The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization
2006; Springer Nature; Volume: 25; Issue: 13 Linguagem: Inglês
10.1038/sj.emboj.7601193
ISSN1460-2075
AutoresAngélique Levoye, Julie Dam, Mohammed Akli Ayoub, Jean‐Luc Guillaume, Cyril Couturier, Philippe Delagrange, Ralf Jockers,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoArticle15 June 2006free access The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization Angélique Levoye Angélique Levoye Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, France Search for more papers by this author Julie Dam Julie Dam Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, France Search for more papers by this author Mohammed A Ayoub Mohammed A Ayoub Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, FrancePresent address: Institut de Génomique Fonctionnelle, Département de Pharmacologie Moléculaire, CNRS-UMR 5203, INSERM U 661, Universités Montpellier 1 et 2, Montpellier, France Search for more papers by this author Jean-Luc Guillaume Jean-Luc Guillaume Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, France Search for more papers by this author Cyril Couturier Cyril Couturier Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, FrancePresent address: CNRS UMR 8090, Institut de Biologie de Lille—Institut Pasteur de Lille, Lille, France Search for more papers by this author Philippe Delagrange Philippe Delagrange Institut de Recherches SERVIER, Suresnes, France Search for more papers by this author Ralf Jockers Corresponding Author Ralf Jockers Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, France Search for more papers by this author Angélique Levoye Angélique Levoye Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, France Search for more papers by this author Julie Dam Julie Dam Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, France Search for more papers by this author Mohammed A Ayoub Mohammed A Ayoub Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, FrancePresent address: Institut de Génomique Fonctionnelle, Département de Pharmacologie Moléculaire, CNRS-UMR 5203, INSERM U 661, Universités Montpellier 1 et 2, Montpellier, France Search for more papers by this author Jean-Luc Guillaume Jean-Luc Guillaume Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, France Search for more papers by this author Cyril Couturier Cyril Couturier Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, FrancePresent address: CNRS UMR 8090, Institut de Biologie de Lille—Institut Pasteur de Lille, Lille, France Search for more papers by this author Philippe Delagrange Philippe Delagrange Institut de Recherches SERVIER, Suresnes, France Search for more papers by this author Ralf Jockers Corresponding Author Ralf Jockers Department of Cell Biology, Institut Cochin, Paris, France Inserm U567, Paris, France CNRS, UMR 8104, Paris, France Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, France Search for more papers by this author Author Information Angélique Levoye1,2,3,4, Julie Dam1,2,3,4, Mohammed A Ayoub1,2,3,4, Jean-Luc Guillaume1,2,3,4, Cyril Couturier1,2,3,4, Philippe Delagrange5 and Ralf Jockers 1,2,3,4 1Department of Cell Biology, Institut Cochin, Paris, France 2Inserm U567, Paris, France 3CNRS, UMR 8104, Paris, France 4Université Paris 5, Faculté de Médecine René Descartes, UM 3, Paris, France 5Institut de Recherches SERVIER, Suresnes, France *Corresponding author. Institut Cochin, INSERM U567, CNRS 8104, Department of Cell Biology, 22 rue Méchain, Paris 75014, France. Tel.: +33 1 40 51 64 34; Fax: +33 1 40 51 64 30; E-mail: [email protected] The EMBO Journal (2006)25:3012-3023https://doi.org/10.1038/sj.emboj.7601193 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info One-third of the ∼400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT1 and MT2 melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT2 did not modify MT2 function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT1 protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT1 without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT1. Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs. Introduction Although the formation of functional protein complexes via dimerization or oligomerization is a common theme in biology, G protein-coupled receptors (GPCRs) were assumed to exist as monomers for many years since the heterologous expression of a single GPCR was usually sufficient to produce the expected pharmacology and function. However, biochemical and structural data accumulated over the last 15 years indicate that most, if not all, GPCRs exist as functional dimers or higher oligomeric units (Bouvier, 2001; Milligan, 2004). GPCR dimerization includes the formation of homodimers (between two identical receptor protomers) and heterodimers (between two protomers of different receptors). GPCR heterodimerization has been reported for more than 40 receptor combinations (Prinster et al, 2005) and in most of these cases heterodimerization had a marked effect on GPCR function. Some GPCRs function as obligatory heterodimers as shown for the GABAB and taste (T1R1−3) receptors. Other GPCRs may form heterodimers allowing for mutual regulation (trafficking, desensitization) between the two specific promoters within the heterodimer as observed in most of the known cases. Finally, heterodimerization may profoundly modify the pharmacological properties of the receptor. Recent genome sequencing projects indicated that approximately 370 sequences belong to the nonodorant GPCR family in the human genome (Joost and Methner, 2002; Fredriksson et al, 2003; Vassilatis et al, 2003). More than 220 of these receptors have been matched with known ligands. However, over 140 receptors (≈40%) still remain as orphans despite the vast and long-standing effort of academic and industrial research to pair these receptors to potential ligands (Vassilatis et al, 2003). Whereas a considerable amount of data has been accumulated on the homo- and heterodimerization of GPCRs with known ligands, nothing is known about the dimerization of orphan receptors. Heterodimerization between orphan and nonorphan GPCRs opens the interesting possibility that orphan receptors regulate the ligand binding, signaling and/or trafficking of GPCRs with known ligands. We specifically addressed this issue by studying the dimerization of the orphan X-linked GPR50 receptor whose function is unknown (Reppert et al, 1996; Drew et al, 1998; Drew et al, 2001). A deletion mutant of GPR50 has been recently shown to be genetically associated with mental diseases such as bipolar affective disorder and major depressive disorder (Thomson et al, 2005). Moreover, some GPR50 variants are associated with higher triglyceride levels and lower HDL-cholesterol levels (Bhattacharyya et al, 2006). We show that GPR50 homodimerizes and forms heterodimers with MT1 and MT2 melatonin receptors that share the highest sequence homology with GPR50 among all GPCRs. Importantly, MT1 loses its ability to bind to melatonin receptor-specific agonists and to couple to G proteins when engaged into GPR50/MT1 heterodimers. Our results shed light on a previously unappreciated role of orphan receptors in the regulation of nonorphan GPCRs with known function. Results Homo- and heterodimerisation of GPR50 The formation of GPR50 homodimers was first explored in Western blot experiments. In HEK 293 cell extracts expressing a fusion protein between GPR50 and the yellow fluorescent protein (YFP) (GPR50-YFP), anti-GFP antibodies detected two immunoreactive species with apparent molecular weights of approximately 90 and 180 kDa, likely corresponding to the monomeric and dimeric form of GPR50-YFP (Figure 1A). To confirm GPR50 homodimerization, we performed co-immunoprecipitation experiments with differentially tagged receptors. When immunoprecipitating GPR50-YFP, co-precipitated Flag-GPR50 was revealed in SDS–PAGE as a monomer at 70 kDa and as a SDS-resistant dimer at 140 kDa (Figure 1B). Figure 1.Detection of GPR50 homo- and heterodimers by SDS–PAGE and co-immunoprecipitation. (A) Lysates from HEK 293 cells stably expressing Flag-GPR50-YFP were separated by SDS–PAGE and analysis was performed by Western blot using an anti-GFP antibody. (B–D) Crude membranes were prepared from HEK 293 cells transiently expressing GPR50-YFP alone or with Flag-GPR50 (B), Flag-MT1 (C) or Myc-MT2 (D). GPR50-YFP was immunoprecipitated with a monoclonal anti-GFP antibody. Membranes and immnunoprecipitates were then separated by SDS–PAGE and analysis was performed by Western blot using polyclonal anti-Flag (B,C) or anti-Myc (D) antibodies. Similar results were obtained in three additional experiments. mb=membrane; IP=immunoprecipitation, M=monomer; D=dimer. Download figure Download PowerPoint GPR50 belongs to the melatonin receptor GPCR subfamily that in humans comprises two further subtypes, MT1 and MT2 (Reppert et al, 1996). To explore GPR50 heterodimerization with MT1 and MT2, GPR50-YFP was coexpressed with the N-terminally Flag-tagged MT1 and Myc-tagged MT2. Anti-GFP antibodies were indeed able to specifically pull down MT1 and MT2 proteins (Figure 1C and