©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinct Mechanisms Regulate 5-HT and Thrombin Receptor Desensitization (*)

(Received for publication, September 29, 1994; and in revised form, November 18, 1994)

Valérie Vouret-Craviari Patrick Auberger (1) Jacques Pouysségur Ellen Van Obberghen-Schilling

From the Centre de Biochimie, CNRS-UMR134, Parc Valrose, 06108 Nice, Cédex 2, France and INSERM-U364, Faculté de Médecine, Avenue de Vallombrose, 06107 Nice, Cédex 2, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have compared the desensitization of two receptors, the thrombin receptor which displays dual coupling to both pertussis toxin-sensitive (G(i)) and -insensitive (G(q)) proteins and the serotonin type 2 (5-HT(2)) receptor which selectively couples to G(q). In the case of the thrombin receptor, cleavage induces activation and irreversible receptor modification followed by rapid (T = 3 min) and extensive desensitization of the receptor's ability to modulate phospholipase C (G(q)). 5-HT-induced desensitization of its receptor is markedly slower (T = 10 min) and by 60 min only 50% of the phospholipase C response is lost. This effect occurs with a parallel disappearance of 5-HT receptors from the cell surface. Whole cell phosphorylation studies showed that the thrombin receptor is rapidly phosphorylated upon activation. In contrast, the 5-HT(2) receptor displays a low basal level of phosphorylation which is not increased upon agonist treatment. The cytoplasmic tail of the 5-HT(2) receptor which contains several protein kinase consensus sequences was found not to be involved in receptor activation or desensitization. However, a chimeric receptor having the core of the 5-HT(2) receptor and the cytoplasmic tail of the thrombin receptor was able to undergo 5-HT-induced desensitization and phosphorylation. These results indicate that (i) both 5-HT(2) and thrombin receptors have unique shut-off mechanisms, and (ii) that sequences in the carboxyl terminus of the thrombin receptor are sufficient to trigger rapid uncoupling of the receptor from its G protein(s) and downstream effector(s).


INTRODUCTION

Loss of the biological response to a ligand despite its constant presence is termed desensitization. Mechanisms involved in desensitization of G protein-coupled receptors have been elucidated in part using the beta2-adrenergic receptor as a prototype. It has been shown that at least three different phenomena underlie the desensitization process (reviewed in (1) ). (i) Following activation, the receptor is phosphorylated by either a beta2-adrenergic receptor kinase which belongs to the G protein receptor kinase family (2) or by the cAMP-dependent protein kinase A (termed heterologous phosphorylation) as a consequence of receptor-mediated stimulation of cAMP formation. In the case of receptors coupled to phospholipase C activation, the kinase implicated in heterologous phosphorylation is protein kinase C. Receptor phosphorylation is a rapid event, since it can be detected as early as 1 min after agonist treatment. Phosphorylated receptors are subsequently recognized by an arrestin-like protein (1) which binds to the receptor and inhibits G protein interaction. (ii) The receptor is internalized/sequestered in an intracellular compartment where it is inaccessible to ligand. This phenomenon also occurs within minutes to hours of agonist exposure and induces an impairment of receptor G protein coupling. Recently, a consensus sequence for sequestration of the beta2-adrenergic receptor has been identified(3) . This sequence, which encompasses a tyrosine residue highly conserved among members of the G protein-coupled receptor family, is located in close proximity to the seventh transmembrane domain and is present in both the 5-HT(2) and thrombin receptors. Recent observations of Yu et al.(4) demonstrate that serine and threonine residues in the carboxyl-terminal tail of the beta2-adrenergic receptor are important for sequestration. In fact, the sequestration pathway has been proposed to be an important mechanism by which cells re-establish their normal responsiveness to agonists following removal of the stimulus(4, 5) . (iii) Finally, after prolonged agonist exposure, the receptor is down-regulated, a phenomenon which leads to a dramatic decrease in the total cellular receptor level. Although the mechanisms which result in down-regulation are not precisely defined, the presence of tyrosine residues in the carboxyl-terminal tail appears to be necessary.

To study the desensitization process of G protein-coupled receptors involved in mitogenic signaling, we selected two receptors, 5-HT(2) and thrombin receptors, which have been shown to stimulate growth in a line of hamster fibroblasts (CCL39 cells) (for review, see (6) ). cDNA clones corresponding to both receptors have previously been isolated in the laboratory. In CCL39 cells, the 5-HT(2) receptor is coupled to the activation of phospholipase C via a G(q) protein(7, 8) . The thrombin receptor is coupled to both activation of phospholipase C and inhibition of adenylate cyclase, implicating at least two G proteins (G(q) and G(i))(9, 10, 11, 12) .

It is known from previous reports that the 5-HT(2) receptor undergoes ligand-induced desensitization in P11 cells and transfected Swiss 3T3 cells(13, 14) . However, in these studies, only long term desensitization was addressed since the effect of 5-HT was examined from 1 to 8 h following agonist addition. In the present study, we have examined rapid ligand-dependent 5-HT(2) receptor desensitization. In vivo labeling experiments were performed to determine the possible role of phosphorylation in desensitization of this receptor. Further, the role of the receptor carboxyl-terminal extension was analyzed using a truncated receptor mutant.

