©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Post-translational and Activation-dependent Modifications of the G Protein-coupled Thrombin Receptor (*)

(Received for publication, December 27, 1994; and in revised form, February 6, 1995)

Valérie Vouret-Craviari Dominique Grall Jean-Claude Chambard Ulla B. Rasmussen (1) Jacques Pouysségur Ellen Van Obberghen-Schilling (§)

From the Centre de Biochimie, CNRS UMR134, Parc Valrose, 06108 Nice Cedex 2, France and Transgene, S.A., 11 rue de Molsheim, 67082 Strasbourg, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(2) 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(2)-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(2)-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(2)-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 beta2-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.


EXPERIMENTAL PROCEDURES

Materials

Highly purified human alpha-thrombin (3209 NIH units/mg) was a generous gift of Dr. J. W. Fenton II (New York State Department of Health, Albany, NY). Hirudin was kindly provided by Transgene (Strasbourg, France), and human plasmin (American Diagnostica, Greenwich, CT) was provided by Dr. J. Maraganore (Biogen, Cambridge, MA). Bovine trypsin and chymotrypsin were from Sigma. The 7-residue hamster thrombin receptor peptide agonist (SFFLRNP) was synthesized by NEOSYSTEM (Strasbourg, France). Triton X-100 was from Pierce, and N-glycosidase F was from New England Biolabs. [P]Orthophosphate and myo-[2-^3H]inositol were purchased from Amersham. The monoclonal antibody P4D5 directed against an epitope of the vesicular stomatitis virus glycoprotein (12) was a kind gift from Dr. B. Goud (Institut Pasteur, Paris), peroxidase-conjugated anti-mouse and anti-rabbit IgGs were from Sigma, and fluorescein isothiocyanate-labeled sheep anti-mouse IgG was purchased from Amersham. The polyclonal antibody, TG262, was raised against the peptide corresponding to residues 42-55 of the human thrombin receptor (SFLLRNPNDKYEPF).

Plasmid Constructs

The epitope-tagged human thrombin receptor construct (pTRV) was made by replacing the stop codon and entire 3`-non-coding region of the human receptor cDNA (kindly provided by Dr. S. Coughlin, University of California, San Francisco) (1) with an EcoRV restriction site by polymerase chain reaction mutagenesis. Subsequently, the modified receptor cDNA was ligated to a sequence encoding the VSVG (^1)epitope (12) and subcloned into the pRK5 expression vector(13) . The resulting cDNA codes for all 427 residues of the receptor followed by a COOH-terminal extension composed of an 8-residue spacer (GWEGPPGP) and the VSVG epitope of 11 residues (YTDIEMNRLGK).

Cells and Culture Conditions

The human embryonic kidney 293 cell line was cultivated in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing penicillin (50 units/ml), streptomycin (50 µg/ml), and 8% heat-inactivated fetal calf serum. Stably transfected cells were grown in the presence of hirudin (0.1 thrombin-inactivating units/ml). To obtain clones expressing the epitope-tagged thrombin receptor, 293 cells were cotransfected with the plasmids pTRV and pcDNAINEO (encoding the selectable marker) using the calcium phosphate method. Selection was performed in presence of 500 µg/ml geneticin (G418 from Life Technologies, Inc.). Receptor expression was verified by indirect immunofluorescent staining of cells using anti-VSVG antibodies.

Immunocytochemistry

Cells plated in 6-well plates on glass coverslips were fixed in a 1:1 mixture of methanol:formaldehyde (15 min at -20 °C and then 10 min at room temperature). For staining, fixed cells were incubated for 1 h at room temperature with anti-VSVG antibodies diluted 1:500 in phosphate-buffered saline containing 1% bovine serum albumin and 0.2% gelatin. Fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (1:100 dilution, 45 min at room temperature) was used to reveal the primary antibodies. Cultures were analyzed by fluorescence microscopy with a Nikon Diaphot microscope (fluor 40/1.3 oil Ph4 DL).

