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INTRODUCTION |
Activation of platelets in response to exposed collagen is a
critical step in response to vascular injury and the formation of
intravascular thrombi associated with stroke and myocardial infarction.
A necessary step in the activation of platelets by collagen is
signaling by the glycoprotein VI
(GPVI)1 receptor (1-3). The
GPVI receptor is homologous to immune receptors and associates
non-covalently with Fc R
(3, 4), a transmembrane protein that
mediates signaling by several immune receptors through an
immunoreceptor tyrosine activation motif (ITAM) (5). ITAM signaling is
initiated by tyrosine phosphorylation mediated by Src family tyrosine
kinases (6), a process associated with receptor movement to specialized
regions of the cell membrane known as lipid rafts (7, 8). In platelets
and other hematopoietic cells ITAM activation ultimately results in
phospholipase C
activation (9) and intracellular calcium release
through a series of signaling proteins including the non-receptor
tyrosine kinase SYK (3, 10) and the adaptors SLP-76 (11) and LAT (12,
13).
We and others (14, 15) have shown that mutation of a single
transmembrane arginine in GPVI (GPVI R272L) is sufficient to uncouple
the receptor from the Fc R
chain. GPVI R272L is unable to activate
the release of intracellular calcium in RBL-2H3 cells (14), a
hematopoietic cell model in which heterologous expression of GPVI
confers collagen-dependent calcium signaling (16).
Unexpectedly, and unlike previously studied Fc R
partners (17, 18),
truncation of the GPVI intracellular domain also abrogated GPVI
signaling despite preservation of the critical transmembrane arginine
(14, 15). These results suggest that the GPVI intracellular domain might play an important role in GPVI signaling, an idea recently confirmed by the identification of an interaction between the Src
family kinase Lyn and the GPVI intracellular domain (19). Biochemical
studies have also shown that the GPVI intracellular domain interacts
with calmodulin (20). The functional importance of these interactions
for signal transduction by GPVI, however, is unknown.
To further investigate the role of the GPVI intracellular domain during
signal transduction we have stably expressed a series of receptor
truncation mutants and amino acid substitution mutants in RBL-2H3 cells
to analyze receptor signaling and protein-protein interactions. These
studies identify two critical functional domains within the GPVI
intracellular tail, a highly basic region that mediates interaction
with calmodulin and a proline-rich region that mediates interaction
with Src family kinases. Interruption of either one of these domains
significantly impairs GPVI signaling despite normal association with Fc
R
. In addition, the function of these domains appeared autonomous,
i.e. loss of calmodulin binding, Lyn association or Fc R
coupling had little effect on GPVI association with the other two
interacting proteins. Our results reveal an important independent role
for the GPVI intracellular tail in the regulation of receptor signaling
and suggest that the ligand-binding subunit of this receptor functions
as an adaptor to bind downstream signaling proteins. The extent to
which the intracellular domains of similar multisubunit receptors also
function to modulate receptor signaling in an adaptor-like fashion
remains to be investigated.
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EXPERIMENTAL PROCEDURES |
Materials--
All reagents were from Sigma unless stated.
Convulxin was purified from the venom of the South American rattlesnake
(Crotalus durissus terrificus) by gel filtration as
described (14). Mouse monoclonal anti-calmodulin and anti-FcR
antibody were from Upstate Biotechnology, Inc. (Lake Placid, NY).
Rabbit anti-Lyn polyclonal antibody was from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). Anti-GPVI monoclonal antibody HY101 was made as
described (16) and affinity-purified from hybridoma supernatants by
HiTrap Sepharose-protein G affinity chromatograhy (Amersham
Biosciences).
Generation of GPVI Mutants--
All GPVI truncation mutants were
generated using PCR to insert a premature STOP codon in place of the
indicated amino acid as previously described (14). Point mutations in
the basic domain of GPVI were generated using sequential, overlapping
oligonucleotide linkers to replace the region between the BspE1 site
(nucleotide 812 of coding sequence) and the STOP codon of FLAG-tagged
human GPVI. This was accomplished with the following 5 overlapping
oligonucleotides (S, sense oligonucleotide; AS, antisense
oligonucleotide; all shown 5'-3') to generate wild-type and RK mutant
receptors: S1, GTCCGGATATGCCTAGGGGCTGTGATCCTAATAATCCTGGCGGGGTTTCTGGCAGAGGAC; AS2, CCCCTGTGCCGCAATCTTTTCCTCCGGCTGTGCCAGTCCTCTGCCAGAAACCC; S3, AGATTGCGGCACAGGGGGCGCGCCGTGCAGAGGCCGCTTCCGCCCCTGCCGCCCCTC; AS4, ACCCCCATGGGATTTCCGGGTCTGCGGGAGGGGCGGCAGGGGCG; S5,
CGGAAATCCCATGGGGGTCAGGATGGAGGCCGACAGGATGTTCACAG CCGCGGG; AS2-RK1,
CCCCTGTGCCGCAACGCAGCGGCTGCGCTGTGCCAGTCCTCTGCCAGAAACCC; AS2-RK2,
CCAGCGTGTGCCAATCTTTTCCTCCGGCTGTGCCAGTCCTCTGCCAGAAACCC; S3-RK2,
AGATTGGCACACGCTGGGG- CCGCCGTGCAGAGGCCGCTTCCGCCCCTGCCGCCCCTC.
