(Received for publication, December 27, 1994; and in revised form, February 6, 1995)
From the
The purpose of the present study was to analyze the post-translational and activation-dependent modifications of the G protein-coupled thrombin receptor. A human receptor cDNA was engineered to encode an epitope tag derived from the vesicular stomatitis virus glycoprotein at the COOH terminus of the receptor and expressed in human embryonic kidney 293 cells. We show here that the mature receptor is a glycosylated protein with an apparent molecular mass ranging from 68 to 80 kDa by SDS-polyacrylamide gel electrophoresis. Removal of asparagine-linked oligosaccharides with N-glycosidase F leads to the appearance of a 36-40-kDa receptor species. The current model for receptor activation by thrombin involves specific hydrolysis of the arginine-41/serine-42 (Arg-41/Ser-42) peptide bond. Cleavage of the receptor by thrombin was demonstrated directly by Western analyses performed on membranes and glycoprotein-enriched lysates from transfected cells. Whereas thrombin treatment of cells results in increased mobility of the receptor in SDS-polyacrylamide gel electrophoresis, we found that their treatment with the thrombin receptor agonist peptide leads to a decrease in thrombin receptor mobility due, in part, to phosphorylation. The serine proteases trypsin and plasmin also cleave and activate the receptor similar to thrombin, whereas chymotrypsin cleaves the receptor at a site distal to Arg-41, thus rendering it unresponsive to thrombin while still responsive to thrombin receptor agonist peptide.
Thrombin initiates the majority of its biological effects on
cells via a surface receptor for which the corresponding cDNA has been
cloned (1, 2) . The thrombin receptor cDNA sequence
encodes a member of the G protein receptor superfamily with 8
hydrophobic domains, including one at the extreme NH terminus, which presumably corresponds to a signal peptide
sequence. Five potential sites for asparagine-linked (N-linked) glycosylation are present on the human receptor
(and four on the hamster receptor), as well as numerous serine,
threonine, and tyrosine residues that are potential sites of
phosphorylation. On the NH
-terminal external domain, a
putative thrombin cleavage site has been identified which is
characterized by an arginine (Arg-41) flanked on the carboxyl side by a
cluster of negatively charged residues displaying sequence similarity
to the thrombin-binding domain in the tail of the thrombin inhibitor
hirudin (see (2) and (3) and references therein). The
presence of this cleavage site on the receptor, along with the
observation that thrombin stimulation of cellular effects requires a
proteolytically active enzyme, has led to the following model of
receptor activation (1) . It has been proposed that thrombin
specifically recognizes its receptor via the acidic hirudin-like
domain, located approximately 10-20 residues downstream of the
proposed cleavage site. Ensuing cleavage of the Arg-41/Ser-42 bond
exposes a sequence (SFLLRN) that is capable of activating the receptor
by interacting with an undefined ligand-binding domain. Several lines
of evidence favor this hypothesis, including the fact that the cellular
effects of thrombin can be mimicked by synthetic peptides corresponding
to the unmasked ligand sequence(1, 4, 5) .
Also, antibodies to the new NH
-terminal sequence are
capable of inhibiting thrombin activation of
cells(6, 7, 8) . Finally, treatment of cells
with thrombin is accompanied by the release of the
NH
-terminal activation peptide from the cell surface, as
determined by the loss of binding to cells of antibodies specific to
the cleaved off sequence (9) .
Studies addressing
post-/co-translational modifications and structure-function
relationships have been carried out on other G protein-coupled receptor
family members, in particular the 2-adrenergic receptor and
rhodopsin (for reviews, see Refs. 10 and 11). Using biochemical and/or
genetic approaches, it has been demonstrated that these receptors can
undergo glycosylation and palmitylation during biosynthesis.
Activation-dependent phosphorylation, which plays a role in receptor
desensitization, has also been shown. The high degree of sequence
similarity observed among serpentine receptors would indicate that they
undergo similar post-translational and activation-dependent
modifications. However, little is known at present about the
post-/co-translational and activation-dependent modifications of the
thrombin receptor. Characterization of this receptor has been difficult
due, in part, to the lack of an efficient stable expression system and
appropriate antibodies. In the present study, we have obtained high
expression levels of the receptor in human 293 cells. The addition of
an epitope tag on the receptor has allowed us to analyze the protein
and to directly demonstrate its glycosylation, phosphorylation, and
cleavage by thrombin. To our knowledge, this report represents the
first illustration of receptor proteolysis.