D). Formation of MT1/GPR50 heterodimers was further supported by direct Western blot experiments (see Supplementary Figure 1). Taken together, Western blot and co-immunoprecipitation experiments suggest that GPR50 form homo- and heterodimers. To exclude possible artefacts associated with co-immunoprecipitation of membrane-bound receptors solubilized in detergent, we studied GPR50 dimerization in intact cells using the Bioluminescence Resonance Energy Transfer (BRET) approach. The GPR50-Rluc construct was used as BRET donor and was coexpressed with the BRET acceptor YFP fused to the C-terminus of different receptors (Figure 2A). Significant energy transfer was observed in cells coexpressing GPR50-Rluc with similar amounts of GPR50-YFP, MT1-YFP or MT2-YFP. These BRET signals were comparable to those obtained for the MT1 homodimer expressed at similar levels. Stimulation of cells with melatonin did not modify the basal BRET signals (not shown). The specificity of the assay was illustrated by the absence of significant transfer between GPR50-Rluc and YFP fusion proteins of control GPCRs (β2-adrenergic receptor (β2-AR) and CCR5) expressed in similar amounts to those of melatonin receptor YFP fusions. Taken together, BRET experiments show that GPR50 is engaged into constitutive homo- and heterodimeric complexes with MT1 and MT2 in intact cells. Figure 2.Constitutive dimerization of GPR50 in living HEK 293 cells. (A) GPR50-Rluc was transiently coexpressed in HEK 293 cells with the indicated C-terminal YFP fusion proteins expressed at comparable amounts as determined by direct fluorescence measurements (20–30 fmol of YFP fusion receptor per mg of protein as estimated from curves correlating YFP fluorescence with the number of ligand-binding sites (Ayoub et al, 2002)). BRET signals generated for the MT1 homodimer (MT1-Rluc/MT1-YFP), when expressed at comparable amounts, were used as internal control. BRET measurements were performed in living cells by adding 5 μM coelenterazine. Data are means±s.e.m. of at least three independent experiments each performed in duplicate. (B–D) BRET donor saturation curves were generated by transfecting transiently HEK 293 cells with a constant DNA amount of GPR50-Rluc and increasing quantities of the indicated YFP-tagged receptors. The BRET, total luminescence and total fluorescence were measured. The curves represent 3–5 individual saturation curves. Curves obtained for the BRET acceptors GPR50-YFP, MT1-YFP and MT2-YFP were best fitted with a nonlinear regression equation assuming a single binding site, those obtained for β2-AR-YFP and CCR5-YFP were best fitted with a linear regression equation. Download figure Download PowerPoint The relative propensity of GPR50 to form homodimers or to engage into heterodimers with MT1 or MT2 was then studied with the recently developed BRET donor saturation assay (Mercier et al, 2002; Couturier and Jockers, 2003; Ramsay et al, 2004). Cells were cotransfected with constant amounts of the BRET donor (receptor fused to Rluc) and increasing quantities of the BRET acceptor (receptor fused to YFP). In this assay, the amount of acceptor required to obtain the half-maximal BRET (BRET50) for a given amount of donor reflects the relative affinity of the two partners (Mercier et al, 2002). Specific BRET signals increased as a hyperbolic function and reached an asymptote with increasing acceptor/donor ratios. Comparable BRET50 values of 0.36±0.08, 0.35±0.08 and 0.41±0.06 (n=3–5) were obtained for GPR50 homodimers, GPR50/MT1 heterodimers and GPR50/MT2 heterodimers, respectively (Figure 2B–D), indicating that the propensity of GPR50 heterodimerization with MT1 and MT2 is similar to that of GPR50 homodimerization. For control receptors (β2-AR and CCR5), BRET signals increased linearly and were not saturable as expected for nonspecific interactions. Consequences of GPR50 heterodimerization on ligand binding to MT1 and MT2 To assess the functional consequences of GPR50 heterodimerization with MT1 and MT2, we generated a HEK 293 cell line stably expressing the GPR50-YFP fusion protein (HEK-GPR50). Incubation of these cells with a saturating concentration of 2(125I)-iodomelatonin (125I-MLT) for MT1 and MT2 confirmed that GPR50 has no apparent affinity for this radioligand as previously reported (Reppert et al, 1996) (Figure 3A). The effect of GPR50 on 125I-MLT binding to MT1 and MT2 was studied by expressing MT1-Rluc and MT2-Rluc fusion proteins in HEK-GPR50 cells and wild-type HEK 293 cells (HEK-wt). Despite the presence of similar quantities of MT1-Rluc in both cell