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• W2023766647 abstract "The inducible serotonergic 1C115-HT cell line expresses a defined set of serotonergic receptors of the 5-HT2B, 5-HT1B/D, and 5-HT2A subtypes, which sustain a regulation of serotonergic associated functions through G-protein-dependent signaling. 1C115-HT cells have been instrumental to assign a signaling function to the cellular prion protein PrPC. Here, we establish that antibody-mediated ligation of PrPC concomitant to agonist stimulation of 5-HT receptors modulates the couplings of all three serotonergic receptors present on 1C115-HT cells. Specific impacts of PrP antibodies were monitored depending on the receptor and pathway considered. PrPC ligation selectively cancels the 5-HT2A-PLC response, decreases the 5-HT1B/D negative coupling to adenylate cyclase, and potentiates the 5-HT2B-PLA2 coupling. As a result, PrPC ligation disturbs the functional interactions occurring between the signaling pathways of the three receptor subtypes. In 1C115-HT cells, antagonizing cross-talks arising from 5-HT2B and 5-HT2A receptors control the 5-HT1B/D function. PrPC ligation reinforces the negative regulation exerted by 5-HT2B on 5-HT1B/D receptors. On the other hand it abrogates the blocking action of 5-HT2A on the regulatory loop linking 5-HT1B/D receptors. We propose that the ligation of PrPC affects the potency or dynamics of G-protein activation by agonist-bound serotonergic receptors. Finally, the PrPC-dependent modulation of 5-HT receptor couplings is restricted to 1C115-HT cells expressing a complete serotonergic phenotype. It critically involves a PrPC-caveolin platform implemented on the neurites of 1C115-HT cells during differentiation. Our findings define PrPC as a modulator of 5-HT receptor coupling to G-proteins and thereby as a protagonist contributing to the homeostasis of serotonergic neurons. They provide a foundation for uncovering the impact of prion infection on serotonergic functions. The inducible serotonergic 1C115-HT cell line expresses a defined set of serotonergic receptors of the 5-HT2B, 5-HT1B/D, and 5-HT2A subtypes, which sustain a regulation of serotonergic associated functions through G-protein-dependent signaling. 1C115-HT cells have been instrumental to assign a signaling function to the cellular prion protein PrPC. Here, we establish that antibody-mediated ligation of PrPC concomitant to agonist stimulation of 5-HT receptors modulates the couplings of all three serotonergic receptors present on 1C115-HT cells. Specific impacts of PrP antibodies were monitored depending on the receptor and pathway considered. PrPC ligation selectively cancels the 5-HT2A-PLC response, decreases the 5-HT1B/D negative coupling to adenylate cyclase, and potentiates the 5-HT2B-PLA2 coupling. As a result, PrPC ligation disturbs the functional interactions occurring between the signaling pathways of the three receptor subtypes. In 1C115-HT cells, antagonizing cross-talks arising from 5-HT2B and 5-HT2A receptors control the 5-HT1B/D function. PrPC ligation reinforces the negative regulation exerted by 5-HT2B on 5-HT1B/D receptors. On the other hand it abrogates the blocking action of 5-HT2A on the regulatory loop linking 5-HT1B/D receptors. We propose that the ligation of PrPC affects the potency or dynamics of G-protein activation by agonist-bound serotonergic receptors. Finally, the PrPC-dependent modulation of 5-HT receptor couplings is restricted to 1C115-HT cells expressing a complete serotonergic phenotype. It critically involves a PrPC-caveolin platform implemented on the neurites of 1C115-HT cells during differentiation. Our findings define PrPC as a modulator of 5-HT receptor coupling to G-proteins