Figure 1: Top, epitope-tagged thrombin receptor cDNA construct. The blackbox at the 3`-extremity of the coding sequence indicates the VSVG epitope. Bottom, immunocytochemistry of the epitope-tagged thrombin receptor. Indirect immunofluorescence microscopy of the human thrombin receptor in clone 293TRV2J (A) or control 293 cells (B) is shown. Cells were fixed and stained using the P4D5 antibody raised against the COOH-terminal tag sequence of the transfected receptor as described under ``Experimental Procedures.''
Interestingly, upon inspection of several independent G418-resistant clones, we observed the presence of both positively staining cells and unstained cells in each clone. Cells expressing the thrombin receptor were generally rounder and less adherent to the culture dish than cells devoid of receptor expression. The staining of positive cells in all clones examined was quite intense and uniform, as if the cells were expressing similar numbers of receptors. However, the percentage of labeled cells varied from one clone to another, resulting in different levels of functional expression. As indicated in Fig. 2, the percentage of fluorescent cells in different clones correlated well with the extent of functional receptor expression by each clone, as determined by phospholipase C activation in response to thrombin or thrombin receptor agonist peptide (TRP). Note that basal rates of phosphatidylinositol hydrolysis remain low in clones expressing high levels of thrombin receptor, indicating that the non-liganded receptor is tightly controlled. Further, the dose dependence of thrombin-stimulated phospholipase C activation in transfected cells was similar to that observed in fibroblasts, which express only the endogenous receptor (not shown). Altogether, these results indicate that the VSVG sequence at the COOH terminus of the receptor does not impair ligand-induced receptor activation, regulation, or G protein coupling. It can also be seen in Fig. 2that low levels of endogenous thrombin receptor activity can be detected in nontransfected 293 cells. Consistent with this observation, we have observed the 3.5-kilobase thrombin receptor mRNA transcript in parental 293 cells by Northern blot analysis (not shown).
Figure 2:
Phospholipase C activation in parental 293
cells and in G418-resistant clones transfected with pTRV encoding the
tagged thrombin receptor. Total H-labeled inositol
phosphates accumulated during a 10-min incubation with 20 mM LiCl and no addition (0), thrombin (10
M), or TRP (30 µM) are shown. Results
represent the mean of duplicate determinations; fold-stimulation of
inositol phosphate formation above basal levels for each clone are
indicated (above bars), as well as the estimated % of
positively staining cells determined by immunocytochemistry using P4D5
antibody.
Figure 3:
Western analysis of the epitope-tagged
thrombin receptor. A, immunoblot analyses using P4D5
(anti-VSVG) or TG262 (anti-TRP) antibodies were performed on crude
membranes prepared from control, 293 cells, or from cells transfected
with the tagged human receptor (clone 293TRV18K). B, cells
expressing the tagged receptor (clone 293TRV2K) were treated for 15 min
in serum-free medium with no addition (0), thrombin
(10M) (Th), or TRP (30
µM) prior to preparation of membranes and immunoblot
analysis using the tag sequence-specific P4D5 antibody. The positions
of molecular mass markers are indicated.
Five potential
sites for N-linked glycosylation (NX(S/T), X P) are present on the human thrombin receptor(1) . To
determine whether the receptor is glycosylated on asparagine residues,
freshly prepared membranes from cells expressing the tagged receptor
were treated with N-glycosidase F, and Western analyses were
performed using anti-VSVG antibodies. Removal of N-linked high
mannose and hybrid and complex oligosaccharides with N-glycosidase F led to the appearance of a receptor species
ranging from 36-40 kDa (Fig. 4). In addition to N-linked glycosylation, it is likely that the receptor
contains O-linked carbohydrate, since we observed a slight
decrease in its apparent M
by immunoblot analyses
following treatment of membranes with neuraminidase and O-glycosidase (not shown).
Figure 4: Removal of N-linked glycosaccharides from the thrombin receptor. Equivalent amounts of membranes from 293 cells or from the transfected 293TRV cells (clone 293TRV18K) were treated with N-glycosidase F (PNGase) and analyzed by Western blot analysis using P4D5 antibody.
Since the protein was found to
be highly glycosylated, we performed lectin affinity purification of
cell lysates to increase the yield of receptor for further analysis.
