The Fc receptor c-chain is necessary and sufficient to initiate
signalling through glycoprotein VI in transfected cells by the snake
C-type lectin, convulxin
Oscar Berlanga
1,2
, David Tulasne
1
, Teresa Bori
3
, Daniel C. Snell
1
, Yoshiki Miura
4
, Stephanie Jung
4
,
Masaaki Moroi
4
, Jonathan Frampton
2
and Steve P. Watson
1
1
Department of Pharmacology, University of Oxford, UK;
2
Weatherall Institute of Molecular Medicine, John Radcliffe Hospital,
Oxford, UK;
3
Division of Medical Sciences, The Medical School, University of Birmingham, UK;
4
Department of Protein

mucin-like Ser/Thr region, followed by a transmembrane
and short cytoplasmic tail. GPVI forms a complex with
the Fc receptor c-chain (FcR c-chain), which is respon-
sible for signalling through GPVI [2–4]. Previous reports
on two receptors sharing homology with GPVI, namely
FcaR and paired immunoglobulin-like receptor A (PIR-
A), revealed that they are expressed on the surface of the
membrane independently of the FcR c-chain in cell lines
[5,6], but coexpression with FcR c-chain increases the
level of expression of the receptor at the surface [5,7].
The interaction between FcaRorPIR-AandtheFcR
c-chain occurs in the transmembrane region as the result
of oppositely charged amino-acid residues [6,8]. The
immune receptor tyrosine-based activation motif domain
within the cytoplasmic tail of the FcR c-chain is
responsible for signalling after engagement of the recep-
tor complex [9].
The interaction between platelets and collagen involves
adhesion and activation leading to increased strength of
adhesion, secretion and ultimately aggregation [10–12]. It is
accepted that the integrin a2b1 is the major receptor
supporting strong platelet adhesion to collagen, whereas
GPVI mediates activation [3,13]. The multimeric nature of
collagen means that the development of specific ligands to
either receptor is essential for understanding their relative
contribution to the overall mechanism of platelet–collagen
interaction. Among these, collagen-related peptide (CRP) is
thought to signal specifically through GPVI, as demonstra-
ted by the lack of response to the peptide in GPVI-deficient
platelets [13]. Convulxin, a C-type lectin from the venom of

UK). Convulxin was supplied by Drs Leduc and Bon
(Institute Pasteur, Paris, France). Anti-Syk N-19 was from
Santa Cruz (Insight Biotechnology Ltd, Wembley
Middlesex, UK). Fluorescein isothiocyanate (FITC)-
conjugated anti-(human CD36) was from Serotec
(Kidlington, Oxfordshire, UK). FITC-conjugated anti-
rabbit IgG (Fab¢)
2
fragments were from Sigma (Poole,
Dorset, UK). All other reagents were from previously
described sources [3,4,14].
Cell culture and platelet preparation
K562 and Jurkat cells were grown in RPMI-1640
medium, and COS-7 cells were grown in Dulbecco’s
modified Eagle’s medium. Both cell lines were supple-
mented with 1 m
M
glutamine, 100 UÆmL
)1
penicillin,
100 lgÆmL
)1
streptomycin and 10% heat-inactivated fetal
bovine serum under 5% CO
2
/95% air in a humidified
incubator. Cells were kept at exponential phase of growth.
Differentiation of K562 cells was induced with phorbol
myristate acetate (PMA) as previously described [14].
Platelets were obtained from drug-free volunteers on the

EGTA, 5 m
M
MgCl
2
,
pH 7.6 adjusted with KOH) supplemented on the day of
experiment with 5 m
M
glutathione and 2 m
M
ATP, and
resuspended in 400 lL cytomix buffer, which was added in
an electroporation cuvette already containing 5–10 lg
plasmid DNA. After electroporation, cells were diluted
to a final volume of 5 mL with complete prewarmed
medium and placed in the incubator. The calcium phos-
phate method was used with COS-7 cells. A mixture
containing 500 lL sterile distilled water, 186 lL1
M
CaCl
2
and 20 lg plasmid DNA was prepared. After 5 min,
750 lL2· HBS buffer [250 m
M
NaCl, 10 m
M
KCl,
1.5 m
M
Na

