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Institut de Geeneetique et de Biologie Moleeculaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Santee et de la Recherche Meedicale, Universitee L. Pasteur de Strasbourg, Illkirch, France (M.D., A.J., S.G., L.M.)
IFR6, Services de Biochimie et d'Angio-Heematologie, Hpital Lariboisieere, Paris, France (P.M., J.C., L.D., J.-M.L.)
Abstract
Although potentially implicated in several physiological functions, few functional mutations have been identified in the human 5-hydroxytryptamine (HT)2B receptor gene. A heterozygous mutation R393X in the 5-HT2B receptor was recently identified in a patient diagnosed with pulmonary hypertension after intake of the anorexigenic dexfenfluramine. Although reported to generate a lack of function, this C terminus-truncated 5-HT2B receptor should somehow affect transduction pathways relevant to pulmonary hypertension. In our study, we investigated putative modifications in transduction of the R393X-mutated 5-HT2B receptor. In stably transfected cells, we confirmed the loss of inositol 1,4,5-trisphosphate stimulation caused by the Gq uncoupling, despite conserved ligand affinity between the normal and mutated receptors. We also observed a partial loss of nitric-oxide synthase stimulation. However, the truncated R393X receptor presented 1) a strong gain of efficacy in cell proliferation as assessed by mitogen-activated protein kinase activity and thymidine incorporation, 2) a preferential coupling to G13 as shown by blocking antiserum, and 3) an apparent lack of internalization upon agonist stimulation as observed by confocal microscopy. This work demonstrates that, in the 5-HT2B receptor, the C terminus, including the palmitoylation and phosphorylation sites, is absolutely required for proper transduction and internalization. For the first time, we show that the lack of C terminus can generate a switch of coupling to G13, a reduced NO synthase activation, and an increase in cell proliferation. All these modifications are relevant in pathophysiological vasoconstriction.
Knowledge of 5-HT2B receptor genetic variants is of the utmost interest in view of the involvement of these receptors in various pathologies (Roth and Shapiro, 2001). Few studies have investigated mutations in the 5-HT2B receptor gene (MIM 601122 ). It was identified as a candidate gene in obsessive-compulsive disorder: one single nucleotide polymorphism was described in intron 1, but no evidence for functional mutation was found in the coding regions of 5-HT2B receptor (Kim et al., 2000). More recently, by investigating the 5-HT2B receptor gene in patients who developed pulmonary hypertension after intake of fenfluramines, a heterozygous mutation was found in one female who 5 years before followed a 9-month anorexigenic treatment (Blanpain et al., 2003). The reported mutation consists of a C/T transition resulting in the substitution of a stop codon instead of Arg393 of the predicted protein sequence (R393X). This mutation truncates the complete carboxy-terminal tail of the receptor, removing 1) putative palmitoylation and phosphorylation sites essential for the internalization process (Parker et al., 2003), and 2) the PSD-95, Dlg, ZO-1 (PDZ) binding motif involved in the coupling of nitric-oxide synthase (NOS) (Manivet et al., 2000) or other scaffold proteins (Becamel et al., 2004).
The 5-HT2B receptor transduction pathways may greatly depend on cell types, but cell transfection seems to be an indispensable tool to solve the complexity of 5-HT2B receptor-coupling mechanisms. Blanpain et al. (2003) described, by transient transfection, that despite normal expression at the plasma membrane and normal agonist binding, this R393X mutation causes loss of 5-HT2B receptor function as measured by IP3 production and calcium-dependent aequorin luminescence. In this study, the mutated receptor displayed no constitutive activity compared with the wild-type receptor, and it did not produce dominant negative effects over the IP3/Ca2+ pathway in cotransfection experiments.