First, the time course of receptor cleavage was examined using wheat
germ-enriched fractions of solubilized cells. By Western analysis using
anti-VSVG antibodies, specific labeling of the receptor was observed in
transfected 293 cells, whereas no bands could be detected in
nontransfected cells. As shown in Fig. 5, cleavage of the
receptor by thrombin (10M) occurs within 1
min and appears to be maximal 2 min after treatment of cells with the
protease. Upon short exposure of the blot, the receptor migrates as
three sharp bands (of which the lowest is the major species) within a
broad diffuse band. Following cleavage, a smaller M
band appears, and the trailing smear migrates faster. Treatment
with TRP, which activates the receptor independent of cleavage, induces
an upward shift of the diffuse band and more intense staining of the
sharp band of higher M
.
Figure 5:
Western analysis of the tagged thrombin
receptor in nontransfected 293 cells and in a G418-resistant clone
(293TRV2J) transfected with pTRV. Cells were treated for the indicated
times with thrombin (10M) (Th) or
TRP (30 µM) prior to Western analysis of wheat
germ-enriched cell lysates using P4D5
antibody.
We next investigated
whether the upward shift in mobility of the receptor activated by TRP
was due to some post-translational modification, such as
polyubiquitination or phosphorylation. Ligand-induced receptor
ubiquitination of growth factor receptors has previously been
demonstrated. In the case of the platelet-derived growth factor
receptor, modification was maximal 7 min following platelet-derived
growth factor addition, as determined by immunoblotting of
lectin-enriched cell lysates using anti-ubiquitin
antiserum(17) . In TRV cells, we were unable to detect any
addition of ubiquitin to the thrombin receptor upon activation by
thrombin or TRP (results not shown). Concerning phosphorylation, it has
recently been reported that the thrombin receptor expressed in rat 1
cells is phosphorylated following thrombin
treatment(18, 19) . To determine the phosphorylated
state of the tagged receptor in transfected 293 cells, we performed in vivo labeling experiments using
[P]orthophosphate followed by
immunoprecipitation of the receptor with anti-VSVG antibodies and
autoradiography. The results in Fig. 6show that under
non-stimulated conditions, receptor phosphorylation could not be
detected. After thrombin addition, we observed phosphorylation of a
protein that migrated with the expected M
of the
thrombin receptor and that was absent in immunoprecipitates from
nontransfected 293 cells. TRP also induced receptor phosphorylation (Fig. 6). Thus, activation-dependent phosphorylation could
account, at least in part, for the decreased mobility of the receptor
observed following TRP stimulation of cells. Indeed, phosphatase
treatment was able to slightly increase mobility of the activated
receptor (results not shown).
Figure 6:
Phosphorylation of the thrombin receptor. P-Labeled 293 cells or 293TRV2K cells stably expressing
the tagged receptor were treated with thrombin (Th) or TRP for
the indicated times and immunoprecipitated with P4D5 antibodies as
described under ``Experimental
Procedures.''
Figure 7:
Western analysis of protease-treated human
thrombin receptor. TRV2K cells were treated for 15 min with
10M thrombin (Th), 4
10
M trypsin (Tr), 3
10
M plasmin (Pl), 10
M chymotrypsin (Ch), or 30 µM TRP.
Protease treatments (with the exception of thrombin) were carried out
in serum-free medium containing hirudin (1 thrombin-inactivating
unit/ml). Wheat germ-enriched cell lysates were separated on a 10%
acrylamide gel prior to Western analysis with P4D5 antibodies, as
described under ``Experimental Procedures.'' Positions of
molecular mass markers are shown.
The functional consequences of thrombin receptor cleavage in TRV cells were determined by evaluating the effect of these proteases on phospholipase C activation. As shown in Fig. 8, trypsin is able to induce efficient thrombin receptor coupling to phospholipase C, at approximately 10-fold higher doses than those required for thrombin to stimulate the effect. We found that plasmin induced a weak but significant response as well. These results are consistent with the ability of these proteases to induce a mobility shift in the receptor that is similar to the one induced by thrombin. Chymotrypsin, which cleaves the receptor at a position different than thrombin, impaired the ability of thrombin to activate the receptor in TRV cells, presumably due to removal of the thrombin recognition and cleavage sites on the receptor. By contrast, cleavage of the receptor by chymotrypsin did not destroy its responsiveness to TRP, which is independent of cleavage, suggesting that the chymotrypsin-truncated receptor is able to assume a functional conformation and interact with G proteins.