site 1. pEGFP plasmid, coding for the green fluorescent
protein (GFP), was from Clontech (Basingstoke,
Hampshire, UK). A chimeric protein GPVI–GFP was
made by inserting the cDNA of GPVI into the EcoRI site of
EGFP plasmid.
Immunoprecipitation and affinity precipitation
Cells [(2–4) · 10
6
] were lysed in buffer [10 m
M
Tris 150 m
M
NaCl, 5 m
M
EDTA, 1% (v/v) Triton X-100, 0.5 m
M
phenylmethanesulfonyl fluoride, 1 m
M
Na
3
VO
4
,5lgÆmL
)1
leupeptin, 5 lgÆmL
)1
aprotinin, 0.5 lgÆmL
)1
pepstatin A,
pH 7.3] and rotated at 4 °C for 30 min; they were then

M
convulxin
dissolved in Tris/NaCl/Tween [20 m
M
Tris, 137 m
M
NaCl,
0.1% (v/v) Tween 20, pH 7.6] for 1 h at room temperature;
they were then washed and incubated with convulxin
antibody and secondary antibody, both dissolved in Tris/
NaCl-T.
Flow cytometry
Cells were resuspended in Tyrodes/Hepes or NaCl/P
i
buffer
containing 1% human serum albumin and 0.02% sodium
azide. For some experiments, cells were fixed with 3.7%
formaldehyde for 30 min and platelets with 1% formalde-
hyde for 1 h, followed by 10 min incubation with 50 m
M
2952 O. Berlanga et al.(Eur. J. Biochem. 269) Ó FEBS 2002
NH
4
Cl. All incubation times were 30 min unless otherwise
indicated. GPVI was detected as previously described [14].
Briefly, cells were incubated with 20 n
M
convulxin, washed,
andincubatedwith0.4lgÆmL
)1

converted into a concentration of Ca
2+
by measuring R
max
(maximal fluorescence in lysed, labelled cells) and R
min
(minimum fluorescence in lysed, labelled cells in the presence
of 5 m
M
EGTA).
Adhesion assay
Ninety-six-well plates were coated with 20 lgÆmL
)1
colla-
gen, convulxin, CRP, or BSA in NaCl/P
i
, and incubated
overnight at 4 °C, then saturated with 1% BSA for 1 h at
room temperature and washed with Tyrodes/Hepes buffer
(pH 7.3). Then 2 · 10
5
cells in Tyrodes/Hepes buffer were
added per well and allowed to adhere for 1 h at 37 °C. Cells
were washed at least three times with Tyrodes/Hepes buffer
and fixed with 3% formaldehyde. Assays were performed in
triplicate for each condition, and at least 10 different fields
of cells per experiment were counted under the microscope.
RESULTS
GPVI is expressed at the surface independently of FcR
c-chain in COS-7 and K562 cells

GPVI–GFP. Magnification of the latter revealed colocali-
zation between GPVI–GFP and convulxin, confirming
surface expression of GPVI independently of FcR c-chain.
These results demonstrate surface expression of GPVI
independently of the FcR c-chain, but the possibility
remained that FcR c-chain was necessary for stable surface
expression of the receptor. To address this, GPVI was stably
transfected into the erythroleukaemic cell line K562, which
Fig. 1. GPVI is surface-expressed on the membrane independently of the
FcR c-chaininCOS-7cells.(A) COS-7 cells were transiently trans-
fected with wild-type GPVI (F1), GPVI mutated in the transmembrane
arginine to alanine (FA), or depleted of the cytoplasmic tail (C1),
together (bottom panels) or not (upper panels) with the FcR c-chain.
Control cells were transfected with empty plasmid (pRc). Surface-
expressed GPVI was detected by flow cytometry using convulxin or an
antibody to convulxin and indirectly labelled with FITC-conjugated
anti-rabbit IgG (filled histogram). Background fluorescence was
obtained in the absence of convulxin (empty histogram). (B) Whole cell
lysates from the above cells were subjected to SDS/PAGE under
nonreducing conditions using 10% polyacrylamide gels. GPVI was
detected by ligand blotting using convulxin and FcR c-chain by
Western blotting using a specific antibody. When 10% polyacrylamide
gels were used, a shift in mobility of C1 was observed relative to F1 or
FA, according to its lower molecular mass (not shown). Different
bands possibly corresponding to differently phosphorylated forms of
FcR c-chain are observed in platelets and F1-c cells, but not the others.
n ¼ 2.
Ó FEBS 2002 FcR c-chain is sufficient for GPVI signalling (Eur. J. Biochem. 269) 2953
lacks detectable expression of FcR c-chain ([16] and Fig. 4).
Mock-transfected cells (K562/pRc) did not express GPVI as