lines, as determined by luminescence measurements, the number of 125I-MLT-binding sites was decreased by more than 50% in HEK-GPR50 cells (Figure 3A). No such effect was observed when comparable quantities of MT2-Rluc were expressed (Figure 3B). Hence, GPR50 specifically decreases 125I-MLT binding to MT1 but not to MT2. In addition, decreased 125I-MLT binding is unlikely to be due to receptor relocalization into intracellular compartments as the lipophilic 125I-MLT easily penetrates cell membranes. To further investigate this point, GPR50-YFP was transfected in a HEK 293 cell clone stably expressing approximately 100 fmol of Flag-MT1 per mg of protein (Figure 3C). Once again, 125I-MLT binding was significantly decreased in cells expressing GPR50-YFP. A similar decrease in 125I-MLT binding was observed for the untagged GPR50 showing that the YFP moiety is not responsible for this effect (Figure 3C). The decreased binding effect was dependent on the GPR50 concentration as expression of increasing amounts of GPR50-YFP progressively decreased the amount of 125I-MLT-binding sites (Figure 3D). The amount of surface-expressed Flag-MT1 in this stable cell clone remained unchanged by GPR50-YFP coexpression when monitored by flow cytometry using an antibody directed against the Flag epitope. Figure 3.125I-MLT binding to MT1 and MT2 in the presence of GPR50. (A, B) HEK 293 cells stably expressing GPR50-YFP (HEK-GPR50) transiently expressed 5–10 fmol of MT1-Rluc per mg of protein (A) or 10–15 fmol of MT2-Rluc per mg of protein (B). Expression was monitored with the luciferase assay and by 125I-MLT binding (500 pM). (C) HEK 293 cells stably expressing 70–80 fmol of Flag-MT1 per mg of protein were transfected with untagged GPR50 or C-terminal YFP fusion constructs of GPR50, MT2 or MT2C113A. Expression of YFP fusion proteins was monitored by measuring YFP fluorescence, relative expression of GPR50 and GPR50-YFP was detected by Western blot using anti-GRP50-specific antibodies (not shown). Expression of Flag-MT1 was monitored by flow cytometry using anti-Flag antibodies (not shown) and by 125I-MLT binding. (D) Increasing concentrations of GPR50-YFP were expressed in HEK 293 cells stably expressing Flag-MT1, and YFP fluorescence, 125I-MLT binding and MT1 surface expression were determined. (E) HEK 293 cells transiently expressing MT1-Rluc in the absence (▪) or presence of GPR50-YFP (▵) were incubated with increasing concentrations of 125I-MLT. Data are means±s.e.m. of at least three independent experiments each performed in duplicate (A–C) or are representative of three further experiments (D, E) (***P 0.05). Download figure Download PowerPoint Dimerization between MT1 and its known heterodimerization partner MT2 (Ayoub et al, 2002) was used to assess the specificity of the inhibitory effect of GPR50 on 125I-MLT binding to MT1. In cells expressing Flag-MT1 and MT2-YFP, the number of 125I-MLT-binding sites was higher than in cells expressing the same amount of Flag-MT1 alone, consistent with the fact that the radioligand may bind both MT1 and MT2 (Figure 3C). Moreover, an MT2-C113A-YFP mutant, which has lost the capacity of 125I-MLT binding (Mseeh et al, 2002), still heterodimerized with MT1 (Supplementary Figure 2) without blocking 125I-MLT binding of the MT1 protomer (Figure 3C). This suggests that 125I-MLT binding to MT1 is not modified in the MT1/MT2 heterodimer. Accordingly, the inhibitory effect of GPR50 on 125I-MLT binding to MT1 appears to be specific for the GPR50/MT1 heterodimer. We next performed 125I-MLT saturation experiments to determine the precise impact of GPR50 on the Bmax and Kd of 125I-MLT binding to MT1 (Figure 3E). The 125I-MLT saturation curves were best fitted using a nonlinear regression equation with a single binding site indicating the presence of a single pharmacological species. Comparable Kd values of 290±64 pM and 335±56 (n=3) were obtained in cells expressing MT1-Rluc in the presence and absence of GPR50-YFP, respectively. Bmax values decreased to 51±9.4% in the presence of GPR50-YFP. The pharmacological profile of MT1 was then determined in the presence and absence of GPR50 in 125I-MLT competition binding experiments. In cells expressing MT1 alone, the Ki values obtained for six melatonin receptor-specific ligands were in accordance with previously reported values (Dubocovich et al, 1997; Audinot et al, 2003; Ayoub et al, 2004) (Table I). Very similar Ki values were obtained for the remaining 125I-MLT-binding sites in the presence of GPR50. Our data suggest that the 125I-MLT-binding sites