Noteworthy, coupling of 5-HT2B receptors to IP3 stimulation has never been observed in vivo despite its contractile effects in rat stomach (Cox and Cohen, 1995) or vasculature (Ellis et al., 1995), or its proliferative effect in carcinoid tumor cells (Launay et al., 1996). Moreover, 5-HT2B receptor can trigger IP3-independent Ca2+ release from ryanodine-sensitive intracellular stocks in lung endothelial cells (Ullmer et al., 1996). Most mutations in G protein-coupled receptor (GPCR) leading to loss of function are autosomal recessive, whereas most disease caused by GPCR as gain of function mutations are inherited in an autosomal dominant manner (Spiegel and Weinstein, 2004). If the heterozygous R393X 5-HT2B receptor mutation is related to the pulmonary hypertension, it must trigger transduction alterations relevant to this pathology.
Previous studies established that, in addition to its role in phosphoinositide hydrolysis, the 5-HT2B receptor can transactivate growth factor receptors that control mitogen-activated protein kinase (MAPK) signaling and cell cycle progression (Launay et al., 1996; Nebigil et al., 2000). The 5-HT2B receptor protein is overexpressed in lung arteries of pulmonary hypertension patients as well as in mice chronically submitted to hypoxia, a model that shares several features with pulmonary hypertension (Launay et al., 2002). The 5-HT2B receptor is required for smooth muscle cell proliferation and structural remodeling of arteries, consistent with the ability of 5-HT2B receptor to initiate cell cycle progression in mouse fibroblasts (Nebigil et al., 2000). The 5-HT2B receptor can also elicit a reversible endothelium-dependent relaxation of pulmonary arteries, associated with an increase in cyclic GMP (Glusa and Pertz, 2000), through coupling to endothelial NOS. The 5-HT2B receptor-dependent NOS activation requires the PDZ motif at the C terminus (Manivet et al., 2000). Because the R393X mutation abolishes IP3 release and removes the C terminus PDZ motif, Blanpain et al. (2003) suggested that the mutated receptor had lost its Gq coupling and probably its ability to stimulate NOS. Besides endothelium-dependent vasodilatation, the 5-HT2B receptor could participate in pulmonary arterial smooth muscle cells proliferation or remodeling (Launay et al., 2002). If this 5-HT2B receptor-myoproliferative effect is relevant to smooth muscle cells in pulmonary hypertension patients, we hypothesize that it should be affected by the mutated R393X allele. In this study, we therefore investigated a putative gain of function of the R393X 5-HT2B receptor and showed that despite its loss of IP3 stimulation as a result of Gq uncoupling and its loss of endothelial NOS stimulation because of the absence of the PDZ binding domain, this receptor retains some NO coupling and presents higher efficacy for cell proliferation via a nearly exclusive G13 coupling. In addition to the switch in coupling pathways, R393X 5-HT2B receptor showed no apparent internalization upon agonist stimulation.
Materials and Methods
Reagents. Neurochemicals were provided by Roche (Basel, Switzerland), Eli Lilly (Indianapolis, IN), and GlaxoSmithKline (Welwyn Garden City, Hertfordshire, UK) companies (Table 1 for chemical makeup of compound numbers). All other chemicals were reagent grade and purchased from commercial sources. The radioactive compound (6)-[125I]1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl ([125I]DOI; 81.4 TBq/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Rabbit antisera against rodent Gs (C-18), Gq/11 (C-19), and G13 (A-20) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Ten concentrations of each competing drug were used. Reported values are the means of four independent experiments performed in triplicates and are expressed in nanomolar or percentage of wild type (Emax) ± S.E.M. IP3 levels were determined 5 min, NOS activities 10 min, and MAPK activities 20 min after agonists addition.
Mutagenesis. The different constructs of the human wild-type 5-HT2B and truncated 5-HT2B receptor cDNA were performed by polymerase chain reaction. The mutated reading frames were subcloned into enhanced green fluorescent protein variants, pEYFP fused at the N terminus to generate wild-type 5-HT2B receptor-pEYFP and R393X 5-HT2B receptor-pEYFP. All the constructs were verified by sequencing before transfection.
Cell Culture. LMTK- cell lines were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. For transfection experiments, cells were seeded at a density of 1.5 x 106 cells/10-mm dish and cultured for 24 h. Transient transfection was then performed using Jet Pei according the manufacturer's recommendations. The cells were then cultured in the same medium for 48 h. The stable expressing human 5-HT2B receptor and R393X 5-HT2B receptor were grown with 0.8 mg/ml G418 (Geneticin).