Thrombin is a serine protease with multiple cellular effects, including growth stimulation in CCL39 hamster fibroblasts, reviewed in Refs. 15 and 16. Thrombin activates its receptor by a mechanism which involves cleavage of the amino-terminal ectodomain of the receptor to expose a new amino-terminal sequence which functions as a built-in ligand(17) . This irreversible modification of the receptor by thrombin is accompanied by rapid attenuation of signaling in cells. In CCL39 fibroblasts, it has previously been demonstrated that thrombin induces homologous desensitization of phosphoinositide breakdown(9) . Studies on the premegakaryocytic HEL cell line indicate that desensitization of the receptor involves both proteolysis of the receptor and phosphorylation(18) . More recently, it has been shown that rapid internalization and recycling of thrombin receptors occurs in HEL cells subsequent to stimulation by thrombin or thrombin receptor peptide agonist. Interestingly, the recycled receptors are refractory to thrombin since they are already cleaved(19, 20) . Finally, Ishii et al.(21) demonstrated that the inhibition of thrombin receptor signaling can be induced by co-expression of the receptor and the beta-adrenergic receptor kinase 2 in a Xenopus oocyte expression system(21) . They further showed that removal of the thrombin receptor carboxyl-terminal tail, containing several potential serine and threonine phosphorylation sites, prevented betaadrenergic receptor kinase 2-mediated desensitization of the thrombin-stimulated Ca response.

The present studies were designed to explore the distinct regulatory mechanisms that exist between different serpentine receptors. Whereas thrombin receptor desensitization is rapid and nearly complete, desensitization of the 5-HT(2) receptor is slower, and only 50% of the response is lost. We show for the first time in a thrombin-responsive cell line that ligand-dependent desensitization of the thrombin receptor correlates well with ligand-induced phosphorylation of the receptor. By contrast, phosphorylation of the 5-HT(2) is not stimulated by ligand treatment. Rather, loss of 5-HT responsiveness parallels receptor sequestration. Finally, since the last 72 amino acids of the 5-HT(2) receptor are not required for G protein coupling or desensitization, we used a chimeric receptor composed of the truncated 5-HT(2) receptor and the cytoplasmic tail of the thrombin receptor to examine the role of the thrombin receptor cytoplasmic tail in the desensitization process.


EXPERIMENTAL PROCEDURES

Materials

5-HT was purchased from Sigma and human alpha-thrombin (3209 NIH units/mg) was kindly provided by Dr. J. W. Fenton II (New York State Department of Health, Albany, NY). The thrombin receptor peptide corresponding to the hamster receptor sequence (SFFLRNP) was synthesized by NEOSYSTEM (Strasbourg, France). Triton X-100 was from Pierce and phorbol 12,13-dibutyrate (PDBu), (^1)from Sigma. [P] orthophosphate and myo-[2-^3H]inositol were from Amersham (les Ulis, France). 12Ca5 monoclonal antibody directed against influenza virus hemagglutinin (HA) epitope (22) was a gift of Dr. M. Levis (University of California, San Francisco). The P4D5 monoclonal antibody directed against an epitope of the vesicular stomatitis virus glycoprotein (23) was kindly provided by Dr. B. Goud (Institut Pasteur, Paris, France). Biotinylated rabbit anti-mouse IgG and strepavidin-conjugated phycoerythrin were from Immunotech (Marseille, France).

Plasmids

The plasmid encoding the Chinese hamster 5-HT(2)R (peSR) described previously (8) corresponds to a 3.5 kilobase cDNA encoding the 5-HT(2)R subcloned in the EcoRI site of the pECE expression vector. The epitope-tagged pHA-5-HT(2)R was constructed by exchanging the cDNA sequence corresponding to the 5`-noncoding region and 11 NH(2)-terminal amino acids of the 5-HT(2)R, with a sequence coding for 11 residues (including the initiation methionine) of the HA epitope. The HA-5-HT(2)R cDNA was subcloned in the pcDNAneo expression vector (Invitrogen). pHA-5HT(2)RDelta1244 which encodes a truncated HA-tagged 5-HT(2)R was constructed by deleting the sequence corresponding to the last 72 residues and 3`-noncoding region of pHA-5-HT(2)R. pTRV, encoding the epitope-tagged human thrombin receptor, was constructed by subcloning the 5`-noncoding region and the entire coding sequence of the human thrombin receptor cDNA (17) (kindly provided by Dr. S. R. Coughlin, University of California, San Francisco) in the pRK5 expression vector (24) . A sequence encoding the VSVG epitope tag was inserted in-frame at the 3` extremity of the thrombin receptor cDNA. pHA-5-HT(2)/Thr-VSVG R, encoding a chimeric 5-HT(2)/Thr receptor was constructed by cloning the cDNA encoding the cytoplasmic tail of the epitope-tagged thrombin receptor (residues 374 to 427) in-frame with the HA-5-HT(2) Delta1244 receptor.

Cells and Culture Conditions

PS200 cells represent a Na/H antiporter-deficient variant of CCL39 Chinese hamster lung fibroblasts. These cells were chosen for the present studies since they have an undetectable level of 5-HT(2)R expression, as described(8) . Nontransfected PS200 cells and stably transfected subclones (5-HT(2) cells which express the 5-HT(2)R, HA-5-HT(2) cells which express the HA epitope-tagged receptor, HA-5-HT(2)Delta1244 cells which express the truncated 5-HT(2)R, and HA-5-HT(2)/Thr-VSVG cells which express the chimeric receptor) were cultivated in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 7.5% fetal calf serum, penicillin (50 units/ml), and streptomycin (50 µg/ml). Human embryonic kidney 293 cells and cells stably transfected with the VSVG epitope-tagged human thrombin receptor (293TRV2J cells) were cultivated in the same medium containing 8% heat-inactivated fetal calf serum.

Cell Transfection and Selection

To obtain clones stably expressing receptors, PS200 cells (1 times 10^6 cells/10-cm dish) were transfected 24 h after plating with 25 µg of plasmid DNA (in the presence of 2.5 µg of pcDNAneo when the pRK5 expression vector was used) according to a modified calcium phosphate precipitation procedure(25) . Clones resistant to G418 (400 µg/ml) were selected after 2-3 weeks and screened for their ability to increase phosphoinositide breakdown in response to 5-HT or thrombin. Transient transfection of 293 cells (3 times 10^6 cells/10-cm dish or 5 times 10^5 cells/well of 6-well plates) was performed by using 25 µg or 8 µg of plasmid DNA.