Fig. 3. Surface expression of GPVI in K562 cells. (A) Cells mock-
transfected (K562/pRc) or transfected with wild-type GPVI (K562/F1)
or a cytoplasmic-tail-deleted mutant (K562/C1) were differentiated
with PMA for 1 and 3 days, and expression of GPVI at the surface
detected by flow cytometry using convulxin (filled histogram). Back-
ground fluorescence was obtained in the absence of convulxin (empty
histograms). (B) The above cells were stably cotransfected with the
FcR c-chain and GPVI expression at the surface detected as above.
Expression of GPVI after 3 days of differentiation is higher than at
1 day. Mock-transfected cells display a low level of GPVI expression,
in both the presence (K562/pRc-c) and absence (K562/pRc) of FcR
c-chain. Extensive cellular death is detected in the cultures after 3 days
of differentiation, and expression of GPVI is not homogeneous within
the clonal population of cells (see K562/F1 and K562/F1-c). n ¼ 3.
Fig. 2. Convulxin colocalizes at the membrane with the chimeric protein
GPVI–GFP in the absence of FcR c-chain. COS-7 cells growing on
coverslips were transient transfected with a plasmid encoding either
GFP or GPVI–GFP. After 48 h, cells were fixed. GPVI–GFP was
visualized by incubation with convulxin or an antibody to convulxin,
and indirectly labelled with R-phycoerythin-conjugated anti-rabbit
IgG. Fluorescent GFP is directly visualized in the microscope. Upper
panel shows cytoplasmic localization of GFP (top left), and absence of
binding of convulxin to those cells (top right). The chimeric protein
GPVI–GFP localizes to the membrane (bottom left) and is recognized
by convulxin (bottom right). Initial magnification · 20. Bottom panels
show a magnification of cells transfected with GPVI–GFP and incu-
bated with convulxin (right). Arrows indicate spots of colocalization
between GPVI–GFP and convulxin. The bright cytoplasmic spot of
GFP or GPVI–GFP (upper and low panels, respectively) probably
corresponds to the site of synthesis/accumulation of the protein. Bar

these cells with 2 and 20 n
M
convulxin led to an increase in
tyrosine phosphorylation of Syk (Fig. 6B). The increase was
evident after 30 s of stimulation and maximum after 90 s,
phosphorylation remaining for up to 270 s. Stimulation with
20 n
M
convulxin produced slightly stronger phosphorylation
than 2 n
M
after 30 s of stimulation, although longer stimu-
lation using both concentrations of convulxin rendered a
similar intensity of response (Fig. 6B). Phosphorylation of
Syk was also detected in K562/F1-c cells nondifferentiated
with PMA, which expressed a lower level of the GPVI–FcR
c-chain complex (Fig. 7A). The increase in signal in PMA-
treated cells was due to an increase in the number of receptors
at the surface of the cells. A similar set of experiments was
carried out with K562 cells that had been stably transfected
with the cytoplasmic-tail-deleted form of GPVI together with
FcR c-chain (K562/C1-c). No increase in phosphorylation
of Syk was detected in these cells upon convulxin stimulation
(Fig. 7A), demonstrating that deletion of the cytoplasmic tail
Fig. 6. Phosphorylation of FcR c-chainandSykinK562cells.(A) K562
cells stably expressing FcR c-chain (pRc/c), or cotransfected with
either wild-type GPVI and FcR c-chain (F1/c) or a cytoplasmic-tail-
deleted GPVI and FcR c-chain (C1/c) were stimulated for 90 s with
20 n
M

