McGregor, J. L., James, E., Dechavanne, M., and Luscher, E. F. (1982) J. Clin. Invest. 70, 304-311[Medline] [Order article via Infotrieve] |
9. | Sugiyama, T., Okuma, M., Ushikubi, F., Sensaki, S., Kanaji, K., and Uchino, H. (1987) Blood 69, 1712-1720[Abstract] |
10. | Moroi, M., Jung, S. M., Okuma, M., and Shinmyozu, K. (1989) J. Clin. Invest. 84, 1440-1445[Medline] [Order article via Infotrieve] |
11. | Moroi, M., Okuma, M., and Jung, S. M. (1992) Biochim. Biophys. Acta 1137, 1-9[Medline] [Order article via Infotrieve] |
12. |
Jandrot-Perrus, M.,
Lagrue, A. H.,
Okuma, M.,
and Bon, C.
(1997)
J. Biol. Chem.
272,
27035-27041 |
13. |
Polgar, J.,
Clemetson, J. M.,
Kehrel, B. E.,
Wiedemann, M.,
Magnenat, E. M.,
Wells, T. N. C.,
and Clemetson, K. J.
(1997)
J. Biol. Chem.
272,
13576-13583 |
14. | Watson, S. P. (1999) Thromb. Haemostasis 82, 365-376[Medline] [Order article via Infotrieve] |
15. |
Poole, A.,
Gibbins, J. M.,
Turner, M.,
van Vugt, M. J.,
van de Winkel, J. G.,
Saito, T.,
Tybulewicz, V. L.,
and Watson, S. P.
(1997)
EMBO J.
16,
2333-2341 |
16. |
Morton, H. C.,
van den Herik-Oudijk, I. E.,
Vossebeld, P.,
Snijders, A.,
Verhoeven, A. J.,
Capel, P. J.,
and van de Winkel, J. G.
(1995)
J. Biol. Chem.
270,
29781-29787 |
17. |
Taylor, L. S.,
and McVicar, D. W.
(1999)
Blood
94,
1790-1796 |
18. |
Ono, M.,
Yuasa, T.,
Ra, C.,
and Takai, T.
(1999)
J. Biol. Chem.
274,
30288-30296 |
19. | Strouse, R. J., and Daniel, J. L. (1996) Thromb. Res. 82, 485-493[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Chiang, T. M.,
Rinaldy, A.,
and Kang, A. H.
(1997)
J. Clin. Invest.
100,
514-521 |
21. | Morton, L. F., Hargreaves, P. G., Farndale, R. W., Young, R. D., and Barnes, M. J. (1995) Biochem. J. 306, 337-344[Medline] [Order article via Infotrieve] |
22. |
Kahn, M. L.,
Nakanishi-Matsui, M.,
Shapiro, M. J.,
Ishihara, H.,
and Coughlin, S. R.
(1999)
J. Clin. Invest.
103,
879-887 |
23. |
Hamawy, M. M.,
Swieter, M.,
Mergenhagen, S. E.,
and Siraganian, R. P.
(1997)
J. Biol. Chem.
272,
30498-30503 |
24. | Connolly, A. J., Ishihara, H., Kahn, M. L., Farese, R. V., and Coughlin, S. R. (1996) Nature 381, 516-519[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Bakker, A. B.,
Baker, E.,
Sutherland, G. R.,
Phillips, J. H.,
and Lanier, L. L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
9792-9796 |
26. | Posner, R. G., Subramanian, K., Goldstein, B., Thomas, J., Feder, T., Holowka, D., and Baird, B. (1995) J. Immunol. 155, 3601-3609[Abstract] |
27. |
Nieswandt, B.,
Bergmeier, W.,
Schulte, V.,
Rackebrandt, K.,
Gessner, J. E.,
and Zirngibl, H.
(2000)
J. Biol. Chem.
275,
23998-24002 |
28. | Barsumian, E. L., Isersky, C., Petrino, M. G., and Siraganian, R. P. (1981) Eur. J. Immunol. 11, 317-323[Medline] [Order article via Infotrieve] |
29. | Francischetti, I. M., Saliou, B., Leduc, M., Carlini, C. R., Hatmi, M., Randon, J., Faili, A., and Bon, C. (1997) Toxicon 35, 1217-1228[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Nakamura, T.,
Jamieson, G. A.,
Okuma, M.,
Kambayashi, J.,
and Tandon, N. N.
(1998)
J. Biol. Chem.
273,
4338-4344 |
31. | Monnet, E., Sizaret, P., Arbeille, B., and Fauvel-Lafeve, F. (2000) Thromb. Res. 98, 423-433[CrossRef][Medline] [Order article via Infotrieve] |
32. | Nieuwenhuis, H. K., Akkerman, J. W., Houdijk, W. P., and Sixma, J. J. (1985) Nature 318, 470-472[Medline] [Order article via Infotrieve] |
33. |
Tsuji, M.,
Ezumi, Y.,
Arai, M.,
and Takayama, H.
(1997)
J. Biol. Chem.
272,
23528-23531 |
34. |
Alber, G.,
Miller, L.,
Jelsema, C. L.,
Varin-Blank, N.,
and Metzger, H.
(1991)
J. Biol. Chem.
266,
22613-22620 |
35. |
Lanier, L. L., Yu, G.,
and Phillips, J. H.
(1991)
J. Immunol.
146,
1571-1576 |
36. | Le Coniat, M., Kinet, J. P., and Berger, R. (1990) Immunogenetics 32, 183-186[Medline] [Order article via Infotrieve] |