Phosphoinositol Lipid Breakdown

Inositol phosphate formation was measured on cells grown to confluence in 12-well plates and rendered quiescent by serum deprivation in Dulbecco's modified Eagle's medium for 24 h in the presence of myo[2-^3H]inositol (2 µCi/ml, Amersham). For assays, cells were incubated for 4 min in 20 mM LiCl, and agonists were added for the indicated times. The reaction was stopped by rapid aspiration of the medium and addition of 10 mM formic acid; accumulation of total inositol phosphates was measured following anion exchange chromatography and liquid scintillation counting, as described(26) .

In Vivo Labeling and Immunoprecipitation

Immunoprecipitation of the 5-HT(2) Receptor

48 h after transfection of the pHA-5-HT(2)R, pHA-5-HT(2)RDelta1244, or pHA-5-HT(2)/Thr-VSVG plasmids, 293 cells were washed and incubated in phosphate-free Dulbecco's modified Eagle's medium containing 200 µCi/ml [P]orthophosphate for 3.5 h. At the end of the labeling period, agonist was added for 10 min. Cells were then washed with ice-cold PBS and lysed in 600 µl of Triton X-100 lysis buffer (50 mM Tris/HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 200 µM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 4 µg/ml leupeptin, 1 µg/ml aprotinin, and 1% Triton X-100) for 20 min at 4 °C. The lysates were incubated for 45 min at 4 °C with 2 µl of nonimmune mouse serum coupled to Protein A-Sepharose, then clarified by centrifugation at 12,000 times g for 5 min. Precleared lysates were incubated for 2 h at 4 °C with 12Ca5 antibody coupled to Protein A-Sepharose. After five washes in lysis buffer, two washes in high salt buffer (lysis buffer containing 0.5 M LiCl), two washes in low salt buffer (lysis buffer without NaCl), and two additional washes in lysis buffer, 50 µl of Laemmli buffer containing 2 M urea was added to the pellet and samples were analyzed by SDS-PAGE in 10% acrylamide gels.

Immunoprecipitation of the Thrombin Receptor

293 cells stably expressing the thrombin receptor were plated on 6-well plates. After reaching confluence, cells were labeled for 3.5 h in phosphate-free Dulbecco's modified Eagle's medium containing 200 µCi/ml [P]orthophosphate, treated with thrombin for 10 min, and lysed in 300 µl of Triton X-100 lysis buffer, and immunoprecipitation was performed as described above with 5 µl of anti-VSVG antibody.

Western Blot Analysis

Transfected 293 cells were solubilized in Triton X-100 lysis buffer for 20 min at 4 °C and clarified as described above. Clarified lysates were incubated for 1 h at 4 °C with wheat germ lectin-Sepharose 6MB (Pharmacia, Uppsala, Sweden), then washed four times in lysis buffer. Wheat germ-enriched fractions were resuspended in Laemmli sample buffer containing 2 M urea and separated by SDS-PAGE on a 10% polyacrylamide gel. Proteins were electrophoretically transferred to Hybond-C membrane (Amersham) and immunoprobed as described previously(27) .

Cell Surface Staining and Flow Cytometric Analysis on Transfected PS200 Cells

Cell monolayers were detached in PBS and treated with serotonin (10M) for the indicated times. For triple staining, cells (1 times 10^7/ml) were first labeled with a saturating concentration of anti-HA 12Ca5 antibody (1:3000 dilution) at 4 °C for 30 min in 100 µl of PBS containing 0.1% bovine serum albumin. Following one wash in PBS, 0.1% bovine serum albumin, cells were labeled with biotinylated rabbit anti-mouse IgG for 30 min, then incubated with strepavidin-conjugated phycoerythrin (1:100 dilution) for an additional 30 min. After a final wash, cells were fixed in PBS, 0.3% formol buffer. Analyses were performed by flow cytometry with a dual laser Facs Star Plus (Beckton Dickinson). Side and forward scatter of blue laser was used to gate out debris and damaged cells.


RESULTS

Since specific anti-receptor antibodies are required to assess the role of phosphorylation in desensitization and antibodies which immunoprecipitate 5-HT(2) and thrombin receptors were not available, we added an epitope tag to each receptor sequence. In the case of the 5-HT(2) receptor, the amino-terminal 11 amino acids of the receptor were exchanged with 11 residues encoding an immunodominant peptide from influenza hemagglutinin (HA). For the thrombin receptor, we added an epitope tag corresponding to a sequence from the vesicular stomatitis virus glycoprotein to the carboxyl terminus of the full-length receptor. This position was chosen for two reasons. First, a signal peptide sequence is present at the extreme amino terminus of the receptor sequence. Second, and more important, the receptor is cleaved after residue Arg-41 by thrombin upon activation. Schematic representations of the HA epitope-tagged 5-HT(2) receptor, the VSVG epitope-tagged thrombin receptor, and the chimeric 5-HT(2)/Thr receptor, which encodes both epitopes, are shown in Fig. 1. The residues in the cytoplasmic tails of the receptors which are potential phosphorylation sites for G protein receptor kinases are indicated. Consensus phosphorylation sites for PKC are also present in both receptors. One putative PKC phosphorylation site is present in the third intracellular loop of the thrombin receptor, and five sites were identified in the 5-HT(2) receptor by inspection of the sequence (two in the third intracellular loop and three in the carboxyl tail). No consensus protein kinase A phosphorylation sites exist in the 5-HT(2) receptor sequence, whereas two are present in the third intracellular loop of the thrombin receptor(28) .


Figure 1: Representation of the epitope-tagged 5-HT(2), thrombin, and chimeric 5-HT(2)/Thr receptors. The PstI restriction site was used to construct the truncated 5-HT(2) receptor. Potential phosphorylation sites for G protein receptor kinases (circles) and protein kinase C (triangles) are indicated. Potential sites of palmitoylation are shown.