Mutant C-tails were constructed by annealing the 5 oligonucleotides
simultaneously followed by ligation and cloning into the BspE1-NotI site of the original FLAG-tagged human
GPVI (14). GPVI receptor amino acids reported correspond to the
sequence predicted by the open reading frame of GenBankTM
accession number AB035073 starting at nucleotide number 13. All GPVI
mutations were expressed in pcDNA3.0 (Invitrogen).
Generation of Mutant GPVI-expressing RBL-2H3 Cell
Lines--
RBL-2H3 cells were electroporated and stable cell lines
generated as previously described (16). Surface expression of the mutant receptors was determined using flow cytometry with
FITC-conjugated anti-FLAG (M2, Sigma) antibody.
Purification of GPVI from GPVI-expressing RBL-2H3
Cells--
Human FLAG-tagged GPVI was purified from GPVI-expressing
RBL 2H3 cells using the general method of Clemetson et al.
(21), with minor changes. Cells were lysed in an equal volume of 20 mM Tris, pH 7.4, 200 mM NaCl and 4% nonanoyl
N-methylglucamide (MEGA-9, Dojindo Molecular Technologies
Inc, Gaithersburg, MD) containing protease inhibitors added from 100×
final concentration (Sigma, mammalian protease inhibitor).
Detergent-insoluble cellular debris was pelleted at 10,000 × gav for 15 min, and 20% v/v convulxin (CVX)-Sepharose beads or HY101-conjugated beads used to precipitate GPVI from the supernatant in an overnight incubation. CVX is a C-lectin
type snake venom derived from the South American rattlesnake C. durissus terrificus that binds GPVI with high
affinity and activates GPVI signaling (22). The beads were loaded into
a disposable 0.7 × 10-cm column and washed with 2% nonanoyl
N-methylglucamide buffer containing high (300 mM) and low (100 mM) salt to remove proteins
associated with GPVI or CVX through ionic interaction. GPVI was eluted
from CVX beads using a solution of 0.10% w/v SurfectAmps SDS
(Pierce-Endogen), 10 mM Tris-HCl, pH 7.4. Aliquots of the elution fractions were checked for protein by gold staining and for
GPVI using HY101 and/or anti-FLAG. Selected fractions were concentrated
(Amicon 30; Amicon, Beverly, MA) for electrophoresis (8% gel,
non-reducing conditions). Preparative recovery of GPVI was achieved
using a model 422 electroeluter according to the manufacturer's
instructions (Bio-Rad, Hercules, CA). The purified protein was
lyophilized, weighed, and re-hydrated in 10 mM Tris, pH
7.4, 100 mM NaCl, pH 7.4, 0.1% nonanoyl
N-methylglucamide. For gold staining membranes were washed
3× for 10 min in phosphate-buffered saline, 0.5% Tween and then
developed using membragold (Bioworld, Dublin, OH)
Fluorescence Spectroscopy--
Emission spectra were obtained at
25 °C using a Varian Cary Eclipse Fluorescence spectrophotometer
with well-plate attachment (Varian, Walnut Creek, CA). Measurements
were taken at an excitation wavelength of 295 nm (5-nm slit) and with
scanning emission (1.5-nm slit) under various experimental conditions.
Buffer conditions were kept constant at 10 mM Tris-HCl, 100 mM NaCl, pH 7.4, and 0.1% nonanoyl
N-methylglucamide. For all assays 10 µM
purified GPVI was incubated with increasing concentrations of bovine
brain calmodulin (Molecular Probes, Eugene, OR) as outlined in the
text. Magnesium, calcium, or EDTA was added to their final
concentrations from stock. The final volume was kept constant at 200 µl.
Each data point was determined by two individual measurements and was
plotted as the fraction of a maximum emission shift. Data were
transformed from fluorescence titration experiments and values
calculated by linear regression (SigmaPlot 2002). Determination of
KD for the association was obtained from
transforming Equation 1,
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(Eq. 1)
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into Equation 2,
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(Eq. 2)
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where f is the fraction of the maximal emission
change and [X] is the protein concentration. Data were fit using a
one and two site Hill plot for the determination of the dissociation
constant (KD) and coefficient of binding
(slope). The two-site model was more appropriate for the data as judged
by the fitting coefficients and the residuals between the experimental
and expected values.