and thereby as a protagonist contributing to the homeostasis of serotonergic neurons. They provide a foundation for uncovering the impact of prion infection on serotonergic functions. Some aspects of serotonin (5-hydroxytryptamine (5-HT) 1The abbreviations used are: 5-HT, 5-hydroxytryptamine; DOI, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; GPCR, G-protein coupled receptor; PLC, phospholipase C; IP3, inositol 1,4,5-trisphophate; NO, nitric oxide; NOS, NO synthase; PLA2, phospholipase A2; AA, arachidonic acid; PrPC, cellular prion protein; SERT, 5-HT transporter; 5-CT, 5-carboxamidotryptamine; d4, day 4; FScA, forskolin-stimulated cAMP accumulation.)homeostasis may relate to the plasticity of serotonergic neurons, which continually adapt their phenotype in response to 5-HT as well as to a variety of neuronal and non-neuronal factors (1Azmitia E.C. Neuropsychopharmacology. 1999; 21: 33S-45SCrossref PubMed Scopus (153) Google Scholar). In addition to releasing 5-HT, serotonergic neurons of the raphe nuclei contribute to the integration of 5-HT signals by expressing 5-HT receptors, including 5-HT1A, 5-HT1B/D, 5-HT2A, and 5-HT2B subtypes (2Gaspar P. Cases O. Maroteaux L. Nat. Rev. Neurosci. 2003; 4: 1002-1012Crossref PubMed Scopus (1011) Google Scholar). These receptors most likely behave as autoreceptors modulating 5-HT-related cellular functions (3Hoyer D. Clarke D.E. Fozard J.R. Hartig P.R. Martin G.R. Mylecharane E.J. Saxena P.R. Humphrey P.P. Pharmacol. Rev. 1994; 46: 157-203PubMed Google Scholar, 4Mouillet-Richard S. Mutel V. Loric S. Tournois C. Launay J.M. Kellermann O. J. Biol. Chem. 2000; 275: 9186-9192Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). However it is unclear whether the same neurons or distinct subpopulations harbor these receptor subtypes. The complexity of serotonergic signaling networks is far from being solved. Indeed, the targets recruited by 5-HT receptor-mediated signals are poorly characterized. Besides, still little is known about cross-talks between signaling cascades coupled with a single 5-HT receptor subtype and/or between pathways related to distinct receptor subclasses. Taking advantage of the 1C11 murine neuroectodermal cell line, which is endowed with the capacity to undergo serotonergic differentiation upon appropriate induction (4Mouillet-Richard S. Mutel V. Loric S. Tournois C. Launay J.M. Kellermann O. J. Biol. Chem. 2000; 275: 9186-9192Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), we previously underlined the existence of functional interactions between three 5-HT receptor subtypes (5Tournois C. Mutel V. Manivet P. Launay J.M. Kellermann O. J. Biol. Chem. 1998; 273: 17498-17503Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Within 4 days of treatment with dibutyryl cyclic AMP and cyclohexane carboxylic acid, nearly 100% of 1C11 neuroprogenitor cells are converted into 1C115-HT neuronal cells, which express a complete serotonergic phenotype including 5-HT metabolism, storage, and transport (4Mouillet-Richard S. Mutel V. Loric S. Tournois C. Launay J.M. Kellermann O. J. Biol. Chem. 2000; 275: 9186-9192Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). This serotonergic differentiation program is accompanied by the selective and time-dependent onset of three serotonergic receptors. 1C115-HT cells become sensitive to external 5-HT at day 2 of differentiation with the induction of 5-HT1B/D (1500 GTI binding sites/cell) and 5-HT2B (2200 DOI binding sites/cell) receptors. At day 4, the repertoire of neurotransmitter receptors extends to 5-HT2A receptors (400 DOI binding sites/cell), which further contribute to the integration of 5-HT signals. In mature 1C115-HT cells, these autoreceptors take a critical part in the regulation of the intensities of 5-HT-related functions, namely synthesis, storage, and transport (4Mouillet-Richard S. Mutel V. Loric S. Tournois C. Launay J.M. Kellermann O. J. Biol. Chem. 2000; 275: 9186-9192Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). All three receptors belong to the G-protein coupled receptors (GPCRs) family (see Ref. 6Hoyer D. Hannon J.P. Martin G.R. Pharmacol. Biochem. Behav. 