Figure 8:
Phospholipase C activation by proteases.
Phospholipase C activity was determined by measuring total H-labeled inositol phosphate formation in 293 cells or in
293TRV2K cells, following protease addition for 10 min. Protease
concentrations were 10
M for thrombin (Th) and 10
M for trypsin (Tr), plasmin (Pl), and chymotrypsin (Ch).
In the case of chymotrypsin pretreatment (+Ch), cells
were incubated for 10 min in serum-free Dulbecco's modified
Eagle's medium in absence or presence of 10
M chymotrypsin prior to rinsing one time and treatment
with 30 µM TRP or 10
M thrombin for 10 min. The mean of duplicate determinations are
expressed as -fold stimulation above basal
values.
In the present study, stable expression of the thrombin
receptor was obtained using 293 human embryonic kidney cells.
Heterologous expression of the receptor was considerably higher in this
system than in other fibroblastic cell lines that we have examined to
date for reasons that are not clear at the present time. Since
immunocytochemical analyses of transfected cells revealed that thrombin
receptor expression was heterogeneous in clonal populations, we did not
attempt to determine receptor numbers expressed by the different TRV
clones. Interestingly, we observed a similar heterogeneity of
expression for other receptors (e.g. the 5-HT and
2-adrenergic receptors) stably transfected in 293 cells and
fibroblasts. These results indicate that ligand binding assays on
transfected clones may not accurately determine receptor numbers per
cell. Indeed, this should be kept in mind when interpreting functional
studies on clones thought to express low, moderate, or high receptor
numbers per cell. Nonetheless, we found that functional receptor
expression by the different transfected clones, as measured by
thrombin- or TRP-induced phospholipase C stimulation, correlated well
with the number of positively staining cells. Analysis of the
receptor-mediated phospholipase C response in transfected cells
indicated that the epitope tag attached to the COOH terminus of the
receptor does not significantly modify receptor activity.
The VSVG sequence, corresponding to an epitope that is virtually absent in nontransfected cells, has proved to be very useful for characterization of the receptor protein. Although Western blots of the thrombin receptor from platelet membranes or transfected cells have previously been published(6, 20) , no visualization of receptor cleavage has been shown to our knowledge. In the present investigation, we were able to directly demonstrate thrombin receptor cleavage by immunoblot analysis. In addition to thrombin, we found that trypsin, plasmin, and chymotrypsin are able to cleave the receptor expressed at the surface of 293 cells. This system should be useful to determine whether the receptor is a substrate for other proteases as well. Recently, Suidan et al.(21) reported that granzyme A, a serine protease stored in cytoplasmic granules of T lymphocytes, is capable of cleaving and activating the thrombin receptor. In their study, a synthetic 32-mer peptide spanning the activation site of the receptor was cleaved by the enzyme. It remains to be determined whether receptor cleavage by granzyme A occurs in intact cells.
Trypsin is a
full agonist of the phospholipase C response in cells that express the
thrombin receptor, although thrombin is 10-fold more potent than
trypsin for this effect. The enhanced efficiency of thrombin to cleave
and activate its receptor has been attributed to the presence of a
hirudin-like sequence adjacent to the cleavage site on the receptor
that binds to the anion-binding exosite of thrombin(3) . In
contrast to trypsin, phospholipase C activation by plasmin in TRV cells
was found to be weak. Although this observation is not fully understood
at present, it is possible that cleavage of the receptor by plasmin is
not restricted to the arginine-41/serine-42 bond since additional bands
are occasionally visible on immunnoblots of the plasmin-cleaved
receptor. It is clear from our immunoblot analyses that chymotrypsin
cleaves the receptor and that the chymotrypsin cleavage site is
distinct from the thrombin cleavage site. This result provides an
explanation for the finding over 10 years ago that chymotrypsin
modifies the platelet response to thrombin(22, 23) .
This effect of chymotrypsin on human platelets is specific to
thrombin-induced activation and can be overcome by high doses of
thrombin. Although the site(s) of chymotrypsin cleavage has not been
defined, chymotrypsin treatment of the receptor results in the loss of
approximately 7 kDa, indicating that cleavage may remove up to 60 amino
acids from the NH terminus. The fact that this truncation
by chymotrypsin does not lead to receptor activation provides further
evidence supporting the proposal by Chen et al.(24) that receptor activation does not occur by release of
the receptor from tonic inhibition imposed by its
NH
-terminal sequences.