Measurement of Cytoplasmic Calcium in RBL-2H3
Cells--
Adherent cells were detached from culture plates using 5 mM EDTA and resuspended in RHB medium (RPMI 1640 medium
containing 25 mM HEPES and 1 mg/ml bovine serum albumin) at
2 × 107 cells/ml. Fura-2/AM (Molecular Probes, Inc.)
was added to 4 µg/ml, and cells were incubated at 37 °C for 30 min. Excess Fura-2/AM was removed by washing in RHB medium.
Fluorescence was measured using an Aminco-Bowman Series-2 luminescence
spectrometer (SLM-Aminco, Urbana, IL). Fluorescence was measured at 340 and 380 nm for excitation and at 510 nm for emission. Cells (2 × 106) were stirred continuously during the fluorescence
recording. The data were recorded as the relative ratio of fluorescence
excited at 340/380 nm and the concentration of mobilized calcium using a dissociation constant of 224 nmol/liter for
Fura-2/Ca2+.
Immunoprecipitation from Detergent-solubilized
Lysates--
RBL-2H3 cells were lysed for 2 h at 4 °C in
ice-cold lysis buffer (1% w/v digitonin (Calbiochem), 0.12% v/v
SurfectAmps Triton X-100 (Pierce-Endogen), 150 mM NaCl,
0.01% w/v sodium azide, 20 mM triethanolamine, pH 7.8).
Detergent-insoluble cellular debris was pelleted at 10,000 × gav for 15 min, and CVX-Sepharose beads were
used to immunoprecipitate GPVI and associated proteins from the
supernatant in another 2-h incubation period. For live cell immunoprecipitations, HY101 was used to saturate the extracellular domain of GPVI prior to lysis and recovered from cell lysate using protein A-protein G beads (Amersham Biosciences). Of note, binding of
HY101 to GPVI is non-activating and does not compete with or block
subsequent CVX binding to the receptor (16). 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). 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 dithiothreitol, 4% w/v SDS, 0.004% bromphenol blue,
20% glycerol), and 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).
Calmodulin Affinity Chromatography--
A crude membrane pellet
was obtained from RBL 2H3 GPVI cells by Dounce homogenization (~30
times on ice) in 10 mM Tris-HCl, pH 7.4, 1 mM
EGTA, containing protease inhibitors. The homogenate was centrifuged at
1500 × gav for 10 min at 4 °C and the
supernatant stored on ice. The pellet was resuspended in half the
original volume of homogenization buffer supplemented with 5 mM magnesium chloride, pH 8.0 and disrupted by Dounce
homogenization. The homogenate was centrifuged at 1500 × gav for 10 min at 4 °C. The pooled
supernatants were centrifuged at 109,000 × gav for 1 h at 4 °C. The supernatant was
discarded and the pellet resuspended to a protein concentration of
~10 mg/ml in 250 mM sucrose, 50 mM potassium
chloride, 0.1 mM calcium chloride, 20 mM
MOPS-Tris, pH 7.2.
Affinity chromatography of cell membrane lysates on
calmodulin-Sepharose (Amersham Biosciences) was performed using a
method described by Klaerke et al. (23). CHAPS was added to
the crude membrane preparation from a 0.5 M stock to a
detergent protein ratio of 5:1 (w/w). After 60 min the unsolubilized
protein was removed by centrifugation. Sepharose-calmodulin beads,
which had been equilibrated with 50 mM HEPES pH 7.4, 1 µM magnesium chloride, 1 mM dithiothreitol,
10 mM CHAPS, were added to the supernatent to 30% (v/v).
Beads-lysate were supplemented with 1 mM calcium chloride
and incubated at 30 °C for 120 min. The beads were loaded into a
disposable 0.7 × 10-cm column, washed in 10 mM CHAPS
buffer containing calcium and 200 mM sodium chloride, and
proteins eluted from the beads by 10 mM EDTA. Elution
fractions were analyzed for GPVI by Western blotting.
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RESULTS |
Calmodulin Associates with GPVI in a Specific and High Affinity
Manner--
A theoretical calmodulin-binding region in GPVI was
identified by screening for a basic amphipathic sequence between the
transmembrane and cytoplasmic receptor domains. A 14 amino acid
sequence between amino acids Trp-292 and Val-306 shares homology to
classical calmodulin-binding motifs and to the recently identified
calmodulin-binding site on glycoprotein Ib
(GPIb
, Fig.
1). As for GPIb
, helical wheel projection of the membrane-proximal region of the GPVI C-tail suggested
that these charged residues might be arranged along a single face of a
helix in a manner similar to that recently reported for GPIb
(Fig. 1
and Ref. 24). Previous studies using purified peptides corresponding to
this region of GPVI (His-293-Pro-311, amino acid numbering from the
start methionine of the immature protein) have also suggested that GPVI
may interact with bovine calmodulin, an observation confirmed by
co-immunoprecipitation of these two proteins (20).

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Fig. 1.