2002; 71: 533-554Crossref PubMed Scopus (1586) Google Scholar for review). Various cell systems and notably the 1C11 cell line have allowed several of their coupling mechanisms to be characterized. 5-HT1B/D receptors elicit their response through Gi-mediated inhibition of adenylate cyclase (6Hoyer D. Hannon J.P. Martin G.R. Pharmacol. Biochem. Behav. 2002; 71: 533-554Crossref PubMed Scopus (1586) Google Scholar). 5-HT binding to either 5-HT2 subclass activates Gq protein and stimulates phospholipase C (PLCβ), thereby initiating a rapid release of inositol 1,4,5-trisphosphate (IP3) and a rise in intracellular calcium level (6Hoyer D. Hannon J.P. Martin G.R. Pharmacol. Biochem. Behav. 2002; 71: 533-554Crossref PubMed Scopus (1586) Google Scholar). The 5-HT2B receptor is also coupled to the ras/mitogen-activated protein kinase cascade (7Launay J.M. Birraux G. Bondoux D. Callebert J. Choi D.S. Loric S. Maroteaux L. J. Biol. Chem. 1996; 271: 3141-3147Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and cross-talks with tyrosine kinase pathways to promote cell proliferation (8Nebigil C.G. Launay J.M. Hickel P. Tournois C. Maroteaux L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2591-2596Crossref PubMed Scopus (154) Google Scholar). In addition, the 5-HT2B receptor recruits both cellular and inducible nitric-oxide (NO) synthase (NOS) transduction pathways through a type I PDZ domain (9Manivet P. Mouillet-Richard S. Callebert J. Nebigil C.G. Maroteaux L. Hosoda S. Kellermann O. Launay J.M. J. Biol. Chem. 2000; 275: 9324-9331Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Finally, a coupling of the 5-HT2B receptor to the phospholipase A2 (PLA2)/arachidonic acid (AA) pathway was evidenced in 1C115-HT cells and shown to sustain a negative regulation of the 5-HT1B/D receptor function at day 2 of 1C11 serotonergic differentiation (5Tournois C. Mutel V. Manivet P. Launay J.M. Kellermann O. J. Biol. Chem. 1998; 273: 17498-17503Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The inhibitory effect exerted by 5-HT2B receptors on the 5-HT1B/D coupling to adenylate cyclase however is lifted in 1C115-HT day 4 cells by the concomitant activation of the 5-HT2A receptor. 5-HT2A receptors are also coupled with the PLA2 cascade. The signaling pathway through which 5-HT2A receptors counterbalance the antagonizing action of 5-HT2B receptors on the 5-HT1B/D receptor function, necessarily distinct from PLA2, is still unclear. Understanding how serotonergic neurons manage 5-HT-related inputs with respect to the diversity of other signals constitutes another important challenge. Recently, the 1C11 cell line allowed us to identify transduction pathways coupled with the cellular prion protein (PrPC). This protein is the normal counterpart of a pathogenic protein termed scrapie prion protein, which is involved in a group of fatal neurodegenerative disorders known as transmissible spongiform encephalopathies. PrPC is expressed in all cell types and is particularly represented at the surface of neurons to which it is anchored by a glycosylphosphatidylinositol moiety. Whatever the state of differentiation of 1C11 cells, PrPC is endogenously expressed at roughly similar levels (10Mouillet-Richard S. Laurendeau I. Vidaud M. Kellermann O. Laplanche J.L. Microbes Infect. 