It has been reported for many G protein-coupled receptors, including the thrombin receptor(21) , that the carboxyl-terminal receptor tail is involved in the desensitization process. To elucidate the role of the 5-HT(2) receptor's cytoplasmic tail in mediating this response, we have constructed a truncated receptor (using a PstI restriction enzyme site in the cDNA denoted in Fig. 1) in which 72 out of 86 carboxyl-terminal amino acids were removed. In this mutant, the potential G protein-coupled receptor kinase phosphorylation sites present in the cytoplasmic tail of the receptor and three of the PKC consensus sites are eliminated; two possible PKC sites remain in the intracellular loops.

Epitope-tagged 5-HT(2) Receptors Stimulate Phospholipase C Activity in a Manner Similar to Wild Type Receptors in Stably Transfected Cells

In order to compare the desensitization of thrombin and 5-HT(2) receptors in the same system, cell lines which display an equivalent biological response to their respective ligands were constructed. To do this, we used the PS200 subclone of CCL39 fibroblasts which expresses a high endogenous level of thrombin receptors, as determined by the robust response of cells to thrombin and elevated receptor mRNA levels. The 5-HT(2) receptor is not expressed at detectable levels in these cells(8) . Therefore, we transfected PS200 cells with either the wild type 5-HT(2) receptor (5-HT(2)R), the epitope-tagged 5-HT(2) receptor (HA-5-HT(2)R), or the truncated epitope-tagged 5-HT(2) receptor (HA-5-HT(2)RDelta1244), and stably transfected clones were analyzed for their expression of the different 5-HT(2) receptors by measuring phospholipase C activation. Clones with similar responses to 5-HT were selected for further studies. As shown in Fig. 2A, 5-HT activates phospholipase C in a dose-dependent manner in each of the selected clones. Addition of the HA tag did not significantly alter the pharmacological properties of the receptor, as determined by EC values for 5-HT in each of the transfected clones. Estimated values are 2.1 times 10, 1.5 times 10, and 1.3 times 10M for the 5-HT(2)R, HA-5-HT(2)R, and HA-5-HT(2)RDelta1244 transfected clones, respectively. Further, these data indicate that truncation of the receptor cytoplasmic tail does not affect the ability of the receptor to stimulate phospholipase C activity. Thus, the last 72 amino acids of the 5-HT(2) receptor do not appear to be critical for coupling of the receptor to the G(q) protein. Although similar levels of agonist-stimulated inositol phosphate formation were observed in all three clones, differences in receptor/G protein coupling efficiencies between the tagged full-length and truncated receptors could exist. Therefore, the level of tagged receptors expressed at the surface of the clones was examined by performing flow cytometric analyses using the anti-HA antibodies. Results of these experiments are shown in Fig. 2B. Nontransfected PS200 cells and PS200 cells transfected with the 5-HT(2) receptor (without the HA tag) were used as negative controls since they are not recognized by the 12Ca5 antibody. The intensity of fluorescence is 5-fold higher in HA-5-HT(2)R and HA-5-HT(2)RDelta1244 cells than in control cells (mean at 150 arbitrary units instead of 30), indicating that the mature receptor protein is present on their surface. Furthermore, the peaks of fluorescence observed for the two lines expressing tagged receptors are nearly superimposable, indicating that they express a comparable number of receptors.


Figure 2: Functional expression of the 5-HT(2) and epitope-tagged 5-HT(2) receptors in PS200 cells. A, PS200 cells were transfected with plasmids encoding the 5-HT(2)R (circle), HA-5-HT(2)R (box), and HA-5-HT(2)RDelta1244 (Delta) receptors. The dose-dependent activation of phospholipase C by 5-HT in each of the transfected clones is presented. Values are expressed as a percent of maximal stimulation. Minimal and maximal values are 500-4468, 663-10,353, and 636-13,260 cpm for 5-HT(2)R (circle), HA-5-HT(2)R (box), and HA-5-HT(2)RDelta1244 (up triangle), respectively. Error bars reflect the variation between duplicate determinations of inositol phosphate formation over the 10-min period of stimulation. B, flow cytometric analysis of cell surface staining with anti-HA antibodies. Nontransfected (PS200) and transfected cells are shown. The fluorescence intensity determined for 3000 cells is represented as a function of cell number. Mean fluorescence values are 4.2 for PS200 and 5-HT(2)R cells, 13.1 for HA-5-HT(2)RDelta1244 cells, and 16.1 for HA-5-HT(2)R cells.



5-HT- and Thrombin-induced Desensitization of Their Respective Receptors Follows a Different Time Course

Agonist-dependent desensitization of the transfected 5-HT(2) receptor constructs was analyzed by comparing the effect of 5-HT on phosphoinositide breakdown in each of the transfected cell lines pretreated for various times with the ligand. Fig. 3A shows that 5-HT induces a similar time course of desensitization (T = 10 min) in each of the three clones. After 1 h of 5-HT treatment, approximately 50% of the response is lost. It is important to note that similar results are obtained with the endogenous 5-HT(2) receptor in parental CCL39 cells (data not shown). As expected, addition of the HA tag to the amino terminus of the receptor does not alter its ability to activate phospholipase C. All three clones display similar rates of desensitization as well. The fact that we did not detect a notable difference in the time course of desensitization between the wild type and the truncated receptors suggests that the last 72 amino acids of the 5-HT(2) receptor are not implicated in this process.