Comparison of the predicted
calmodulin-binding sites within the GPVI and GPIb
cytoplasmic domains. a, hydropathy plots
(Macvector) of the GPIb and GPVI receptors demonstrates strongly
hydrophilic regions within the membrane-proximal regions of both
receptor's cytoplasmic tails. b, amino acid alignment of
the proximal regions of the GPIb and GPVI cytoplasmic tails reveals
a conserved stretch of basic residues. The residue numbers refer to
amino acid residues numbered from the start methionine. c,
helical wheel projection of the membrane-proximal region of the GPVI
intracellular tail (Trp-292-Val-306).
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To more precisely define the interaction between GPVI and calmodulin we
measured the intrinsic tryptophan fluorescence of GPVI before and after
addition of calmodulin. Because calmodulin has no tryptophan residues,
this technique provides information about the environment surrounding
tryptophan residues of calmodulin-binding proteins. Affinity
chromatography of GPVI-expressing RBL-2H3 cell lysates on CVX-Sepharose
yielded a major protein component identified by Western blotting as
GPVI, migrating at 66 kDa under reducing conditions (Fig.
2a). Minor protein components
were removed by extensive salt washing and were further eliminated by
non-denaturing preparative PAGE (data not shown). Purified GPVI had an
emission peak at 352 nm after excitation of tryptophan at 295 nm.
Calmodulin titrations were performed in the presence of calcium,
magnesium, and/or EDTA. In the presence of calcium, addition of bovine
calmodulin to purified GPVI resulted in a decrease on the emission
maxima (a "blue shift") to 344 nm that did not occur in the
presence of EDTA or when magnesium was present in place of calcium
(Fig. 2b and data not shown). Titration experiments (Fig. 2,
c and d) revealed approximately molar saturation
between the two proteins and a high affinity interaction between GPVI
and calmodulin (KD of 35 nM). Thus
calmodulin interacts with GPVI in living cells in a high affinity and
specific manner.

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Fig. 2.
Calmodulin binds GPVI with high
affinity. a, isolation of GPVI from GPVI-expressing
RBL-2H3 cells by affinity chromatography on CVX-Sepharose. Lane
1, material bound to a CVX-Sepharose column from GPVI-expressing
RBL-2H3 cell lysates. Lanes 2 and 3, material
remaining after washing with high and low salt buffers respectively.
Lane 4, material eluted from the column using high pH buffer
containing detergent. The identity of the purified 66-kDa protein was
established as GPVI was confirmed by Western blotting using anti-GPVI
MAb HY101 (not shown). b, intrinsic tryptophan fluorescence
of GPVI before and after addition of calmodulin. Measurements were
taken at an excitation wavelength of 295 nm and scanning emission with
10 µM GPVI (closed triangle) or 10 µM GPVI + 10 µM calmodulin (open
circle), both with the addition of Ca2+ to saturation.
c, calmodulin titration of GPVI. GPVI (10 µM)
was titrated with calmodulin and the change in emission calculated as
described under "Experimental Procedures"). Each data point was
determined by two individual measurements and was plotted as the
fraction of the maximum emission shift. Excitation and emission
parameters were as described in b. d,
determination of KD and the Hill coefficient of
slope for the interaction of purified GPVI with calmodulin. Data were
transformed from fluorescence titration experiments and values
calculated by linear regression (SigmaPlot 2002). Titration data are
representative of Fig. 2c.
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GPVI-Calmodulin Interaction Is Mediated Exclusively by the Basic
Amino Acids in the GPVI Intracellular Tail--
The basic amino acids
within the GPVI intracellular domain are predicted to form the
calmodulin-binding site if arranged in the shape of an
-helix (Fig.
1). To directly test the role of these amino acids for calmodulin
binding and for signal transduction by GPVI we generated GPVI mutants
in which: (i) the four amino-terminal basic residues were mutated to
alanines (GPVI RK1), (ii) the four carboxyl-terminal basic residues
were mutated to alanines (GPVI RK2), and (iii) all eight basic amino
acids were mutated to alanines (GPVI RK1/2). Significantly, all
mutations of this domain were made in the context of the full-length
receptor rather than in cognate peptides or through receptor
truncation. Mutant receptors were expressed stably at roughly
equivalent levels in RBL-2H3 cells and co-immunoprecipitation studies
used to identify the residues critical for interaction with calmodulin
in live cells. Mutation of either the amino-terminal or
carboxyl-terminal basic amino acids alone (GPVI RK1 or GPVI RK2) did
not interrupt calmodulin binding, but mutation of all eight basic amino
acids (GPVI RK 1/2) abrogated the interaction of these two proteins
(Fig. 3b). GPVI truncation
mutants lacking residues carboxyl to these basic amino acids (A303STOP
and T318STOP) or the transmembrane domain critical for Fc R
coupling
(R272L) bound calmodulin normally, but a truncation mutant lacking the
basic domain (R295STOP) did not. These results demonstrate that the
basic amino acids between Trp-292 and Val-306 in the GPVI intracellular
domain mediate calmodulin interaction and that calmodulin interaction
is independent of either Fc R
or Lyn association (discussed further
below).