1999; 1: 969-976Crossref PubMed Scopus (35) Google Scholar). Moreover, antibody-mediated ligation of PrPC, which is assumed to mimic the interaction with a yet to be identified ligand, induces NADPH oxidase-dependent reactive oxygen species production and activation of the extracellular regulated kinases 1/2 (ERK1/2), two members of the mitogen-activated protein kinase family (11Schneider B. Mutel V. Pietri M. Ermonval M. Mouillet-Richard S. Kellermann O. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13326-13331Crossref PubMed Scopus (156) Google Scholar). In cells harboring a fully differentiated phenotype exclusively, PrPC associates with the membrane protein caveolin and the tyrosine kinase Fyn within a signaling platform that governs the downstream effectors, i.e. reactive oxygen species and ERK1/2 (11Schneider B. Mutel V. Pietri M. Ermonval M. Mouillet-Richard S. Kellermann O. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13326-13331Crossref PubMed Scopus (156) Google Scholar, 12Mouillet-Richard S. Ermonval M. Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Crossref PubMed Scopus (678) Google Scholar). The implementation of the PrPC-caveolin-Fyn complex depends upon the expression of neuronal and neurotransmitter-associated functions. It may relate to the proper structural organization of the partners within subcellular microdomains. Noticeably, the PrPC-Fyn coupling occurs on the neurites of 1C11-derived neuronal progenies where bioaminergic receptors are most likely localized (13Kellermann O. Lafay-Chebassier C. Ermonval M. Lehmann S. Mouillet-Richard S. C. R. Acad. Sci. (Paris). 2002; 325: 9-15Google Scholar). We assume that the PrPC-dependent signaling activity may contribute to cell homeostasis and take part to the fine-tuning of neuronal and neurotransmitter-associated functions, notably in 1C115-HT fully differentiated serotonergic cells. In an attempt to shed some light on the mechanisms sustaining the homeostasis of serotonergic neurons, we took interest in relations between 5-HT-mediated pathways and PrPC. Our initial goal was to monitor in 1C115-HT serotonergic cells the functionality of 5-HT receptors under PrPC ligation. We show here that PrPC modulates the couplings and cross-talks of the 5-HT1B/D, 5-HT2A, and 5-HT2B receptors in 1C115-HT serotonergic cells. Materials—Dibutyryl cyclic AMP and cyclohexane carboxylic acid were from Sigma-Aldrich Chimie (St-Quentin Fallavier, France). Neurochemicals were from RBI (Natick, MA). All other chemicals, of the purest grade available, were from classical commercial sources. [125I]DOI (81.4 TBq/mmol) and [14C]AA (57 mCi/mmol) were from PerkinElmer Life Sciences. Forskolin was purchased from Calbiochem. Agonists and antagonists of 5-HT2 receptor subtypes were selected according to Porter et al., Cussac et al., and Jerman et al. (14Porter R.H. Benwell K.R. Lamb H. Malcolm C.S. Allen N.H. Revell D.F. Adams D.R. Sheardown M.J. Br. J. Pharmacol. 1999; 128: 13-20Crossref PubMed Scopus (324) Google Scholar, 15Cussac D. Newman-Tancredi A. Quentric Y. Carpentier N. Poissonnet G. Parmentier J.G. Goldstein S. Millan M.J. Naunyn-Schmiedeberg's Arch. Pharmacol. 2002; 365: 242-252Crossref PubMed Scopus (76) Google Scholar, 16Jerman J.C. Brough S.J. Gager T. Wood M. Coldwell M.C. Smart D. Middlemiss D.N. Eur. J. Pharmacol. 2001; 414: 23-30Crossref PubMed Scopus (85) Google Scholar). All tissue culture reagents and Hank's balanced salt solution were from Invitrogen. PrP monoclonal antibodies (SAF61, SAF32, and BAR221, all IgG) with distinct binding epitopes (11Schneider B. Mutel V. Pietri M. Ermonval M. Mouillet-Richard S. Kellermann O. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13326-13331Crossref PubMed Scopus (156) Google Scholar) were obtained from the Service de Pharmacologie et d'Immunologie, Commissariat à l'Energie Atomique (Saclay, France). Polyclonal goat IgG antibodies against the 5-HT transporter (SERT) were from Santa Cruz Biotechnology. Cell Cultures—1C11 cells were grown and induced to differentiate toward the serotonergic pathway in the presence of 1 mm dibutyryl cyclic AMP and 0.05% cyclohexane carboxylic acid as in Ref. 5Tournois C. Mutel V. Manivet P. Launay J.M. Kellermann O. J. Biol. Chem. 1998; 273: 17498-17503Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar. Radioligand Binding Experiments—Radioligand binding experiments were as described previously (17Loric S. Maroteaux L. Kellermann O. Launay J.M. Mol. Pharmacol. 1995; 47: 458-466PubMed Google Scholar, 18Kellermann O. Loric S. Maroteaux L. Launay J.M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar). Experiments were performed using 1C115-HT cells or mouse LMTK– fibroblasts stably transfected with cDNA encoding the mouse 5-HT2A or 5-HT2B receptor. Determination of Phospholipase C Activity—Cells were washed twice with fresh serum-free medium and exposed to various agonists or antagonists of the 5-HT2 receptors family and in some experiments to PrP antibodies. After 10 min of incubation, cells were washed twice in cold phosphate-buffered saline and scraped with a rubber policeman. Intracellular IP3 levels were then measured radioimmunologically as detailed previously (17Loric S. Maroteaux L. Kellermann O. Launay J.M. Mol. Pharmacol. 1995; 47: 458-466PubMed Google Scholar). Measurement of NOS Activity—NOS activity was evaluated by direct electrochemical measurement of NO as described by Kanai et al. (19Kanai A.J. Pearce L.L. Clemens P.R. Birder L.A. VanBibber M.M. Choi S.Y. de Groat W.C. Peterson J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14126-14131Crossref PubMed Scopus (322) Google Scholar). Cells were washed twice and incubated at 37 °C in perfusate as described (19Kanai A.J. Pearce L.L. Clemens P.R. Birder L.A. VanBibber M.M. Choi S.Y. de Groat W.C. Peterson J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14126-14131Crossref PubMed Scopus (322) Google Scholar). NO production was directly measured in a porphyrinic microsensor positioned on the surface of 1C115-HT cells 10 min after 5-HT2 agonist and/or anti-PrP antibody addition by a nanoinjector (20Malinski T. Taha Z. Nature. 1992; 358: 676-678Crossref PubMed Scopus (1029) Google Scholar). Measurement of Phospholipase A2 Activity—PLA2 activity was assessed through measurement of [14C]arachidonic acid release 10 min after receptor and/or PrP ligation as in Ref. 5Tournois C. Mutel V. Manivet P. Launay J.M. Kellermann O. J. Biol. Chem. 1998; 273: 17498-17503Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar. Measurements of 5-HT1B/D Receptor Response—The functionality of the 5-HT1B/D receptor was assessed as in Ref. 5Tournois C. Mutel V. Manivet P. Launay J.M. Kellermann O. J. Biol. Chem. 1998; 273: 17498-17503Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar. The 5-HT1B/D receptor-mediated response was followed by measuring (15 min, 37 °C) the 5-carboxamidotryptamine (5-CT)-induced inhibition of cAMP accumulated in the presence of 1 μm forskolin and 0.1 mm rolipram, a phosphodiesterase inhibitor. For each experiment, data were expressed according to the response obtained with 1 μm forskolin (100%). Antibody-mediated PrPC Ligation—Ligation of PrPC at the surface of 1C115-HT cells was carried out using SAF61, SAF32, or BAR221 antibodies at 10 μg/ml as in Ref. 11Schneider B. Mutel V. Pietri M. Ermonval M. Mouillet-Richard S. Kellermann O. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13326-13331Crossref PubMed Scopus (156) Google Scholar. Cell Permeabilization—1C115-HT cells were permeabilized prior to antibody-mediated neutralization of G proteins according to Launay et al. (7Launay J.M. Birraux G. Bondoux D. Callebert J. Choi D.S. Loric S. Maroteaux L. J. Biol. Chem. 1996; 271: 3141-3147Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Caveolin Immunosequestration—1C115-HT cells were bombarded with tungsten microprojectiles coated with antibodies to caveolin-1 as in Ref. 12Mouillet-Richard S. Ermonval M. Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Crossref PubMed Scopus (678) Google Scholar. Uncoated microprojectiles were used as a control. Data Analysis and Statistics—Statistical analyses on small groups were performed using non-parametric tests. The significance criterion of p < 0.001 was adopted. All values are given as arithmetic means ± standard errors of the means (S.E.). PrPC Ligation Modifies PLA2 and PLC (but Not NOS) Activities Coupled to 5-HT2 Receptors—To assess potential cross-talks between PrPC and the signaling pathways coupled with the 1C115-HT serotonergic receptors, we monitored the levels of various second messengers production in response to 5-HT receptor activation combined to antibody-mediated ligation of PrPC. A first set of experiments was centered on the two 5-HT2 subtypes, i.e. 5-HT2A and 5-HT2B receptors. Treatment of 1C115-HT day 4 cells with 100 nm DOI, a selective 5-HT2-receptor agonist, stimulates PLC (18Kellermann O. Loric S. Maroteaux L. Launay J.M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar), PLA2 (5Tournois C. Mutel V. Manivet P. Launay J.M. Kellermann O. J. Biol. Chem. 1998; 273: 17498-17503Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and NOS (9Manivet P. Mouillet-Richard S. Callebert J. Nebigil C.G. Maroteaux L. Hosoda S. Kellermann O. Launay J.M. J. Biol. Chem. 2000; 275: 9324-9331Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) activities. When 1C115-HT d4 cells were incubated with anti-PrP antibodies (SAF61, SAF32, or BAR221) only, no PLC, PLA2, or NOS activation was obtained (Fig. 1, A–C). Now, upon simultaneous exposure to DOI and PrP antibodies, the level of NOS activity did not differ from that obtained in response to DOI alone (Fig. 1A). By contrast, as shown in Fig. 1B, PrPC ligation combined with the addition of DOI caused a significant 1.7-fold increase in PLA2 stimulation as compared with DOI treatment alone. PrPC ligation also had an impact on the DOI-induced PLC activation. Concomitant exposure of 1C115-HT d4 cells to DOI and PrP antibodies reduced by 3-fold the level of IP3 production triggered by the sole addition of 100 nm DOI, thereby remaining at the basal level (Fig. 1C). In all experiments, identical results could be obtained using three distinct antibodies against PrP (SAF61, SAF32, or BAR221). Of note, no effect could be recorded on the DOI-induced activation of NOS, PLA2, or PLC under simultaneous incubation of 1C115-HT d4 cells with irrelevant antibodies to the membranous serotonin transporter (SERT) used as a negative control (Fig. 1, A–C). These overall data argue against the occurrence of functional interactions between PrPC and the 5-HT2B receptor-NO pathway. In turn, they emphasize PrPC-dependent modulations of the PLC and PLA2 couplings associated to 5-HT2A and/or 5-HT2B receptors. PrPC Ligation Preferentially Enhances the Efficacy of PLA2 Coupling to the 5-HT2B Receptor—The effect of PrP ligation on 5-HT2 receptors-induced PLA2 activity was further investigated using SAF61 antibodies. In 1C115-HT day 4 cells, both 5-HT2A and 5-HT2B receptors promote PLA2 activation and subsequent AA release (5Tournois C. Mutel V. Manivet P. Launay J.M. Kellermann O. J. Biol. Chem. 1998; 273: 17498-17503Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Because