Membrane Preparation and Cleavage of Oligosaccharide Chains with N-Glycosidase F

Crude membrane preparations were prepared following a hypotonic shock of cells as follows. Confluent monolayers in 10-cm culture dishes were rinsed once in TE (10 mM Tris, pH 7.5, 1 mM EDTA) and once in H(2)O before harvesting in 0.5 times TE buffer containing protease and phosphatase inhibitors (5 mM Tris, pH 7.5, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM orthovanadate, 50 mM NaF) and incubating on ice for less than 5 min. Subsequently, swollen cells were disrupted by passing them six times through a 26 gauge needle, and nuclei were removed by centrifugation (2000 rpm for 10 min at 4 °C). The nuclei-depleted supernatant was recovered and spun at 12,000 rpm for 30 min at 4 °C to pellet the crude membrane fraction, which was resuspended in 0.5 times TE buffer with inhibitors. Removal of asparagine-linked high mannose, hybrid, and complex oligosaccharides with N-glycosidase F was performed on freshly prepared membranes (5000 NEB units/20 µg of protein) according to the protocol suggested by New England Biolabs, except that all incubations were carried out at room temperature.

Wheat Germ Enrichment and Immunoblotting

Following the indicated treatment, cells were rinsed with phosphate-buffered saline, solubilized in Triton lysis buffer containing protease and phosphatase inhibitors (50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 5 µg/ml aprotinin, and 1% Triton X-100), and centrifuged (12,000 rpm) for 10 min at 4 °C. Clarified lysates were incubated for 1 h at 4 °C with wheat germ Lectin-Sepharose 6MB (Pharmacia, Uppsala, Sweden) and then washed four times in lysis buffer. For immunoblotting, wheat germ-enriched fractions were resuspended in Laemmli sample buffer containing 2 M urea, separated by SDS-PAGE on a 10% gel and transferred to Hybond C nitrocellulose membrane (Amersham). The membranes were blocked using TN buffer (50 mM Tris, 150 mM NaCl) containing 5% non-fat milk prior to incubation for 2 h with the anti-VSVG antibody. Blots were washed with TN containing 0.02% Triton X-100 (TN-Triton) prior to incubation with the peroxidase-conjugated sheep anti-mouse IgG for 1 h. The thrombin receptor was detected using the enhanced chemiluminescence (ECL) detection system (Amersham).

P Labeling and Immunoprecipitation

Cells grown to confluence in 10-cm dishes were washed and incubated for 4 h in phosphate-free Dulbecco's modified Eagle's medium containing 200 µCi/ml [P]orthophosphate. Agonists were added for the indicated times immediately prior to solubilization of cells in 600 µl of Triton lysis buffer containing protease and phosphatase inhibitors (as indicated above) for 20 min at 4 °C. Lysates were incubated for 45 min at 4 °C with non-immune mouse serum coupled to protein A-Sepharose (Pharmacia) and centrifuged prior to incubation of the clarified lysates (2 h at 4 °C) with anti-VSVG antibodies coupled to protein A-Sepharose. Following immunoprecipitation, samples were washed five times in lysis buffer, twice in high salt buffer (lysis buffer containing 0.5 M LiCl), and two times in low salt butter (lysis buffer without NaCl) and then resuspended in Laemmli sample buffer containing urea and separated by SDS-PAGE in 10% resolving gels. Phosphorylation of the thrombin receptor was revealed by autoradiography of dried gels.

Phosphatidylinositol Breakdown

Determination of total polyphosphoinositide formation was performed on cells in 12-well plates essentially as described(14) , except that ^3H-labeled myoinositol (2 µCi/ml) was added to cell culture medium approximately 24 h prior to the experiment.


RESULTS

Expression of the Thrombin Receptor in Transfected 293 Cells

The pTRV plasmid construct containing the human thrombin receptor cDNA (Fig. 1, top) was transfected in 293 human embryonic kidney cells, and G418-resistant cell populations or independent clones were selected. The cDNA was placed under control of the cytomegalovirus promoter, which has been shown to direct high level expression of transfected sequences in these cells(15) . To detect the expressed receptor, an epitope tag that corresponds to a sequence of the VSVG was fused to the COOH terminus of the protein. The VSVG tag sequence is recognized by the specific monoclonal antibody, P4D5. Using P4D5 antibody, G418-resistant clones expressing the human or hamster thrombin receptor were analyzed by indirect immunofluorescence microscopy. Fig. 1(bottom, A) depicts the VSVG-specific staining of a clone (293TRV2J), which expresses relatively high levels of the tagged receptor. Staining is predominantly at the cell surface, both in cells fixed with methanol/formaldehyde (as described under ``Experimental Procedures'') and in cells fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 to render the internal epitope accessible to antibodies. It is noteworthy that a similar pattern of staining was observed with polyclonal antibodies directed against the human receptor agonist peptide sequence immediately adjacent to the thrombin cleavage site of the receptor. (^2)Background staining with anti-VSVG antibodies is virtually absent in nontransfected 293 cells (Fig. 1, bottom, B).