Figure 3: Desensitization rates of agonist-induced phosphoinositide breakdown in transfected cells. A, quiescent [^3H]inositol-labeled cells stably expressing the indicated receptor were stimulated with 5-HT (10M) for various times, then 20 mM LiCl was added and total inositol phosphate formation was determined over a 10-min period. Values are expressed as a percentage of maximal stimulation. Minimal and maximal values are 225-1810, 356-3672, and 347-1939 for 5-HT(2)R (circle), HA-5-HT(2)R (), and HA-5-HT(2)RDelta1244 (up triangle), respectively. B, quiescent 5-HT(2)R transfected cells were pretreated with 1 unit/ml thrombin (Thr) for the indicated times. Then, 20 mM LiCl was added, and total inositol phosphates were measured after 10 min as described under ``Experimental Procedures.'' Error bars reflect variation between duplicate determinations.



Thrombin receptor desensitization was assessed by measuring phospholipase C activity following different times of thrombin treatment in the clone 5-HT(2)R which expresses the wild type 5-HT(2) receptor. As shown in Fig. 3B, the ability of the thrombin receptor to stimulate phospholipase C is rapidly attenuated following receptor activation (T = 3 min). After 1 h of thrombin pretreatment, more than 80% of the response is lost. These results are consistent with previously reported studies of thrombin-induced desensitization of phosphoinositide breakdown carried out in parental CCL39 cells(9) . It is noteworthy that the thrombin receptor peptide agonist induces receptor desensitization with the same time course, and to the same extent, as thrombin thereby indicating that receptor cleavage is not responsible for this effect(11) . Thus, we show that, whereas desensitization of thrombin receptor is rapid and extensive, desensitization of the 5-HT(2) receptor in the same cells displays a markedly slower time course and reduced maximal effect. The difference in desensitization of two G protein-coupled receptors which activate at least one common effector suggests the presence of different regulatory mechanisms.

Protein Kinase C Is Not Implicated in Receptor Desensitization

Activation of phospholipase C triggers the formation of two second messengers, inositol(1, 4, 5) -trisphosphate and diacylglycerol, which in turn stimulate cytoplasmic Ca release and activate PKC, respectively. As mentioned above, both 5-HT(2) and thrombin receptors possess PKC consensus phosphorylation sites, which render them susceptible to modulation by the kinase. Since the 5-HT(2) receptor displays five PKC consensus sequences, we analyzed the effect of PKC on 5-HT(2) receptor desensitization. To do so, PKC was either inhibited by the addition of a specific inhibitor, GF 109203X(29) , or by down-regulation of the kinase following a 24-h pretreatment with phorbol 12,13-dibutyrate (PDBu). The effect of the PKC inhibitor on 5-HT-induced receptor desensitization in HA-5-HT(2)R or HA-5-HT(2)RDelta1244 transfectants is shown in Fig. 4, A and B, respectively. GF 109203X failed to significantly alter either the rate of desensitization or the extent of the response determined after 1 h of 5-HT pretreatment. Similarly, down-regulation of PKC (Fig. 4) had little or no detectable effect on desensitization of the 5-HT(2) receptor in these cells. Therefore, we conclude that desensitization of the 5-HT(2) receptor is independent of PKC. This proposal is further supported by the fact that we did not detect a difference in the time course of receptor desensitization between the wild type and the truncated receptor, in which the three PKC consensus sites present in the cytoplasmic tail are missing.


Figure 4: Comparison of the rate of desensitization of agonist-induced phosphoinositide breakdown in pretreated or nontreated transfected PS200 cells. A, HA-5-HT(2) cells; B, HA-5-HT(2)Delta1244 cells. Quiescent [^3H]inositol-labeled cells were pretreated with the GF 109203X compound, 5 min before agonist addition (open triangles in A and open circles in B) or with 100 ng/ml PDBu for 24 h before agonist addition (filled symbols). Then, 20 mM LiCl was added and production of total inositol phosphates was determined after 10 min. Points represent the mean of duplicate values.



The role of PKC in thrombin-induced desensitization in CCL39 cells has been addressed previously in the laboratory. It was concluded from these studies that desensitization of polyphosphoinositide breakdown induced by thrombin is independent of PKC(9) . The absence of a PKC effect on thrombin receptor desensitization in CCL39 cells has also been observed using the GF 109203X inhibitor in similar experiments as described above. (^2)

The finding that PKC is not a major contributor to 5-HT(2) or thrombin receptor desensitization is further confirmed by the absence of heterologous desensitization of these two receptors which both activate PKC (9) and results not shown.

Phosphorylation of 5-HT(2) and Thrombin Receptors

An increasing body of evidence indicates that G protein receptor kinases play an important role in the desensitization process. To determine whether receptor phosphorylation might be involved in the desensitization of 5-HT(2) and thrombin receptors, we examined agonist-dependent phosphorylation of these two receptors. Initial attempts to detect phosphorylation of tagged 5-HT(2) receptors or thrombin receptors in the PS200 subclones described above were unsuccessful. Therefore, in vivo phosphorylation experiments were carried out in 293 cells transfected, either transiently or stably, with expression plasmids encoding the various receptors. The level of receptor expression obtained in these cells is considerably higher than that observed in PS200 cells.

As shown in Fig. 5A (left panel), expression of both the full-length and truncated 5-HT(2) receptors can be detected in 293 cells by Western blot analysis following transient transfection. The HA-5-HT(2) receptor migrates as a band of approximately 80,000 Da whereas the truncated receptor migrates with an apparent molecular mass of 50,000 Da. We also observe the presence of higher molecular mass bands in immunoblots of the truncated or full-length tagged receptors, which may correspond to dimeric and trimeric forms, as judged by their size. The formation of these larger molecular species presumably occurs following lysis of the cells, and not in intact cells, since the intensity of their labeling increases with prolonged incubation of cell lysates with Sepharose beads.