DOI activates both 5-HT2 receptor subtypes, we selected a series of 5-HT2 agonists with distinct specificities toward the 5-HT2A and 5-HT2B subtypes. The level of AA production monitored in 1C115-HT d4 cells under stimulation by a given agonist and concomitant PrP ligation was compared with the maximal response (Emax) elicited by the agonist alone. As shown in Fig. 1D, PrPC ligation had no significant effect on the PLA2 activity induced by treatment with 100 nm MK212, preferentially targeting 5-HT2A receptors. By contrast, a potentiative (1.5-fold) effect of PrPC was observed on the PLA2 response to 100 nm BW723C86, a selective agonist of 5-HT2B receptors (Fig. 1D, see also supplemental Fig. S1 for other anti-PrP antibodies). Concomitant PrPC ligation also strongly enhanced by 2.4-fold the efficacy of the PLA2 response to 100 nm 1-(m-chlorophenyl)-piperazine with preferential affinity for 5-HT2B over 5-HT2A receptors. These data show that the efficacy of PLA2 response associated to 5-HT2A receptors is not affected by PrPC ligation. They also indicate that the enhancing effect of PrPC on PLA2 activity relates to 5-HT2B receptors. To explore the functional relation between PrP and the 5-HT2B receptor, we investigated whether PrP ligation affects the binding properties of the 5-HT2B receptor. We selected a panel of agonists and antagonists that differentially bind to 5-HT2A or 5-HT2B receptors (14Porter R.H. Benwell K.R. Lamb H. Malcolm C.S. Allen N.H. Revell D.F. Adams D.R. Sheardown M.J. Br. J. Pharmacol. 1999; 128: 13-20Crossref PubMed Scopus (324) Google Scholar, 15Cussac D. Newman-Tancredi A. Quentric Y. Carpentier N. Poissonnet G. Parmentier J.G. Goldstein S. Millan M.J. Naunyn-Schmiedeberg's Arch. Pharmacol. 2002; 365: 242-252Crossref PubMed Scopus (76) Google Scholar, 16Jerman J.C. Brough S.J. Gager T. Wood M. Coldwell M.C. Smart D. Middlemiss D.N. Eur. J. Pharmacol. 2001; 414: 23-30Crossref PubMed Scopus (85) Google Scholar). This set of drugs allows discriminating between both 5-HT2 receptor subtypes and more precisely specifies the 5-HT2B receptor. We determined the pharmacological profile of the 5-HT2B receptor in 1C115-HT day 4 cells by assessing the binding affinities to each drug, used as a competitor of DOI binding. The binding of all drugs to 1C115-HT d4 cells was then followed under simultaneous incubation with antibodies to PrP. As shown in Fig. 2A, the binding constants of the drugs to 1C115-HT cells exposed to PrP antibodies (SAF61) did not significantly differ from those monitored without PrP ligation. In view of the exclusive specificity of this set of drugs toward the 5-HT2B receptor, we conclude that PrP antibodies have no impact on the binding properties of the 5-HT2B receptor. In another set of experiments, we evaluated the effect of the same panel of drugs on PLA2 activity in 1C115-HT d4 cells. In the case of agonists, the pEC50 values were deduced from the dose-response curves. As for antagonists, their capacities to inhibit the DOI-dependent stimulation of PLA2 activity were monitored to calculate pKB values. Comparison between the binding constants of the drugs (pKi) and the apparent equilibrium constants deduced from their effects on PLA2 activity (pEC50 or pKB) yield a correlation ratio of 0.787 (n = 19, p < 0.0001) (Fig. 2B). The dose effect of the drugs on PLA2 activity was then monitored in 1C115-HT day 4 cells submitted to concomitant PrPC ligation using SAF61 antibodies. The pEC50 and pKB values thus obtained were plotted against the pKi values of the drugs determined in 1C115-HT cells exposed to PrP antibodies (Fig. 2C). A highly significant ratio (0.957, n = 19, p < 0.0001) was found between the effects of the agonists and antagonists on PLA2 activity and their binding. This correlation was over one order of magnitude higher than that obtained without PrP antibodies. Because the set of selected drugs specifies the 5-HT2B receptor (14Porter R.H. Benwell K.R. Lamb H. Malcolm C.S. Allen N.H. Revell D.F. Adams D.R. Sheardown M.J. Br. J. Pharmacol. 