Confocal Imaging. LMTK- cells expressing wild-type 5-HT2B receptor-pEYFP or R393X 5-HT2B receptor-pEYFP were grown in a glass-bottomed dish and observed in serum-free medium at room temperature. To visualize the receptor internalization movement after agonist treatment, time-lapse series were taken every 5 min over a 30-min period. To calculate the internalization kinetics of the receptors, we selected more than five regions of interest on the membrane per cell and followed the relative intensity changes of these regions over time, with three individual cells per experiment. Our data represent more than four independent experiments. The kinetic curves were corrected for the bleaching by using the intensity of the whole cell as a normalization factor.
Radioligand Binding Experiments. Radioligand binding experiments were performed as detailed previously (Tournois et al., 1998). Stable LMTK- clones expressed 178 and 128 fmol of 5-HT2B receptor and R393X 5-HT2B receptor/mg protein, respectively, as measured by [125I]DOI binding.
Determination of Intracellular Inositol 1,4,5-Trisphosphate Levels. Cells were washed twice with fresh serum-free medium, incubated for 5 min at 37°C with agonists, and scraped with a rubber policeman. Intracellular IP3 levels were then measured by radioreceptor assay as detailed previously (Tournois et al., 1998). The Emax value for serotonin of the wild-type receptor was 120 pmol/mg protein.
NOS Assay. Cells were incubated with agonists for 10 min. Cell lysate supernatant proteins were assayed for NOS activity by monitoring the conversion of 3H-labeled L-arginine to 3H-labeled L-citrulline as described previously (Manivet et al., 2000). The Emax value for serotonin of the wild-type receptor was 16 pmol/mg protein/min.
Assay of MAPK Activity. Cells were incubated with agonists for 20 min. Cell lysate supernatant proteins were assayed for kinase activity in reaction buffer containing 1 e藽i of [-32P]ATP, 20 e ATP, and 1.5 mg/ml myelin basic protein (Sigma-Aldrich, St. Louis, MO) for 20 min at 30°C, essentially as described previously (Launay et al., 1996). The Emax value for serotonin of the wild-type receptor was 1.15 pmol/mg of protein/10 min.
Thymidine Incorporation. Cells were seeded on 24-well plates at a density of 103 cells per well, grown overnight, and serum-starved for 24 h. Quiescent cells were treated with serotonin at different concentrations for 18 h, and 0.5 e藽i (1 Ci = 37 GBq) of [3H]thymidine was added to the culture during the last 4 h of incubation. The free radioactive thymidine was washed away in 5% trichloroacetic acid, and the incorporated radioactive thymidine was quantified by scintillation counting as described previously (Nebigil et al., 2000).
Cell Permeabilization. The cells were washed twice with phosphate-buffered saline containing 0.1% bovine serum albumin and exposed to 1 hemolytic unit of alveolysin/106 cells at 37°C under agitation as described previously (Manivet et al., 2000).
Statistics. Binding data were analyzed using the iterative non-linear fitting software GraphPad Prism 2.0 (GraphPad Software Inc., San Diego, CA). This allowed to calculate inhibition constants (Ki) according to the Cheng-Prusoff equation (Cheng and Prusoff, 1973). All values represent average of independent experiments ± S.E.M. (n is number of experiments as indicated in the text). Comparisons between groups were performed using Student's unpaired t test or analysis of variance and a Fisher test. Significance was set at P < 0.05.
Results
Because it was not possible to study the mutated 5-HT2B receptor R393X in patients' native tissues, we used stable recombinant expression systems. The two human 5-HT2B receptors cDNA, wild type and R393X, were stably transfected into LMTK- cells, thus ensuring they were studied in an identical cell background. Under the same experimental paradigm, parental nontransfected cells did not exhibit a concentration-dependent rise in second messengers after agonist stimulation. Single clones were chosen for each receptor according their relatively low expression to be as close as possible to physiological levels. Moreover, cells were used at nearly similar passages to ensure stable expression levels. Classic pharmacological binding studies clearly confirmed that the R393X mutation had no significant effect on the binding profile of either agonists or antagonists (Table 1). We also confirmed that the IP3 coupling was lost by this mutated receptor (Table 1). However, because NO coupling and cell proliferation may be more physiologically relevant signaling pathways for this receptor, we asked whether the R393X mutation had differential effect on these two pathways.