observed in the presence of GPR50 most likely correspond to MT1 homodimers, whereas MT1/GPR50 heterodimers are unable to bind 125I-MLT. Table 1. Binding affinities measured in HEK 293 cells expressing MT1 alone or with GPR50 Ligands MT1 MT1/GPR50 Ki (nM) Melatonin 0.73±0.26 0.37±0.28 S20098 0.43±0.23 0.42±0.22 S22153 131±6.24 197±66.9 S20928 254±54.3 358±5.20 S24773 1006±238 1615±612 4P-PDOT 204±6.05 217±10.0 HEK 293 cells expressing MT1 alone or in the presence of Flag-GPR50-Rluc were incubated with 125I-MLT and various concentrations of the indicated compounds. Ki values were calculated as described under ‘Materials and methods’. Data are means±s.e. of three independent experiments each performed in duplicate. Ki values obtained in the absence and presence of GPR50 were not statistically different according to a Student's t test. To further confirm this hypothesis, MT1 homodimers and MT1/GPR50 heterodimers were separated by selective co-immunoprecipitation and 125I-MLT binding of the different receptor pairs was determined. MT1-YFP or GPR50-YFP were coexpressed with Flag-MT1, labeled with a saturating 125I-MLT concentration, solubilized and adjusted to similar YFP fluorescence values. Previous studies have shown that 125I-MLT remains stably bound to MT1 under these experimental conditions (Brydon et al, 1999). Whereas anti-GFP-specific antibodies readily precipitated 12% of the radiolabeled MT1 homodimer (Figure 4, Flag-MT1/MT1-YFP), no significant amounts of 125I-MLT were precipitated from the MT1/GPR50 heterodimer sample (Figure 4, Flag-MT1/GPR50-YFP). Western blot experiments performed with the same samples confirmed that equivalent quantities of Flag-MT1 were co-immunoprecipitated from both samples. As expected, no radioactivity was precipitated in control experiments from cells expressing the Flag-MT1 (−) or GPR50-YFP constructs individually, thus illustrating the specificity of the assay. Our results confirm the hypothesis that MT1 loses its ability to bind to 125I-MLT when engaged into the GPR50/MT1 heterodimer. Figure 4.Absence of 125I-MLT binding to GPR50/MT1 heterodimers. Membranes from HEK 293 cells transiently expressing 30–40 fmol of Flag-MT1 alone or in the presence of the indicated receptors were labeled with 125I-MLT (500 pM), solubilized and immunoprecipitated with a monoclonal anti-GFP antibody. The amount of precipitated 125I-MLT was determined in a γ-counter and precipitates were subsequently separated by SDS–PAGE. The presence of Flag-MT1 was verified by Western blotting using polyclonal anti-Flag antibodies. Data are means of triplicates that are representative of two further experiments. mb=membranes; IP=immunoprecitate. Download figure Download PowerPoint GPR50 antagonizes MT1 signaling To verify whether GPR50 interferes also with melatonin-promoted MT1 signaling, coupling of MT1 to the Gi protein was assessed in the absence and presence of GPR50-YFP. A previously described Gαi/q chimera that couples Gi-coupled receptors to phospholipase C activation was used as a functional read-out for MT1 activation (Goudet et al, 2004). The natural hormone melatonin and the synthetic melatonin receptor-specific agonist S20098 dose-dependently increased the functional response with EC50 values of 57.7±3.5 and 37.0±3.8 pM respectively, in agreement with published values (Godson and Reppert, 1997; Petit et al, 1999) (Figure 5A and B). Coexpression of GPR50-YFP had no major effect on the basal value and the EC50 values (39.6±2.1 and 9.3±3 pM for melatonin and S20098, respectively), but decreased the maximal response by 50 and 45% for melatonin and S20098, respectively. This decrease of the maximal response was not due to decreased expression levels of MT1-Rluc when coexpressed with GPR50-YFP as monitored by measuring the luminescence of the Rluc fusion protein (Figure 5A inset). These results show that GPR50 antagonizes the functional response of the MT1 receptor when stimulated by the synthetic S20098 compound and importantly the natural hormone, melatonin. Figure 5.GPR50 antagonizes MT1 signaling. HEK 293 cells transiently expressing MT1-Rluc alone (▪) or with GPR50-YFP (Δ) were stimulated with increasing concentrations of melatonin (A) or S20098 (B) and inositol phosphate levels were determined. MT1-Rluc expression levels were determined by luminescence measurements (inset). Data are means±s.e.m. of three independent experiments each performed in duplicate. A nonlinear regression equation assuming a single binding site was used to fit the data (GraphPad Prism software). Download figure Download