Figure 5: A, Expression of the transfected 5-HT(2) and 5-HT(2)Delta1244 receptors in transfected cells. Left, 293 cells transiently transfected with 8 µg of vector alone or 5-HT(2) receptor constructs were lysed in 300 µl of Triton X-100 lysis buffer, and wheat germ-enriched fractions were subjected to immunoblotting as described under ``Experimental Procedures.'' Right, 100 µl of P-labeled cell lysates were purified on wheat germ-Sepharose and subjected to immunoblotting analysis. B, phosphorylation of 5-HT(2) receptors. 293 cells transiently transfected with 5-HT(2) receptor constructs were labeled with [P]orthophosphate (200 µCi/ml) for 3.5 h, and 10M 5-HT was added during the last 10 min. The cells were lysed and immunoprecipitated with 12Ca5 antibody coupled to Protein A-Sepharose. Immune complexes were washed and proteins were analyzed by SDS-PAGE in 10% acrylamide gels. Positions of molecular mass markers are shown.



For phosphorylation experiments following transient transfection of 293 cells, the receptor level in each P-labeled cell lysate was followed by Western analysis (Fig. 5A, right panel). It can be seen for the experiment presented in Fig. 5that the same quantity of receptor was present in lysates from control and 5-HT-stimulated cells. The effect of 5-HT on phosphorylation of tagged-5-HT(2) receptors is shown in Fig. 5B. Basal phosphorylation of the wild type 5-HT(2) receptor was observed in some experiments; however, no significant increase in phosphorylation of the tagged-5-HT(2) receptor could be detected following a 10-min incubation of cells with 5-HT. Additional time points also failed to reveal 5-HT-stimulated phosphorylation of the receptor (data not shown). We did detect a labeled band of approximately 80,000 Da in cell lysates; however, we don't believe that this band corresponds to the phosphorylated 5-HT(2) receptor since it is also present in cells which express the truncated receptor form. Concerning the truncated receptor, we were unable to detect its phosphorylation in either control or stimulated cells, despite its presence in the labeled cell lysates (Fig. 5A, right). These results suggest that the residues of the wild type receptor which are phosphorylated in nonstimulated cells are located carboxyl to the site of truncation. Since the T for desensitization of phosphoinositide breakdown is 10 min, and no stimulation of receptor phosphorylation could be detected within 10 min of 5-HT treatment, it appears that receptor phosphorylation is not a key mechanism involved in desensitization of the 5-HT(2) receptor.

To analyze phosphorylation of the thrombin receptor, experiments were carried out in both transiently transfected 293 cells and in 293 cells stably expressing the epitope-tagged thrombin receptor, designated 293TRV2J. (^3)Both stably and transiently transfected cells express high levels of thrombin receptor which can be immunoprecipitated with the anti-VSVG antibody. The thrombin receptor migrates as a broad band with an apparent molecular weight ranging from 62,000-72,000 following cleavage with thrombin, and 72,000-86,000 when cells are stimulated with thrombin receptor agonist peptide or PDBu (Fig. 6). Whereas we were unable to detect receptor phosphorylation in nontreated cells, thrombin treatment was found to stimulate receptor phosphorylation, as shown in Fig. 6. Phosphorylation was observed as early as 1 min following thrombin addition. Therefore, phosphorylation does correlate well with desensitization of the thrombin receptor since similar time courses are observed for both phosphorylation and desensitization of the phospholipase C response.


Figure 6: Phosphorylation of the epitope-tagged thrombin receptor. Stably transfected 293 cells which express the thrombin receptor (TRV2J cells) were labeled with 200 µCi/ml [P]orthophosphate for 3.5 h. At the end of the labeling incubation, thrombin (1 unit/ml), thrombin receptor peptide agonist (TRP) (30 µM), or PDBu (100 ng/ml) was added for 10 min. Cells were lysed and immunoprecipitated with P4D5 antibody coupled to Protein A-Sepharose. Immune complexes were washed and proteins were analyzed by SDS-PAGE in 10% acrylamide gels. Brackets on the right side of the autoradiogram indicate the migration of the receptor from thrombin receptor peptide agonist/PDBu (upper) or Thr (lower) stimulated cells. Positions of molecular mass markers are shown.



Cytoplasmic Extension of the Thrombin Receptor Determines the Desensitization Rate

To determine whether the sequence encoded by the cytoplasmic tail of the thrombin receptor is involved in rapid receptor desensitization, we constructed a chimeric receptor in which the cytoplasmic tail of the 5-HT(2) receptor was replaced by the cytoplasmic tail of the thrombin receptor (see Fig. 1). This mutant receptor was stably expressed in PS200 cells, and a representative clone was chosen for analysis. In this clone, 5-HT stimulates phosphoinositide breakdown to the same extent as in HA-5-HT(2) and HA-5-HT(2)Delta1244 cells. First we evaluated the agonist-dependent desensitization of this receptor. It can been seen from Fig. 7that the time course of desensitization of the chimeric receptor is similar to the time course observed for the thrombin receptor. Thus, sequences present in the carboxyl-terminal region of the thrombin receptor are capable of modifying the rate of 5-HT(2) receptor desensitization. When the phosphorylated state of the chimeric receptor (74,000 Da) was analyzed, we found that the level of receptor phosphorylation is elevated in response to 5-HT (Fig. 8), although stimulation of phosphorylation was lower than that observed with the thrombin receptor. Since all the potential phosphorylation sites present in the carboxyl extension of the thrombin receptor are present in the chimeric receptor, yet the ligand-induced phosphorylation is less marked, it is possible that either the conformation of the chimeric receptor does not lead to phosphorylation of all the residues. In addition, there may be residues present in the cytoplasmic loop of the thrombin receptor which are phosphorylated upon receptor stimulation. Nevertheless, the appearance of ligand-stimulated phosphorylation of the chimeric receptor correlates well with its rapid desensitization in the functional assay.