Pharmacological values obtained for R393X 5-HT2B receptor versus wild-type receptor clearly demonstrate that EC50 values for NO coupling were increased for all agonists (Fig. 1; Table 1; supplemental figure). For some ligands such as serotonin, the EC50 value was increased by a factor 10. The maximal levels of NOS activation (Emax) were all reduced. The Emax value for serotonin was reduced by 40%, with serotonin behaving as a partial agonist. The Emax value for DOI changed from more than 40% of that of serotonin for wild-type receptor to 14% for the mutant receptor, with DOI becoming a nearly silent agonist (Table 1). Most KB values for antagonists were increased (Table 1; supplemental figure). These results indicate a partial loss of NO coupling by the R393X receptor.
In contrast, most EC50 values for the coupling to MAPK activation were decreased. BW 723C86 (a selective 5-HT2B receptor agonist) showed a 7-fold decreased EC50 value. It is interesting that the Emax value of R393X 5-HT2B receptor for serotonin was increased by 27% over the wild-type value, and the Emax value for the active metabolite of dexfenfluramine, norDF, changed from 73 to 86% of that of serotonin for the wild-type receptor (Fig. 1; Table 1). For antagonists, most KB values toward MAPK activation were increased, by 20-fold for ritanserin, for example (Fig. 1; supplemental figure). These results indicate a clear gain of efficacy in MAPK stimulation by the R393X receptor.
Because previous studies have indicated that 5-HT2B receptor-dependent MAPK activation can led to cell proliferation and/or survival, we performed a proliferation assay on both cell lines expressing either wild-type or R393X 5-HT2B receptors. According to these studies, agonist activation of 5-HT2B receptors triggered cell proliferation as assessed by tritiated thymidine incorporation (Fig. 2). In agreement to the pharmacological study on MAPK activation, the cell proliferation triggered by stimulation of the R393X-mutated 5-HT2B receptor was strikingly increased (3-fold at 1 e serotonin), and the stimulation by 100 nM serotonin of this mutated receptor was similar to that of the wild-type receptor by 1 e serotonin (Fig. 3A). Basal thymidine incorporation was also elevated in R393X 5-HT2B receptor (1.7 ± 0.3-fold basal wild type; n = 8). These results indicate a clear gain of efficacy in thymidine incorporation and thus of cell proliferation for R393X 5-HT2B receptor.
We further investigated this gain of cell proliferation using blocking antibodies to various G proteins on alveolysin-permeabilized living cells. We observed that the thymidine incorporation was controlled by both Gq and G13 in the wild-type receptor-expressing cells. Indeed, anti-Gs antiserum was inactive in the same assay. By contrast, the thymidine incorporation seemed almost entirely dependent on G13, but not anymore on Gq (or Gs) in R393X 5-HT2B receptor-expressing cells (Fig. 3B). These results suggest that the gain of efficacy to cell proliferation by the R393X 5-HT2B receptor is subsequent to a change in G protein coupling to G13.
Finally, a previous report indicated that upon agonist stimulation, 5-HT2B receptor is quickly desensitized (Porter et al., 2001), a phenomenon that likely relies, at least in part, on phosphorylation of residues in the C terminus of GPCRs. We therefore compared the wild-type and the R393X 5-HT2B receptor-pEYFP for internalization upon stimulation by 1 e serotonin by confocal time-lapse videomicroscopy. Stimulation of the 5-HT2B receptor wild-type-expressing cells led to a rapid internalization indicated by the quick disappearance of receptors from the plasma membrane with a half-life of 4. 7 ± 0.9 min (n = 7). For R393X 5-HT2B receptor-expressing cells, no significant internalization was detected with half-life longer than 60 min (Fig. 4; supplemental movies). These results indicate that the absence the C terminus-truncated receptor is either partially retained intracellularly or constitutively internalized.