PowerPoint Effect of GPR50 downregulation on MT1 function in hCMEC/D3 cells expressing endogenous receptors The fact that melatonin is known to regulate cerebral blood flow (Regrigny et al, 1998; Yang et al, 2001) and that melatonin receptors are expressed in the vascular system (Savaskan et al, 2001) prompted us to search for the expression of endogenous melatonin receptors in recently immortalized human endothelial cerebral hCMEC/D3 cells (Weksler et al, 2005). RT–PCR experiments revealed the coexpression of MT1 and GPR50 transcripts (Figure 6A). GPR50-selective siRNA duplexes were synthesized to investigate the effect of GPR50 downregulation on MT1 function. The most efficient siRNA decreased the expression of GPR50-YFP in the HEK-GPR50 cells by 80% as monitored by flow cytometry (Figure 6B). Transfection of the GPR50-siRNA in hCMEC/D3 cells decreased the expression of the GPR50 transcript by 60%, whereas an Alexa Fluor 488-labeled control siRNA was without significant effect (Figure 6C). The effect of GPR50 downregulation was tested on 125I-MLT binding. Whereas specific 125I-MLT binding was hardly detectable in nontransfected and in control-siRNA-transfected cells, significant binding was consistently observed in GPR50-siRNA-treated cells (Figure 6D). In agreement with these observations, melatonin and S20098 inhibited forskolin-promoted cAMP accumulation only in GPR50-siRNA-treated cells (Figure 6E). The results obtained in hCMEC/D3 cells expressing both receptors endogenously further support the physiological significance of the inhibitory effect of GPR50 on MT1 function. Figure 6.Downregulation of endogenously expressed GPR50 in hCMEC/D3 cells promotes MT1 function. (A) GPR50 and MT1 transcripts from hCMEC/D3 cells were reverse transcript and amplified by PCR. No amplification was observed when experiments were performed in the absence of reverse transcriptase (not shown). (B) HEK 293 cells stably expressing GPR50-YFP were transfected with GPR50-specific siRNA duplexes (100 nM) and GPR50-YFP fluorescence was measured by flow cytometry 48 h post-transfection. (C–E) Effect of control siRNA and GPR50-specific siRNA duplexes on GPR50 mRNA levels (C), 125I-MLT binding (500 pM) (D), and forskolin-stimulated cAMP accumulation (E) in hCMEC/D3 cells. Stimulation with 1 μM forskolin alone (black bars) or in the presence of 1 μM melatonin (white bars) or 1 μM S20098 (hatched bars) (30 min). NT, nontransfected; bp, base pairs. Data are means±s.e.m. of at least three independent experiments each performed in triplicate (***P<0.001; *P<0.05). Download figure Download PowerPoint Mechanism of the antagonistic effect of GPR50 on MT1 function The decreased responsiveness of MT1 in the presence of GPR50 could be explained by an alteration in the steady-state cell-surface expression of MT1 owing to a change in receptor trafficking as demonstrated for other GPCR heterodimers (Jordan et al, 2001). However, it is not observed for MT1/GPR50 heterodimers, as Flag-MT1 was expressed at the cell surface irrespective of the coexpression or absence of GPR50-YFP, as quantified in a cell surface ELISA (Figure 7F) and visualized by fluorescence microscopy (Figure 7A and C). GPR50-YFP itself localized to the cell membrane when expressed alone or with Flag-MT1 (Figure 7B, D and G). Furthermore, stimulation with melatonin led to the internalization of the MT1 homodimer (Figure 7F), whereas GPR50-YFP remained at the cell surface (Figure 7G). Taken together, these data suggest that MT1 homodimers are internalized upon melatonin stimulation conversely to GPR50 homodimers and MT1/GPR50 heterodimers. Figure 7.Subcellular localization of MT1 and GPR50. (A, B) Confocal images of HEK 293 cells stably expressing Flag-MT1 or GPR50-YFP. (C–E) Localization of Flag-MT1 and GPR50-YFP in HEK 293 cells transiently coexpressing both receptors. Flag-MT1 was detected by immunodetection after permeabilization and GPR50-YFP by measuring the YFP fluorescence. (F, G) ELISA quantification of Flag-MT1 (F) and Flag-GPR50 (G) surface expression in the absence (black bars) and presence of melatonin (white bars). Data are means±s.e.m. of at least three independent experiments each performed in duplicate. Download figure Download PowerPoint Intriguingly, GPR50 displays a noncanonical C-terminal tail of 311 residues (Reppert et al, 1996). To study the potential role of the C-terminus on 125I-MLT binding to the MT1/GPR50 heterodimer, 264 amino acids of the intracellular domain were deleted. The first 55 amino ac
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