therefore lacking FcR c-chain (K562/F1), did not show a
detectable increase in tyrosine phosphorylation of Syk after
20 n
M
convulxin stimulation for 90 s, even when expressing
higher levels of GPVI after PMA treatment (Fig. 7B).
Together, our data indicate that, in K562 cells, cotrans-
fection of GPVI and the FcR c-chain leads to formation of
a receptor complex, which can be activated with the GPVI-
specific ligand convulxin, resulting in phosphorylation of
the protein kinase Syk, therefore reproducing the proximal
events of GPVI signalling in platelets. The FcR c-chain is
crucial for the generation of this signal, as cross-linking of
GPVI in the absence of FcR c-chain was unable to promote
an increase in phosphorylation of Syk.
A transmembrane arginine residue and the cytoplasmic
tail of GPVI are necessary for its association with FcR
c-chain
We next performed experiments to determine whether there
was a physical association between GPVI and FcR c-chain
as described on platelets, and the role of the transmembrane
arginine and cytoplasmic tail of GPVI in this association.
COS-7 cells were cotransfected with the different GPVI
constructs along with FcR c-chain. Affinity precipitation of
GPVI, using convulxin and an antibody to convulxin, and
subsequent immunoblotting to detect FcR c-chain, showed
an association between the FcR c-chain and wild-type
GPVI, but not with the other two mutants of GPVI
(Fig. 8A). This shows that the transmembrane arginine and
the cytoplasmic tail of GPVI are essential for its association

used at a concentration of 100 lgÆmL
)1
, which is 200 times
greater than that required for platelet activation. When
stimulated with CRP under the same conditions in the
absence of GPVI, the cells showed no increase in phos-
phorylation of Syk (data not shown). This demonstrates
that GPVI expression was sufficient to reproduce the
phosphorylation of Syk induced by convulxin and CRP,
but not by collagen.
GPVI-expressing Jurkat cells bind to collagen, CRP
and convulxin, but only convulxin is able to induce
calcium release
The lack of effect of collagen on GPVI in K562 cells led us
to use a second system for expression of GPVI. GPVI was
Fig. 7. FcR c-chain and the cytoplasmic tail of GPVI are necessary to
initiate the GPVI signalling cascade. (A) K562 cells stably transfected
with FcR c-chain and cotransfected with empty vector (pRc/c), wild-
type GPVI (F1/c) or a cytoplasmic-tail-depleted GPVI (C1/c) were
treated or not with PMA for 24 h, then stimulated with convulxin for
90 s and protein lysates subjected to Syk immunoprecipitation.
Immunoprecipitated proteins were separated by SDS/PAGE and
transferred to a poly(vinylidene difluoride) membrane. Phosphorylated
proteins were detected using mAb 4G10. The arrow indicates the
position of Syk. An unidentified phosphorylated band of  100 kDa
coprecipitated with phosphorylated Syk. (B) Cells transfected with the
different mutant versions of GPVI but lacking expression of FcR
c-chain were treated with PMA for 24 h, stimulated with 20 n
M
con-

collagen and CRP, only the snake venom is able to stimulate
the signalling pathway leading to activation.
DISCUSSION
GPVI–FcR c-chain is unique compared with other FcR
c-chain complexes, as it has been reported to act as a
collagen receptor with no apparent function in the immune
response, despite sharing similar structure and signalling
features with other FcRs [1,3,17]. The similarity between
GPVI and different FcRs provides a starting point in the
study of the structure–function relationship of GPVI and
the signalling pathways. Many FcRs and their associated b
and c-chains bind to each other through the transmembrane
domain resulting in a receptor complex that is able to signal
inside the cell. The nature of this binding depends on
oppositely charged amino-acid residues within the trans-
membrane domain of each subunit. Point mutations of these
amino acids have revealed the importance of the arginine
residue in the transmembrane domain of the immunoglo-
bulin receptor. However, the cytoplasmic domain of these
FcRs is not essential for binding to the FcR c-chain. Our
results show that GPVI depends on both the transmem-
brane arginine and at least part of the cytoplasmic tail for its
association with the FcR c-chain, although it is not clear
how the cytoplasmic tail supports the association. The
molecular basis of this interaction is still being studied.
We have shown by transient and stable transfections of
GPVI into COS-7 and K562 cells, respectively, that GPVI is
expressed at the cell surface independently of the FcR
c-chain. This is a property that has previously been
described for the GPVI-related receptors PIR-A and FcaR