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Fig. 3.
Mutational analysis reveals that the basic
domain of the GPVI intracellular tail is required for calmodulin
interaction. a, comparison of the amino acid sequences
of wild-type human GPVI (hGPVI) with those of mouse GPVI
(mGPVI), a series of GPVI truncation mutants and GPVI
receptors with alanine substitution of some or all of the basic
residues in the GPVI C-tail. b, loss of all basic residues
within the GPVI C-tail results in loss of calmodulin association. GPVI
receptors were immunoprecipitated from RBL-2H3 cell lysate of wild-type
and mutant GPVI expressing cells using HY101-Sepharose beads and
co-immunoprecipitated calmodulin detected by Western blotting.
Top lane, immunoprecipitated GPVI receptor; bottom
lane, co-immunoprecipitated calmodulin. Note the loss of
calmodulin association detected only with the RK1/2 and the R295STOP
mutants. c, calmodulin is not co-precipitated with GPVI when
GPVI is precipitated using CVX-Sepharose beads. GPVI receptors were
precipitated from RBL-2H3 cell lysates using CVX-Sepharose beads and
co-immunoprecipitated calmodulin detected by Western blotting.
Top lane, precipitated GPVI receptor; bottom
lane, co-precipitated calmodulin. Note the loss of calmodulin
association with GPVI relative to that seen in b.
d, dissociation of calmodulin from activated GPVI receptors.
RBL-2H3 cells expressing wild-type human GPVI were stimulated with CVX
(10 nM) and lysed 10-120 s following CVX exposure.
GPVI-associated calmodulin was measured as described above for
b. 0 s indicates lysis prior to CVX exposure. Top
lane, immunoprecipitated GPVI receptor; bottom lane,
co-immunoprecipitated calmodulin.
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Calmodulin Is Released by GPVI during Receptor Stimulation--
To
determine the role played by calmodulin during GPVI signaling we
analyzed calmodulin-GPVI interaction before and after receptor
stimulation with CVX, a high affinity GPVI ligand that activates strong
calcium signaling in GPVI-expressing but not wild-type RBL-2H3 cells
(14). In resting cells, calmodulin could be co-immunoprecipitated with
GPVI (Fig. 3, b and d). Following stimulation of
GPVI with CVX, however, calmodulin was released from GPVI within 30-60
s (Fig. 3, b and c). As seen in Fig.
3b, calmodulin could be easily co-immunoprecipitated with
GPVI from resting cell lysate when using HY101, a non-clustering
anti-GPVI antibody (16). When GPVI was precipitated using the
clustering ligand CVX, however, very little calmodulin was
co-precipitated. These results suggest that calmodulin associates with
GPVI in resting cells and calmodulin-GPVI interaction is regulated by receptor-ligand interaction in a manner that may be independent of
downstream GPVI signaling.
GPVI Receptors Unable to Bind Calmodulin Exhibit a Signaling Defect
Despite Preserved Interactions with Fc R
and Lyn--
Although the
interaction of calmodulin with several platelet receptors has recently
been identified, the role of calmodulin during signal transduction by
platelet receptors remains unknown. To detect a role for calmodulin in
GPVI signal transduction we examined the calcium signals stimulated by
the high affinity GPVI ligand CVX in cells expressing wild-type GPVI
(hGPVI), GPVI lacking one-half the basic residues of the
calmodulin-binding domain (GPVI RK1 and GPVI RK2) and GPVI lacking all
basic residues in the calmodulin-binding domain and unable to bind
calmodulin (GPVI RK1/2). As previously observed, expression of GPVI in
RBL-2H3 cells conferred robust calcium signaling in response to CVX
(Fig. 4 and Ref. 14). GPVI receptors
lacking only half the basic residues of the calmodulin-binding domain
but still able to bind calmodulin demonstrated wild-type calcium
responses to CVX with the exception of a lag in the time required to
initiate calcium signaling for the GPVI RK2 mutant receptor
(Fig. 4). GPVI receptors unable to bind calmodulin, however, demonstrated severely reduced calcium responses to CVX despite a
receptor surface expression equivalent to that of wild-type GPVI (Fig.
4). These results suggest that the GPVI receptor domain required for
interaction of calmodulin with GPVI is also required for normal
receptor signaling.

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Fig. 4.
The calmodulin-binding domain of GPVI is
required for normal receptor signaling. Wild-type GPVI
(hGPVI) and GPVI receptors in which some or all basic
residues were mutated to alanines (RK1, RK2, and
RK1/2) were stably expressed in RBL-2H3 cells and the
signaling responses to CVX (10 nM) measured. Left
panels show the projected receptor C-tail helices.
Middle panels show receptor surface expression measured with
FITC-HY101. Right panels show intracellular calcium
signaling responses measured using Fura2 dye.