The presence of N-linked
glycosylation sites within the NH-terminal domain is a
common feature of seven transmembrane domain receptors, with 92%
(211/229) of the receptors surveyed in a recent study displaying this
feature(25) . Glycosylation of the NH
terminus has
been confirmed for several of them (e.g. on residues Asn-6 and
Asn-15 in the
2-adrenergic receptor), using different approaches
(see (10) and references therein). To explain the difference
between the apparent M
of the receptor in SDS-PAGE
and its calculated M
, Brass and colleagues (6) proposed that carbohydrates contribute to as much as
one-third of the mass of the receptor. Indeed, enzymatic
deglycosylation of the receptor with N-glycosidase F presented
here confirms this prediction. In the study by Brass et
al.(6) , the platelet thrombin receptor migrated as a
relatively thin band of M
= 66,000 ±
1000 in SDS-PAGE. The epitope-tagged receptor expressed in 293 cells
migrates in our gel system as a large diffuse band ranging from
approximately 68 to 80 kDa, presumably due to heterogeneity in the
oligosaccharide chains. We have not identified which of the five
potential N-linked oligosaccharide sites on the human receptor
(three on the NH
-terminal extension and two on the second
extracellular loop) are used. However, the first asparagine (position
35) is not conserved in the hamster(2) , rat(26) , or
mouse (Genbank accession no. L03529) thrombin receptor sequences. N-Linked oligosaccharide chains tend to be localized to a
single extracytosolic segment (>30 residues long), and the acceptor
site is often the first (most NH
-terminal) in the protein,
according to a recent survey of glycosylation sites utilized in
mammalian multi-span membrane proteins(25) . The fact that we
never detected the presence of additional bands migrating between the
fully glycosylated protein and the deglycosylated form of the thrombin
receptor, following partial digestion with N-glycosidase F, (
)may suggest that a single site is utilized.
In addition
to N-linked glycosylation sites, serine and threonine residues
in the extracellular domains of the receptor may serve as acceptor
sites for O-linked carbohydrates, since neuraminidase and O-glycosidase treatment of membranes induced a slight shift in M of the receptor (data not shown). As to the
possible role of receptor glycosylation, it may be involved in proper
folding of the polypeptide chain during biosynthesis and/or in its
transport to the cell surface. This was found to be the case in a
recent study on rhodopsin(27) . In addition, N-linked
glycosylation of rhodopsin was found to be important in signal
transduction. Glycosylation has also been shown to protect proteins
from proteolysis (for review, see (28) ). In the case of the
thrombin receptor, the presence of oligosaccharides on the external
receptor surface may modulate thrombin recognition or post-cleavage
conformational changes of the receptor that influence tethered ligand
binding and receptor activation. Mutagenesis studies on the thrombin
receptor will be required to identify the site(s) of glycosylation and
to determine whether their elimination impairs the biosynthesis and/or
function of the receptor.
Activation-dependent phosphorylation of G
protein-coupled receptors by members of the G protein receptor kinase
family has been found to be an important mechanism underlying the
desensitization process (for review, see (11) ). We show here
that thrombin induces phosphorylation of its receptor expressed in 293
cell, and that the effect is mediated by the receptor since the agonist
peptide stimulates phosphorylation in a cleavage-independent manner.
Receptor phosphorylation may account for the apparent increase in
molecular weight of the receptor seen following treatment of cells with
the TRP. Presumably, this phosphorylation is mediated by a G protein
receptor kinase. This question was addressed in a recent study by Ishii et al.(18) , who observed that BARK2 efficiently
attenuated thrombin-induced Ca mobilization when
co-expressed with the receptor in Xenopus oocytes. Findings
from this study and results from our laboratory indicate that sequences
in the COOH terminus of the receptor are targets for G protein receptor
kinase(s)(19) .
In conclusion, we believe that the stable expression system described here should be very useful for structure-activity studies on the thrombin receptor. Downstream signaling events can also be investigated in this system following transient expression of the receptor (or receptor mutants) along with signaling components (i.e. G protein subunits) or regulatory proteins (members of the G protein receptor kinase or arrestin families).