Figure 7: Desensitization rates of agonist-induced phosphoinositide breakdown in 5-HT(2)/Thr transfected cells. Quiescent [^3H]inositol-labeled cells stably expressing the chimeric receptor were stimulated with 5-HT (10M) for various times, then 20 mM LiCl was added and total inositol phosphate formation was determined over a 10-min period. Values are expressed as percentage of maximal stimulation. Minimal and maximal values are 86-445 cpm. Points represent the mean of four independent experiments.




Figure 8: Phosphorylation of the chimeric 5-HT(2)/Thr receptor. 293 cells transiently transfected with the chimeric 5-HT(2)/Thr receptor construct were labeled with [P]orthophosphate (200 µCi/ml) for 3.5 h and 10M 5-HT was added during the last 10 min. The cells were lysed and immunoprecipitated with 12Ca5 antibody coupled to Protein A-Sepharose. Immune complexes were washed as described under ``Experimental Procedures,'' and proteins were analyzed by SDS-PAGE in 10% acrylamide gels. Positions of molecular mass markers are shown.



Sequestration of 5-HT(2) Receptors

In addition to phosphorylation, sequestration is involved in the desensitization of several seven-transmembrane domain receptors. Since the 5-HT(2) receptor is not phosphorylated in response to agonist, we decided to examine whether agonist-induced sequestration of the receptor occurs. Flow cytometric analysis was used to answer this question. As shown in Table 1, 5-HT induces a disappearance of both wild type and truncated 5-HT(2) receptors from the cell surface. After 1 h of 5-HT treatment, 50% of receptors are internalized. This sequestration/internalization is abolished when the incubation is performed at 4 °C. Thus, the time course of receptor sequestration correlates with the time course of desensitization. In the case of the thrombin receptor, immunofluorescence studies in CCL39 cells have revealed that rapid redistribution and internalization of the receptor occurs rapidly (<5 min) upon treatment of cells with thrombin or thrombin receptor peptide agonist. (^4)Similar findings in other cell systems have been reported in detail(19) .




DISCUSSION

The study of G protein receptors and their role in growth stimulation is an area of intense investigation. Although the precise events leading to DNA synthesis stimulation by this family of receptors remain to be elucidated, certain G protein-coupled signaling pathways, notably one or more G(i)-mediated pathways(7, 30, 31) , have been implicated in mitogenic stimulation of cells by a number of different receptors. The CCL39 hamster fibroblast line has proven to be a particularly useful model for these studies (for reviews see Refs. 32 and 33) since these cells express receptors for 5-HT and thrombin coupled to both G(i) and G(q) proteins. cDNAs encoding both 5-HT(2) and thrombin receptors have been cloned from CCL39 cells(34, 35) , and the intracellular signaling pathways activated by their respective ligands have been extensively characterized. It has been proposed that the duration of signal generation by growth factors is a key element in determining cell cycle re-entry(27, 36, 37) ; therefore, it is necessary to characterize the attenuation of cellular responses as well as their activation. In the present paper we have focused on the desensitization of 5-HT and thrombin receptors in an attempt to identify the molecular mechanisms involved in this phenomenon. We found that both the rate and extent of desensitization of these two receptors is quite different. Whereas rapid ligand-induced desensitization of the thrombin receptor correlates well with receptor phosphorylation, the 5-HT(2) receptor does not undergo ligand-induced phosphorylation. The relatively slow loss of 5-HT responsiveness occurs concomitantly with receptor disappearance from the cell surface.

Inspection of the primary sequence of these two receptors indicates that the thrombin receptor may be a good substrate for G protein receptor kinases, since several serine/threonine residues in an acidic environment are present in its cytoplasmic tail(38) . In addition, one consensus phosphorylation site for PKC can be identified in the third cytoplasmic loop of the thrombin receptor(28) . By contrast, the cytoplasmic tail of the 5-HT(2) receptor contains individual rather than clustered serine/threonine residues and thus is not such a good substrate for G protein receptor kinases. However, at least five PKC phosphorylation sites (K/RXXS*/T* or K/RXS*/T* (39) can be found in the 5-HT(2) receptor, including three in the cytoplasmic extension.

Analysis of ligand-stimulated phosphorylation of these receptors revealed that the thrombin receptor is rapidly phosphorylated in response to either thrombin, the thrombin receptor agonist peptide, or the phorbol ester, PDBu. These results obtained on fibroblasts are in agreement with a recent study by Ishii et al.(21) , who also reported that the G protein receptor kinase beta-adrenergic receptor kinase 2 is capable of blunting thrombin receptor signaling when expressed together with the receptor in Xenopus oocytes. The analysis of receptor mutants lacking serine and threonine residues in the intracellular loops or in the cytoplasmic tail led the authors to conclude that the kinase inhibits receptor signaling by phosphorylating serine/threonine residues in the carboxyl tail, rather than in the intracellular loops, as expected by the localization of putative G protein receptor kinase consensus sequences.

In the case of the 5-HT(2) receptor, we were not able to detect any increase in phosphorylation following stimulation with 5-HT. The same results were obtained with the truncated 5-HT(2) receptor in which the potential phosphorylation sites present in the cytoplasmic tail have been removed. These findings support the proposal that the 5-HT(2) receptor is a poor G protein receptor kinase substrate and, therefore, is not subject to short-term desensitization mediated by a member of this kinase family. Indeed, recent studies confirm the presence of G protein receptor kinase phosphorylation sites in G protein-coupled receptors that display rapid agonist-dependent phosphorylation and functional desensitization. For example, the beta2-adrenergic receptor, which contains these sites, is phosphorylated and desensitized following exposure to agonist, whereas the beta3-adrenergic receptor is not(40) . Further, a chimeric receptor with the core of the beta3-adrenergic receptor and the cytoplasmic tail of the beta2 receptor is phosphorylated in response to agonist and can undergo desensitization. In the present study, we demonstrate that the carboxyl tail of the thrombin receptor, which is a G protein receptor kinase substrate, can signal rapid desensitization of a chimeric 5-HT(2) receptor. More detailed mutagenesis analyses should help to further define residues involved in this function.