Discussion
Using stably transfected cell lines expressing human cDNAs encoding wild-type and R393X 5-HT2B receptors, we first confirmed the initial Blanpain et al. (2003) results that the R393X 5-HT2B receptor has kept the same binding affinity for all tested compounds but lost its coupling to Gq and to IP3. We then validated their hypothesis that endothelial NOS coupling is lost in the R393X 5-HT2B receptor. Both of these findings are loss of function.
Moreover, we demonstrate here, for the first time, that the lack of C-terminal tail containing the palmitoylation site in the R393X 5-HT2B receptor is associated with a loss of rapid internalization as shown by confocal analysis of yellow fluorescent protein-tagged receptors. In addition, the R393X 5-HT2B receptor retains its capacity to stimulate NOS activity despite lower efficacy. Furthermore, the R393X 5-HT2B receptor has acquired a striking increase in proliferative capacity, as indicated by two independent criteria, an increase in MAPK Emax value and in thymidine incorporation levels. These latter data seem to be the consequence of a switch from a dual Gq/G13 coupling in wild-type receptor to a nearly exclusive coupling to G13 in the R393X 5-HT2B receptor as shown by blocking antibodies. This switch to G13 coupling and cell proliferation is clear gain of functions for this mutant receptor in transduction pathways, which is relevant for vascular proliferation and remodeling.
In mouse LMTK- fibroblasts stably transfected with the full-length cDNA encoding the 5-HT2B receptor, a serotonin-dependent NO coupling mediated by endothelial and inducible NOS has been observed. The endothelial NOS stimulation is PDZ-dependent, whereas the inducible NOS is controlled by G13 (Manivet et al., 2000). In transfected cells expressing a partial C terminus-truncated form of the 5-HT2B receptor (K403X), DOI-dependent IP3 coupling was retained (Manivet et al., 2000). Combined with our data, this clearly indicates that the Cys397 is required for the formation of the VIII helix and identifies this helix as responsible for the Gq association, because it is destroyed by the absence of the palmitoylation site but retained in the K403X 5-HT2B receptor, as documented previously by structural studies of the rhodopsin/transducin interactions (Sakmar et al., 2002).
A nearly complete lack of apparent internalization was observed in R393X 5-HT2B receptor by time-lapse video confocal microscopy and thus of desensitization that is especially relevant to chronic diseases. This result indicates that the truncated receptor is either partially retained intracellularly or constitutively internalized. Multi-PDZ domain-containing protein 1, a putative scaffolding protein with 13 PDZ domains, was identified as a 5-HT2CR-interacting protein (Ullmer et al., 1998). 5-HT2CR interaction with multi-PDZ domain-containing protein 1 is dynamically regulated by phosphorylation at Ser458 (Parker et al., 2003). Likewise, PSD-95 was shown to modulate 5-HT2AR's activity, intracellular trafficking, and distribution (Xia et al., 2003), and agonist-mediated desensitization of 5-HT2ARs requires the presence of S421 in the C-terminal tail (Gray et al., 2003). In the R393X 5-HT2B receptor, the truncation of most of the carboxy-terminal tail of the receptor removes putative phosphorylation sites and thus PDZ-dependent intracellular trafficking motifs, likely explaining the absence of internalization.
Besides the phosphorylation by second messenger-activated kinases, GPCRs can be also phosphorylated by GPCR kinases, which lead to recruitment of the arrestin proteins. Recent discoveries indicate that the -arrestins play wide-spread roles as scaffolds and/or adapter molecules that organize a variety of complex signaling pathways emanating from GPCRs (Miller and Lefkowitz, 2001). G protein-independent, -arrestin-dependent activation of MAPK has been described previously (Azzi et al., 2003). It was found that inverse agonists for Gs-activated adenylate cyclase could be positive partial agonists for -arrestin-dependent MAPK activation. These authors suggested that multiple active conformations of a receptor may activate different types of effectors such as G proteins and -arrestin (Lefkowitz and Whalen, 2004). Although binding affinities are not affected by the absence of C terminus in R393X 5-HT2B receptors, the efficacy of compounds is differentially affected depending on the transduction pathways. The partial agonist DOI becomes a silent agonist toward NOS activity in the mutant receptor, whereas the partial agonist norDF becomes a full agonist toward MAPK activation. These results clearly reinforce the notion that active conformation of GPCRs differs between different couplings and thus agonist efficacy must be dependent on cellular G protein plethora.