1999; 128: 13-20Crossref PubMed Scopus (324) Google Scholar, 15Cussac D. Newman-Tancredi A. Quentric Y. Carpentier N. Poissonnet G. Parmentier J.G. Goldstein S. Millan M.J. Naunyn-Schmiedeberg's Arch. Pharmacol. 2002; 365: 242-252Crossref PubMed Scopus (76) Google Scholar, 16Jerman J.C. Brough S.J. Gager T. Wood M. Coldwell M.C. Smart D. Middlemiss D.N. Eur. J. Pharmacol. 2001; 414: 23-30Crossref PubMed Scopus (85) Google Scholar), this comparative analysis directly demonstrates that PrPC ligation enhances the coupling potency (EC50) of 5-HT2B receptors to the PLA2 pathway. PrPC Ligation Abrogates the 5-HT2A Receptor-dependent PLC Coupling in 1C115-HT D4 Cells—To further characterize the negative impact of PrPC ligation on the DOI-induced PLC activation, we first sought to evaluate the relative contribution of 5-HT2A and 5-HT2B receptors to the DOI-dependent IP3 production in 1C115-HT day 4 cells. In contrast to the 5-HT2A receptor, a functional coupling of the 5-HT2B receptor to PLCβ has never been evidenced in vivo (21Cox D.A. Cohen M.L. J. Pharmacol. Exp. Ther. 1995; 272: 143-150PubMed Google Scholar, 22Ellis E.S. Byrne C. Murphy O.E. Tilford N.S. Baxter G.S. Br. J. Pharmacol. 1995; 114: 400-404Crossref PubMed Scopus (82) Google Scholar). However, we described previously a 5-HT2B receptor-dependent IP3 accumulation both in LMTK cells stably transfected with a cDNA encoding this receptor or in 1C115-HT cells at day 2 of serotonergic differentiation when 5-HT2A receptors are not yet present (9Manivet P. Mouillet-Richard S. Callebert J. Nebigil C.G. Maroteaux L. Hosoda S. Kellermann O. Launay J.M. J. Biol. Chem. 2000; 275: 9324-9331Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 18Kellermann O. Loric S. Maroteaux L. Launay J.M. Br. J. Pharmacol. 1996; 118: 1161-1170Crossref PubMed Scopus (32) Google Scholar). As already mentioned in Fig. 1C, the addition of 100 nm DOI on 1C115-HT d4 cells promoted a rapid IP3 accumulation reaching a plateau at 15 min with a level of maximal response (Emax) corresponding to a 4-fold increase as compared with the basal level of IP3 production. When the cells were simultaneously exposed to 100 nm DOI and to 100 nm MDL100.907, a selective antagonist of 5-HT2A receptors, IP3 accumulation remained at the basal level (Fig. 3A). Furthermore, as shown in Fig. 3A, treatment of 1C115-HT d4 cells by 100 nm BW723C86, which specifically activates 5-HT2B receptors, did not induce any PLCβ activation either (32 ± 2.5% of the DOI reference level, not significantly different from 26 ± 3.7% corresponding to the basal level). These data indicate that the PLCβ stimulation monitored in 1C115-HT d4 cells in response to 100 nm DOI (Fig. 3A) depends on 5-HT2A receptors only. They also emphasize that between day 2 and day 4 of the serotonergic differentiation program of 1C115-HT cells 5-HT2B receptors lose their ability to recruit the PLCβ/IP3 pathway. As shown in Fig. 3A, IP3 accumulation in 1C115-HT d4 cells remained at a basal level under simultaneous exposure to PrP antibodies and to 100 nm DOI. Again, similar impacts on the DOI-induced IP3 release were recorded using three distinct anti-PrP antibodies (SAF61, SA" @default.
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• W2023766647 title "Modulation of Serotonergic Receptor Signaling and Cross-talk by Prion Protein*" @default.
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