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Loss of Signaling by GPVI Receptors Unable to Bind Calmodulin Is
Not Caused by Loss of Association with Fc R
or Lyn--
Previous
studies have revealed an unexpected role for the intracellular GPVI
C-tail in mediating association of GPVI with Fc R
(14, 15), raising
the possibility that the loss of signaling observed in GPVI
RK1/2-expressing RBL-2H3 cells might be caused by loss of Fc R
association rather than loss of a specific function mediated by the
calmodulin-binding domain of GPVI. To test this possibility we
performed co-immunoprecipitation assays to compare the ability of
wild-type GPVI (WT GPVI) and the GPVI RK mutants to associate with Fc
R
in RBL-2H3 cells. As expected, Fc R
co-precipitated with
wild-type GPVI but not with the GPVI R272L mutant in which a
transmembrane arginine critical for Fc R
association is mutated (Fig. 5). Significantly, Fc R
was also
co-precipitated with all the RK mutants, including the GPVI receptor
lacking all of the basic amino acids (GPVI RK1/2) required for
calmodulin interaction. Similarly co-immunoprecipitation studies also
demonstrated preserved interaction with the Src family kinase Lyn (Fig.
5). These results suggest that the loss of GPVI signaling associated
with loss of the calmodulin-binding domain is not merely the result of
loss of Fc R
or Lyn association and that interaction with calmodulin or an unidentified protein may be required for normal GPVI signal transduction.

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Fig. 5.
Discrete domains within the GPVI C-tail
mediate association with Fc R and with
Lyn. Wild-type GPVI (WT GPVI) and GPVI mutants stably
expressed in RBL-2H3 cells were immunoprecipitated (top
lane) and co-immunoprecipitated Fc R (middle lane)
and Lyn (bottom lane) measured by Western blotting. Note
that the proline-rich region of GPVI deleted in GPVI A303STOP is
required for Lyn but not Fc R association while the transmembrane
arginine (Arg-272) is required for Fc R but not Lyn association.
Neither is required for calmodulin binding (Fig. 3), and loss of the
calmodulin-binding domain does not interfere with Fc R or Lyn
association (GPVI RK1/2).
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GPVI Association with the Src Family Kinase Lyn Is Independent of
GPVI Association with Calmodulin and Fc R
--
Recent studies have
demonstrated that the proline-rich region within the GPVI C-tail
interacts with the SH3 domain of the Src family tyrosine kinase Lyn
(19), but the importance of that interaction for signal transduction by
GPVI is not established. To further dissect the domains of the GPVI
C-tail and test their functional importance for receptor signaling we
stably expressed three GPVI truncation mutants, GPVI T318STOP, GPVI
A303STOP, and GPVI R295STOP, in RBL-2H3 cells. As shown in Fig.
3a, GPVI T318STOP deletes the region of human GPVI without
homology to mouse GPVI, and GPVI A303STOP deletes the proline-rich
domain predicted to interact with Lyn as well as the non-homologous
region. GPVI R295STOP (Fig. 3a and Ref. 14) deletes
virtually all of the intracellular C-tail of GPVI, including the
calmodulin-binding domain, the proline-rich domain, and the
non-homologous domain. To determine whether the functions of these
predicted domains were truly discrete or if there was significant
overlap of the GPVI domains required to mediate interaction with
calmodulin and Lyn we performed co-immunoprecipitation experiments with each receptor in RBL-2H3 cells. As expected, wild-type
GPVI but not GPVI R272L co-precipitated with Fc R
as well as Lyn and
calmodulin (Fig. 5). Surprisingly, all the truncation mutants also
associated with Fc R
, although the association of GPVI R295STOP with
Fc R
was generally weak and could not always be detected (Fig. 5,
Ref. 14, and data not shown). Thus Fc R
association is primarily
mediated by the GPVI transmembrane domain with secondary modulation by
the GPVI intracellular tail.
In contrast to its association with Fc R
, the association of GPVI
with Lyn correlated completely with the presence of the proline-rich
domain in the GPVI intracellular C-tail. Wild-type GPVI associated with
Lyn but GPVI truncation mutants lacking the proline-rich domain (GPVI
R295STOP and GPVI A303STOP) did not (Fig. 5). A GPVI truncation mutant
retaining proline-rich domain (GPVI T318STOP), however, did maintain an
interaction with Lyn (Fig. 5). Surprisingly, the domains of GPVI
required for interaction with Lyn were completely distinct from those
required for interaction with Fc R
or calmodulin. Thus the
transmembrane mutant GPVI R272L interacted normally with Lyn and
calmodulin despite complete loss of Fc R
association (Fig. 5 and
Ref. 14). GPVI RK1/2 was unable to interact with calmodulin but
associated normally with both Lyn and Fc R
(Fig. 5). Finally, GPVI
A303STOP associated normally with both Fc R
and calmodulin despite a
loss of Lyn interaction. Together, these results demonstrate the
presence of discrete functional domains within the GPVI C-tail that
mediate non-overlapping protein-protein interactions.