with mAb 4G10. The arrow indicates the position of phosphorylated
Syk. The membrane was stripped and reblotted to detect Syk.
Ó FEBS 2002 FcR c-chain is sufficient for GPVI signalling (Eur. J. Biochem. 269) 2957
expression of FcaRandFccRI [18,19] possibly through
prevention of their degradation [20]. Consistent with this,
mice depleted of the FcR c-chain did not express detectable
GPVI [21], raising the possibility that in vivo, unlike in cell
lines, GPVI may be degraded in the absence of the FcR
c-chain. This may reflect a specific pathway of degradation
that prevents expression of functionally uncoupled receptors
in certain cell types.
The cotransfection of FcR c-chain with GPVI did not
increase the level of surface expression of the latter relative
to transfection of GPVI alone, either on transient or stable
transfections. The same observation has been reported on
transient transfections of GPVI into COS-7 cells [22]. This is
different from the related PIR-A and Fca-receptor [5,9] and
suggests that, in cell lines, the FcR c-chain is acting as a
signalling and stabilizing subunit for some receptors, such as
FcaR, but only as a signalling subunit for others, such as
GPVI. Conversely, GPVI is required for stable expression
of the FcR c-chain in the absence of other binding partners.
It is well known that the FcR c-chain is essential for
signalling by receptors with which it associates [5,9,19]. Mice
genetically engineered to lack the FcR c-chain do not
express detectable GPVI [21], making it impossible to
determine whether GPVI signals in the absence of FcR
c-chain in vivo. K562 cells do not express detectable levels of
endogenous GPVI or FcR c-chain, even after differentiation
with PMA, making them a suitable system for studying the

(B) Mock-transfected and GPVI-transfected
cells (2 · 10
5
) were incubated for 30 min at
37 °Cina96-wellplatecoatedwithBSA,
convulxin, collagen or CRP. After extensive
washing and fixation, adherent cells were
counted (0.4 mm square). Triplicate samples
were counted for each condition and standard
deviations are shown. (C) Jurkat cells stably
expressing GPVI were stimulated for 90 s with
10 lgÆmL
)1
convulxin (Cvx), 50 lgÆmL
)1
CRP, or 100 lgÆmL
)1
collagen (Coll.). Whole
cell protein lysates were subjected to SDS/
PAGE,thenblottedtodetecttyrosine-phos-
phorylated proteins using mAb 4G10. (D)
Fura-2-loaded mock-transfected cells (pRc)
and GPVI-transfected cells (F1) were stimu-
lated with 10 lgÆmL
)1
convulxin, 50 lgÆmL
)1
CRP and 100 lgÆmL
)1
collageninaspectro-

of GPVI is sufficient to support binding to collagen but not
detectable activation.
There are several possible explanations for the lack of
response to collagen on cell lines transfected with GPVI.
One is the absence of a second receptor such as the integrin
a2b1. This seems unlikely, however, because collagen is also
inactive on a number of megakaryocytic cell lines that
express a low level of GPVI together with other receptors
for collagen [28]. A second explanation is that GPVI
expression may fail to reach the critical level for activation
by collagen. A third possibility is that the interaction of
collagen with GPVI generates a signal that falls below the
detection limits of the assays used in this system. Collagen is
a far weaker stimulus than convulxin in platelets, its
response being heavily dependent on the liberation of
ADP and thromboxanes for activation [29]. However,
collagen still stimulates calcium increases in single platelets
and induces tyrosine phosphorylation in the presence of
ADP and thromboxane receptor antagonists [30] (B. T.
Atkinson & S. P. Watson, unpublished). The most likely
explanation for these observations is that a certain receptor
density is required for functional responses to collagen,
whereas adhesion can be supported by lower receptor
densities. Direct evidence to support this is provided by a
recently published study [31].
In conclusion, we have demonstrated that reconstitution
of GPVI with the FcR c-chain restores its responses to
convulxin and CRP, but that GPVI is unable to signal in
the absence of a protein bearing the immune receptor
tyrosine-based activation motif. In addition, we have

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