Introduction of anti-G13 antibodies in permeabilized cells systematically blocked the 5-HT2B receptor-mediated inducible NOS activation (Manivet et al., 2000). Our present study also provides evidence that the subunit of the G13 protein plays a critical role in the mechanisms of 5-HT2B receptor/NOS coupling. G13 has been reported to activate inducible NOS through a mechanism distinct from other G chains (Kitamura et al., 1996). The Src family kinase (other effectors of HT2BRs) (Nebigil et al., 2000) acts upstream of Rho activation for the G12/13-induced c-Jun NH2-terminal kinase activation (Nagao et al., 1999). The small G protein RhoA and its effector Rho kinase play a major role in the sustained rise in tension induced by vasoconstrictors. Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation, possibly by contributing to both vasoconstriction and vascular remodeling (Fagan et al., 2004). The gain in G13 coupling of the R393X 5-HT2B receptor seems thus relevant to pathological vasoconstriction and remodeling.
Recent work on endothelin B receptor, another Gq-coupled receptor implicated in pulmonary vasoconstriction, showed that although the three intracellular loops of this receptor are involved in coupling to G proteins, residues in the first intracellular loop are specifically required for activation of G13 (Liu and Wu, 2003). It is interesting that these authors have also reported that the deletion of endothelin B receptor C terminus up to residue Cys405, a myristoylation site, increased response of serum response element-mediated transcription to endothelin 1 in both wild-type and Gq/11-mutated fibroblasts. Their conclusion that G13 transduces signals from GPCRs to stimulate growth-promoting pathways via the serum response element seems similar to the coupling of the R393X 5-HT2B receptor to cell proliferation.
A recent report showed that lung tissue samples from several patients with pulmonary hypertension present evidence of infection with Kaposi's sarcoma herpes virus (human herpes virus 8; HHV8), suggesting a pathogenetic role in primary pulmonary hypertension (Cool et al., 2003). The HHV8 open reading frame 74 encodes a constitutively active GPCR for chemokines that leads to cell transformation and vascular overgrowth by constitutive activation of nuclear factor-B (NF-B) and involves activation of G13 and RhoA (Shepard et al., 2001). Mouse lung endothelial cell model in which HHV8-GPCR is stably expressed stimulates phosphatidylinositol 3-kinase and activates NF-B (Couty et al., 2001). Because we previously reported that 5-HT2B receptor via phosphatidylinositol-3 kinase/Akt can activate NF-B (Nebigil et al., 2003), it is tempting to speculate that HHV8-GPCR and 5-HT2B receptor use common G13-dependent pathways leading to vascular remodeling.
Disruption of the association between PDZ proteins and their targets contributes to the pathogenesis of a number of human diseases, most likely because of the failure of PDZ proteins to appropriately target and modulate the actions of the associated proteins. Our results not only confirm the importance of C-terminal tail of GPCR for both desensitization and coupling but also highlight its importance for G protein repertoire selectivity.
Acknowledgements
We thank P. Hickel for excellent technical assistance, Dr. S. Brooks for English corrections, and Drs. F. Mentree and F. Godmard for statistical expertise.
This work has been supported by funds from the Centre National de la Recherche Scientifique, the Institut National de la Santee et de la Recherche Meedicale, the Hpital Universitaire de Strasbourg, the Universitee Louis Pasteur, and by grants from the Fondation de France, the Fondation pour la Recherche Meedicale, the Association pour la Recherche contre le Cancer, the French ministry of research (Actions Concerteees Incitatives), and the European Community.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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