The phorbol ester PDBu is also able to induce thrombin receptor phosphorylation, albeit with a slower time course and a lower maximal effect than that induced by thrombin ( Fig. 6and (21) ). The functional consequence of PDBu-stimulated receptor phosphorylation is not clear in light of the findings in CCL39-derived fibroblasts that PKC activation has no significant effect on desensitization at the receptor level. Rather, inhibitory effects of the phorbol ester analog on phosphatidylinositol turnover stimulated by thrombin and 5-HT (not shown and (9) ) are presumably due to downstream inhibition of phospholipase C by the kinase. Tobin and Nahorski (41) have reported that the M3 muscarinic receptor is phosphorylated in response to 12-O-tetradecanoylphorbol 13-acetate. However, a PKC inhibitor blocks this phosphorylation without affecting carbachol-stimulated receptor phosphorylation(41) . Thus, receptors coupled to phospholipase C activation may be substrates of PKC; however, the role of PKC in the regulation of each of these receptors remains to be elucidated. PKCmediated desensitization of 5-HT(2) receptors was addressed in the present study since this receptor displays five potential PKC phosphorylation consensus sites. We found in our CCL39-derived clones that PKC inhibition did not change the time course of 5-HT(2) receptor desensitization. It has been hypothesized that stimulation of PKC is responsible for acute desensitization of the 5-HT response in platelets(42) . The authors of that report proposed that the PKC activation may desensitize the 5-HT-stimulated IP(1) accumulation and Ca mobilization based on results obtained using a PKC inhibitor (H7 at 300 µM). However, it is not clear whether PKC activation acts at the receptor level or rather distal to the receptor. Since blocking PKC activation in our system (with a specific inhibitor and by down-regulation) was without effect on receptor desensitization and, since we were unable to detect phosphorylation of the 5-HT(2) receptor upon agonist-induced PKC stimulation, we believe that PKC is not involved in desensitization of the 5-HT(2) receptor. Consistent with this, we did not observe any difference in ligand-induced desensitization of the truncated receptor in which three of the five PKC phosphorylation consensus sites were removed.

Even though the 5-HT(2) receptor is not phosphorylated in response to 5-HT, we did observe receptor internalization by flow cytometric analysis. Since this procedure does not distinguish between rapid receptor sequestration, presumably occurring at or near the cell surface, and translocation of receptors to intracellular compartments, we use the term internalization for both processes. A recent report describes a consensus sequence for internalization (NPXXY) of beta2-adrenergic receptors which is present in nearly all G protein-coupled receptors(3) . Mutation of the tyrosine residue completely abolishes agonist-mediated sequestration without affecting G protein coupling, receptor phosphorylation, or down-regulation. Interestingly, this sequence is present in both the thrombin and the 5-HT(2) receptors, as well as in the truncated 5-HT(2) receptor mutant which internalizes as efficiently as the full-length receptor. The role of this tyrosine-containing sequence in sequestration of these two receptors awaits evaluation by mutational analysis. In addition to the presence of this sequence, many reports indicate the importance of serine/threonine residues in either the third intracellular loop or in the receptor's cytoplasmic tail for internalization(4, 43, 44) .

Strikingly different time courses of receptor internalization are displayed by 5-HT(2) and thrombin receptors. In CCL39 cells, the thrombin receptor undergoes very rapid surface redistribution (leq5 min) and nearly complete internalization within minutes (leq15 min) of agonist treatment. (^5)Similar findings have been reported in other cells(19) . Disappearance of the 5-HT(2) receptor occurs more slowly, with maximal effects corresponding to approximately 50% receptor loss, observed 30 min following agonist addition. Many reports indicate that sequestration is an obligatory step for receptor recycling. This could be the case for the serotoninergic receptor, but not for the thrombin receptor, since, following cleavage, de novo receptor synthesis is required for its recognition by thrombin and subsequent activation.

Experiments with the chimeric receptor described in this study have allowed us to clearly demonstrate that the carboxyl-terminal extension of the thrombin receptor is a substrate for one or more protein kinases involved in desensitization. Sequences within this tail appear to be selective, since Ishii et al.(21) have reported that beta-adrenergic receptor kinase 2 (considerably more efficiently than beta-adrenergic receptor kinase 1) blocks thrombin receptor signaling. The 5-HT(2)/Thr receptor should prove to be useful in identifying the specific G protein receptor kinases which mediate in vivo regulation of thrombin receptors, since its use circumvents the problem of endogenous thrombin receptor activation. Further studies should help to define regulatory sites of the receptor involved in recognition of the appropriate kinases.


FOOTNOTES

*
These studies were supported by the Centre National de la Recherche Scientifique (UMR 134), the Institut National de la Santé et de la Recherche Médicale, and the Association pour la Recherche contre le Cancer. 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.

(^1)
The abbreviations used are: PDBu, phorbol 12,13-dibutyrate; HA, hemagglutinin; VSVG, vesicular stomatitis virus; PBS, phosphatebuffered saline; PAGE, polyacrylamide gel electrophoresis; PKC, protein kinase C.

(^2)
F. McKenzie, personal communication.

(^3)
V. Vouret-Craviari, D. Grall, J.-C. Chambard, V. B. Rasmussen, J. Pouysségur, and E. Van Obberghen-Schilling, manuscript in preparation.

(^4)
E. Van Obberghen-Schilling, unpublished observations.

(^5)
D. Grall and E. Van Obberghen-Schilling, unpublished observations.


ACKNOWLEDGEMENTS

We thank D. Grall for expert assistance and C. Cibre for the photographic work. Dr. F. McKenzie is gratefully acknowledged for helpful discussions of this work and critical reading of the manuscript. We thank Dr. J. P. Breittmayer for performing the cytometric analyses.


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