Productive Interaction with Calmodulin, Lyn, and Fc R
Are All
Required for Normal GPVI Signal Transduction--
The biochemical
studies described above suggested that the functional importance of
GPVI interaction with calmodulin, Lyn, and Fc R
could be
distinguished using GPVI mutant-expressing RBL-2H3 cell lines deficient
in only a single interaction. To determine the relative roles of GPVI
interaction with calmodulin, Lyn, and Fc R
on GPVI signaling in
cells we compared the calcium signals initiated by CVX in RBL-2H3 cells
expressing wild-type GPVI and each of the mutant receptors. Surface
expression of all the mutant receptors was equal to or greater than
that of wild-type GPVI (Fig. 6). As
previously reported, loss of Fc R
association resulted in a complete
loss of GPVI signal transduction despite preserved interaction with
calmodulin and Lyn (GPVI R272L, Fig. 6 and Ref. 14). As predicted by
its normal biochemical profile, loss of the non-homologous region of
the human GPVI receptor had no discernable effect on CVX-induced
calcium signals (GPVI T318STOP, Fig. 6). Loss of either the
calmodulin-binding domain (GPVI RK1/2) or the Lyn-binding domain
resulted in significant but not absolute loss of CVX-induced calcium
signaling (Fig. 6), while mutants retaining normal calmodulin and Lyn
binding (GPVI RK1 and GPVI RK2) demonstrated normal signaling responses
to CVX. Interestingly, despite its ability to associate weakly with Fc
R
, the GPVI R295STOP mutant unable to associate with either
calmodulin or Lyn demonstrated a complete loss of function in this
assay (Fig. 6).

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Fig. 6.
The GPVI proline-rich domain is required for
normal GPVI signaling. Signaling by wild-type and mutant GPVI
receptors stably expressed in RBL-2H3 cells was assessed in response to
10 nM CVX. Surface expression of GPVI receptors was
measured with FITC-HY101 (left) and calcium signals measured
using Fura2 dye (right). Note the significant loss of
signaling exhibited by GPVI A303STOP, a mutant unable to associate with
Lyn, despite high levels of receptor expression, as well as the
complete loss of signaling in cells expressing GPVI R272L and GPVI
R295STOP.
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DISCUSSION |
The predicted amino acid sequence of the gene encoding the
platelet collagen receptor GPVI establishes the receptor as a member of
the family of multisubunit receptors that signal through the ITAM
domains of non-covalently associated co-receptors (21). Subsequent
studies have indeed demonstrated that GPVI associates with a signaling
co-receptor, Fc R
, and that Fc R
association is required both for
the receptor's surface expression in platelets (4) and for its ability
to mobilize intracellular calcium (14). Fc R
-dependent
GPVI signaling proceeds through tyrosine phosphorylation of two Fc R
intracellular tyrosines (the Fc R
ITAM), which bind and activate the
non-receptor tyrosine kinase SYK to activate downstream effectors such
as phospholipase C
(13). Like many of the immune receptor
ligand-binding subunits (17, 25), however, GPVI has a sizable
intracellular domain whose role in receptor signaling, if any, is not established.
Recent biochemical studies have suggested that the intracellular domain
of GPVI binds proteins other than Fc R
, which may participate in
downstream GPVI signaling events, strengthening the possibility that
GPVI signaling is regulated in an Fc R
-independent fashion by the
receptor C-tail. Two proteins in particular, calmodulin and the Src
family tyrosine kinase Lyn, have been found to interact directly with
peptides corresponding to the GPVI intracellular C-tail and to
co-immunoprecipitate with GPVI in platelet lysates (20). In the present
study we have extended these observations to address the functional
importance of these interactions during GPVI signaling by addressing
the following questions. (i) What is the stoichiometry and affinity of
GPVI-calmodulin interaction? (ii) Does GPVI interact with Fc R
,
calmodulin, and Lyn through discrete protein domains or does the
receptor interact with these proteins in an interdependent fashion that
requires formation of a complex in which some or all must be present?
(iii) What is the importance of GPVI interaction with each of these
proteins for signal transduction by this receptor? We have approached
these questions through analysis of wild-type and mutant GPVI receptors expressed in RBL-2H3 cells, a cellular system previously demonstrated to confer GPVI-dependent collagen signals in a manner that
accurately reflects platelet responses (16).
Studies of GPVI-calmodulin interaction demonstrate that these proteins
constitutively associate at a 1:1 ratio through a high affinity
interaction. Our results using purified GPVI receptor agree closely
with previous predictions of GPVI-calmodulin interaction based on the
use of GPVI C-tail peptides (20). Our results also confirm reports that
GPVI association with calmodulin is disrupted during receptor
activation in a time course that is consistent with that required to
generate calcium signals. Thus calmodulin interaction with GPVI is tied
to receptor inactivity and calmodulin is either regulated by
receptor-mediated calcium signals or vice versa.