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 ^3H-labeled inositol phosphates accumulated during a 10-min incubation with 20 mM LiCl and no addition (0), thrombin (10M), 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.



The Thrombin Receptor Is a Glycosylated Thrombin Substrate

To visualize the expressed receptor protein and to analyze its presumed cleavage by thrombin, Western blot analyses were performed on freshly prepared membranes from transfected clones expressing the tagged receptor. As shown in Fig. 3, the receptor migrates as an elongated band ranging from approximately 68 to 80 kDa when separated by SDS-PAGE in 10% acrylamide gels. This unique band, seen with the anti-VSVG antibody, is specific to the thrombin receptor since it is absent in parental 293 cells. Furthermore, the same band is observed in immunoblots using antibodies raised against residues 42-55 of the human thrombin receptor, corresponding to the agonist peptide sequence (Fig. 3A). Thrombin treatment of cells prior to membrane preparation induces a shift in mobility, resulting in the appearance of a faster migrating band that corresponds to the cleaved receptor (Fig. 3B). By contrast, treatment of cells with TRP, which activates the receptor without cleaving it, slows down receptor migration. The calculated molecular mass of the VSVG-tagged mature human thrombin receptor is 46.5 kDa (assuming that signal peptide cleavage occurs after alanine-26(16) . Following thrombin cleavage (Arg-41/Ser-42), the receptor loses 2.8 kDa, resulting in a calculated molecular mass of 43.7 kDa. To explain this considerable difference between the calculated molecular mass and the apparent size in SDS-PAGE, we examined the glycosylated state of the receptor.


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(r) 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(r) 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(r).


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(r) 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.''



Cleavage of the Receptor by Other Serine Proteases

To further analyze thrombin receptor cleavage, we examined the electrophoretic mobility of the receptor in glycoprotein-enriched fractions prepared from transfected 293 clones following treatment with serine proteases displaying different specificities, including trypsin, plasmin, and chymotrypsin. By Western analysis (Fig. 7), we observed that trypsin and, to a lesser extent, plasmin treatment both induce a downward shift in apparent M(r) of the receptor similar to that observed with thrombin. These proteases cleave at the carboxylic side of lysine or arginine residues, with plasmin being more selective (lysyl specific) than trypsin. Chymotrypsin, which cleaves following aromatic amino acids (tyrosine, phenylalanine, and tryptophane), induced receptor cleavage, which resulted in a greater increase in mobility than that induced by thrombin. This suggests that chymotrypsin cleavage occurs following some residue distal to arginine-41 where thrombin cleavage occurs.


Figure 7: Western analysis of protease-treated human thrombin receptor. TRV2K cells were treated for 15 min with 10M thrombin (Th), 4 times 10M trypsin (Tr), 3 times 10M plasmin (Pl), 10M 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 ^3H-labeled inositol phosphate formation in 293 cells or in 293TRV2K cells, following protease addition for 10 min. Protease concentrations were 10M for thrombin (Th) and 10M 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 10M chymotrypsin prior to rinsing one time and treatment with 30 µM TRP or 10M thrombin for 10 min. The mean of duplicate determinations are expressed as -fold stimulation above basal values.




DISCUSSION

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(2) and alpha2-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(2) 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(2)-terminal sequences.

The presence of N-linked glycosylation sites within the NH(2)-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(2) terminus has been confirmed for several of them (e.g. on residues Asn-6 and Asn-15 in the beta2-adrenergic receptor), using different approaches (see (10) and references therein). To explain the difference between the apparent M(r) of the receptor in SDS-PAGE and its calculated M(r), 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(r) = 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(2)-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(2)-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, (^3)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(r) 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).


FOOTNOTES

*
These studies were supported by CNRS (UMR134), INSERM, and the Association pour la Recherche contre le Cancer and Boehringer Ingelheim International GmbH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-52-99-25; Fax: 33-52-99-17; vanobber{at}unice.fr.

(^1)
The abbreviations used are: VSVG, vesicular stomatitis virus glycoprotein; PAGE, polyacrylamide gel electrophoresis; TRP, thrombin receptor agonist peptide.

(^2)
D. Grall, V. Vouret-Craviari, and U. Rasmussen, unpublished results.

(^3)
D. Grall and E. Van Obberghen-Schilling, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. S. R. Coughlin for kindly providing the thrombin receptor cDNA. C. Cibre is gratefully acknowledged for photographic work.


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