The juxtamembranous region of GPVI rich in basic amino acids has been
suggested as a calmodulin-binding site based on its homology to a
calmodulin-binding site in GPIb
and on the ability of cognate
peptides to interact with calmodulin (20). We have tested that
interaction directly in the context of the full-length receptor through
the generation and expression in RBL-2H3 cells of mutant GPVI receptors
in which some or all of the charged amino acids in this domain were
replaced by alanines. Co-immunoprecipitation studies demonstrate
clearly that this region is indeed required for calmodulin interaction
but that either the amino-terminal or carboxyl-terminal basic residues
of this domain are sufficient to mediate the GPVI-calmodulin
interaction despite the fact that GPVI and calmodulin associate in a
1:1 ratio.
The release of calmodulin from GPVI during receptor activation suggests
that calmodulin might regulate GPVI signaling and that a GPVI mutant
unable to interact with calmodulin might therefore exhibit altered
signaling. Indeed, a GPVI mutant lacking all the basic residues in this
domain and unable to bind calmodulin exhibited a significant loss of
function. Unexpectedly, this loss of function was not due to a failure
to interact with Fc R
or Lyn, suggesting that this region serves an
independent role in regulating GPVI signal transduction. We cannot
exclude the possibility that the loss of signaling exhibited by GPVI
RK1/2 is due to an effect unrelated to calmodulin binding such as loss
of interaction with an unidentified protein. However, evidence that
GPVI RK1/2 associated normally with Fc R
and Lyn and that calmodulin
binding by GPVI RK1 and GPVI RK2 correlated closely with calcium
signaling suggests that calmodulin binding may regulate GPVI signaling directly.
The ability to dissociate GPVI interaction with calmodulin from that
with Fc R
and Lyn suggested that the GPVI intracellular C-tail might
regulate GPVI signaling through an adaptor-like function, i.e. by mediating interaction with signaling effectors
through discrete and non-overlapping domains. To further test this
model we generated a series of truncation mutants (GPVI T318STOP, GPVI A303STOP, and GPVI R295STOP) and tested them for interaction with the
known GPVI-interacting proteins Fc R
, calmodulin, and Lyn. The
carboxyl-terminal region of human GPVI (deleted in GPVI T318STOP) has
no homologous counterpart in the mouse GPVI receptor, and loss of this
region did not disrupt association with any of these GPVI partners or
have any discernable effect on GPVI-mediated calcium signaling in
RBL-2H3 cells. In contrast, loss of the proline-rich domain (deleted in
GPVI A303STOP) disrupted GPVI interaction with Lyn. Importantly,
despite normal association with Fc R
and calmodulin, GPVI signaling
was clearly impaired in the absence of the proline-rich domain,
suggesting that GPVI-mediated recruitment of Src family tyrosine
kinases to Fc R
may be an important step for ITAM phosphorylation and the initiation of GPVI-Fc R
signaling. Previous studies have shown that GPVI associates with lipid rafts in an
activation-dependent manner and that phosphorylation of Fc
R
occurs exclusively in lipid-raft associated receptors (7),
suggesting that ligand-induced movement of receptors to lipid rafts
regulates the association of GPVI-Fc R
with active Lyn kinase. The
finding that the Lyn-binding domain of GPVI is required for normal
receptor signaling suggests either that receptor association with Src
family tyrosine kinases is regulated in more than one way or that
receptor signaling is regulated by a second, unidentified binding
partner via this domain. Finally, we re-examined a truncation mutant,
GPVI R295STOP, which we previously characterized as exhibiting a
complete loss of calcium signaling associated with loss of Fc R
interaction. While this mutant does indeed exhibit a complete loss of
signaling as well as loss of calmodulin and Lyn association, we were
able to detect a weak association with the Fc R
chain. This suggests
that the GPVI intracellular domain may not be absolutely required for
Fc R
association but may play a supportive role, e.g. to
stabilize the receptor at the cell surface.
Taken together, our studies of GPVI protein-protein interaction and
signaling in RBL-2H3 cells demonstrate an important role for the GPVI
intracellular domain during receptor signaling that is independent of
Fc R
association. In many ways the GPVI intracellular domain appears
to function as a tethered adaptor. The transmembrane domain is critical
for association with Fc R
, a necessary signal-transducing co-receptor. The juxtamembranous basic domain mediates interaction with
calmodulin and the proline-rich domain binds Src family tyrosine kinases and perhaps other SH3 domain-containing proteins. As for many
adaptor proteins, these interactions appear discrete and non-overlapping. Surprisingly, loss of any of these domain functions significantly impairs GPVI receptor signaling, providing a more complex
view of signal transduction by this ligand-binding receptor subunit
than mere coupling to Fc R
. Previous mutational analysis of two
other Fc R
-associated ligand-binding subunits, Fc
RIII and Fc
RIa, have also suggested functional roles for their cytoplasmic domains
(17, 26). Whether these observations reflect modulation of
receptor signaling through discrete protein-protein interactions in a
manner similar to GPVI is a question with important implications for signaling in many circulating cells.