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• W2116715079 abstract "The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT2A receptor in response to hallucinogenic versus nonhallucinogenic agonists, which underlies their distinct capacity to desensitize the receptor. The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT2A receptor in response to hallucinogenic versus nonhallucinogenic agonists, which underlies their distinct capacity to desensitize the receptor. Among the G Protein-Coupled Receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; 5-HT, 5-hydroxytryptamine, serotonin; DOI, 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane; HILIC, hydrophilic interaction liquid chromatography; LSD, lysergic acid diethylamine; PKB, Protein Kinase B; PKC, Protein Kinase C; PLC, phospholipase C; PTX, Pertussis toxin; RSK, Ribosomal S6 kinase; SILAC, stable isotope labeling by amino acids in cell culture. 1The abbreviations used are: GPCR, G protein-coupled receptor; 5-HT, 5-hydroxytryptamine, serotonin; DOI, 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane; HILIC, hydrophilic interaction liquid chromatography; LSD, lysergic acid diethylamine; PKB, Protein Kinase B; PKC, Protein Kinase C; PLC, phospholipase C; PTX, Pertussis toxin; RSK, Ribosomal S6 kinase; SILAC, stable isotope labeling by amino acids in cell culture. activated by serotonin (5-hydroxytryptamine, 5-HT), the 5-HT2A receptor continues to attract particular attention in view of its broad physiological role and implication in the actions of numerous psychotropic agents (1.Meltzer H.Y. Massey B.W. Horiguchi M. Serotonin receptors as targets for drugs useful to treat psychosis and cognitive impairment in schizophrenia.Curr. Pharm. Biotechnol. 2012; 13: 1572-1586Crossref PubMed Scopus (90) Google Scholar, 2.Roth B.L. Berry S.A. Kroeze W.K. Willins D.L. Kristiansen K. Serotonin 5-HT2A receptors: molecular biology and mechanisms of regulation.Crit. Rev. Neurobiol. 1998; 12: 319-338Crossref PubMed Scopus (98) Google Scholar). It is a primary target of widely used atypical antipsychotics such as clozapine, risperidone, and olanzapine, which act as antagonists or inverse agonists (1.Meltzer H.Y. Massey B.W. Horiguchi M. Serotonin receptors as targets for drugs useful to treat psychosis and cognitive impairment in schizophrenia.Curr. Pharm. Biotechnol. 2012; 13: 1572-1586Crossref PubMed Scopus (90) Google Scholar, 3.Gray J.A. Roth B.L. Molecular targets for treating cognitive dysfunction in schizophrenia.Schizophr. Bull. 2007; 33: 1100-1119Crossref PubMed Scopus (191) Google Scholar). The activation of 5-HT2A receptors expressed in the prefrontal cortex has also been implicated in the psycho-mimetic effects of psychedelic hallucinogens, such as lysergic acid diethylamide (LSD), mescaline, and psilocybin, which are often used to model positive symptoms of schizophrenia (4.Aghajanian G.K. Marek G.J. Serotonin and hallucinogens.Neuropsychopharmacology. 1999; 21: 16S-23SCrossref PubMed Scopus (0) Google Scholar, 5.Nichols D.E. Hallucinogens.Pharmacol. Ther. 2004; 101: 131-181Crossref PubMed Scopus (914) Google Scholar, 6.Gonzalez-Maeso J. Sealfon S.C. Psychedelics and schizophrenia.Trends Neurosci. 2009; 32: 225-232Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 7.Gonzalez-Maeso J. Weisstaub N.V. Zhou M. Chan P. Ivic L. Ang R. Lira A. Bradley-Moore M. Ge Y. Zhou Q. Sealfon S.C. Gingrich J.A. Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior.Neuron. 2007; 53: 439-452Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar, 8.Gonzalez-Maeso J. Yuen T. Ebersole B.J. Wurmbach E. Lira A. Zhou M. Weisstaub N. Hen R. Gingrich J.A. Sealfon S.C. Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex.J. Neurosci. 2003; 23: 8836-8843Crossref PubMed Google Scholar). However, these psychoactive effects are not reproduced by structurally-related agonists, such as ergotamine and the anti-Parkinson agent lisuride, despite the fact that they exhibit comparable affinities and efficacies at 5-HT2A receptors (7.Gonzalez-Maeso J. Weisstaub N.V. Zhou M. Chan P. Ivic L. Ang R. Lira A. Bradley-Moore M. Ge Y. Zhou Q. Sealfon S.C. Gingrich J.A. Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior.Neuron. 2007; 53: 439-452Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar, 9.Gonzalez-Maeso J. Sealfon S.C. Agonist-trafficking and hallucinogens.Curr. Med. Chem. 2009; 16: 1017-1027Crossref PubMed Scopus (60) Google Scholar). This paradox was partially resolved by the demonstration that hallucinogens induce a specific transcriptomic signature because of the specific engagement of a Pertussis toxin-sensitive Gi/o/Src signaling pathway which is not activated by nonhallucinogenic agonists (7.Gonzalez-Maeso J. Weisstaub N.V. Zhou M. Chan P. Ivic L. Ang R. Lira A. Bradley-Moore M. Ge Y. Zhou Q. Sealfon S.C. Gingrich J.A. Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior.Neuron. 2007; 53: 439-452Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar, 8.Gonzalez-Maeso J. Yuen T. Ebersole B.J. Wurmbach E. Lira A. Zhou M. Weisstaub N. Hen R. Gingrich J.A. Sealfon S.C. Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex.J. Neurosci. 2003; 23: 8836-8843Crossref PubMed Google Scholar). These findings suggest that hallucinogenic and nonhallucinogenic agonists induce different conformational states of the 5-HT2A receptor, and represent a striking example of functional selectivity that translates into contrasting pattern of mice behavior: induction of head-twitches by hallucinogenic but not by nonhallucinogenic agonists (9.Gonzalez-Maeso J. Sealfon S.C. Agonist-trafficking and hallucinogens.Curr. Med. Chem. 2009; 16: 1017-1027Crossref PubMed Scopus (60) Google Scholar). The differential influence of hallucinogenic versus nonhallucinogenic agonists on signaling pathways suggests that they trigger contrasting patterns of protein phosphorylation. To address this issue, we employed a quantitative phosphoproteomics strategy to directly compare the phosphoproteomes generated in HEK-293 cells by the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) and the nonhallucinogenic 5-HT2A agonist lisuride. We found that DOI, but not lisuride, induced the phosphorylation of a serine residue (Ser280) located in the third intracellular loop of the receptor itself. The hallucinogen-specific phosphorylation of this residue was further validated in vitro and in vivo by using a phosphosite-specific antibody. These findings were followed by a series of experiments to determine the impact of Ser280 phosphorylation upon receptor desensitization and internalization. Human Embryonic Kidney-293 (HEK-293) cells were from the European Collection of Cell Cultures, culture media from Invitrogen (Carlsbad, CA). Lisuride maleate was from Santa Cruz Biotechnologies (Santa Cruz, CA). All other chemicals were from Sigma Aldrich. Isotope-labeled amino acids for SILAC experiments were from Eurisotop (Saint Aubin, France). The rabbit anti-phospho-Thr202/Tyr204-Erk1,2, and anti-total Erk1,2 antibodies were from Cell Signaling Technology (Danvers, MA), the rabbit anti-Hemagglutinin (HA) antibody from Zymed Laboratories Inc. (South San Francisco, CA), the rabbit anti-GFP antibody from Roche Diagnostics, the rabbit anti-5-HT2A receptor antibody from Immunostar (Hudson, MI) and the mouse anti-HA antibody conjugated to agarose beads from Sigma Aldrich. The anti-phosphoSer280-5-HT2A receptor antibody was generated by immunizing rabbits with the synthetic GTRAKLApSFSFL+C peptide coupled to Keyhole Limpet Hemocyanin (KLH, Eurogentec, Liege, Belgium). The construct encoding the HA-tagged 5-HT2A receptor was described elsewhere (10.Becamel C. Gavarini S. Chanrion B. Alonso G. Galeotti N. Dumuis A. Bockaert J. Marin P. The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins.J. Biol. Chem. 2004; 279: 20257-20266Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Following PCR amplification, the receptor cDNA was subcloned into the bicistronic plasmid pIRES2-EGFP (Clontech, Mountain View, CA) using the XhoI/BamhI restriction sites. This construct was transferred to pSinRep5 plasmid for Sindbis virus production (11.Malinow R. Hayashi Y. Maletic-Savatic M. Zaman S.H. Poncer J.C. Shi S.H. Esteban J.A. Osten P. Seidenman K. Introduction of green fluorescent protein (GFP) into hippocampal neurons through viral infection.Cold Spring Harb. Protoc. 2010; (2010, pdb prot5406)Crossref PubMed Scopus (29) Google Scholar). Plasmids encoding HA-tagged 5-HT2A receptor mutants (S280A and S280D) were generated using the Quick Change mutagenesis kit (Stratagene, La Jolla, CA). All constructs were confirmed by DNA sequencing. HEK-293 cells, grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% dialyzed, heat-inactivated fetal calf serum and antibiotics, were transfected at 40–50% confluence using polyethyleneimine (PEI, Sigma-Aldrich), as previously described (12.Dubois F. Vandermoere F. Gernez A.l. Murphy J. Toth R. Chen S. Geraghty K.M. Morrice N.A. MacKintosh C. Differential 14–3-3 affinity capture reveals new downstream targets of phosphatidylinositol 3-kinase signaling.Mol. Cell. Proteomics. 2009; 8: 2487-2499Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and used 48 h after transfection. For stable isotope labeling by amino acids in cell culture (SILAC) experiments (13.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4577) Google Scholar), cells were maintained for 2 weeks in DMEM deficient in lysine and arginine, supplemented with 10% dialyzed serum and either l-lysine/l-arginine for light label (K0R0, L) or l-Lysine-2HCl (2H4, 96–98%)/l-Arginine-HCl (13C6, 99%) for semi-heavy label (K4R6, M) or l-Lysine-2HCl (13C6, 99%; 15N2, 99%)/l-Arginine-HCl (13C6, 99%; 15N4, 99%) for heavy label (K8R10, H) (percentages represent the isotopic purity of the labeled amino acids). Under these conditions, analysis of semi-heavy amino acid incorporation at the protein level indicated a median ratio of 93% (first quartile at 88%, third quartile at 95%). A similar distribution was observed for the incorporation of the heavy amino acids. Primary cultures of cortical neurons were prepared as described previously (14.Weiss S. Pin J.P. Sebben M. Kemp D.E. Sladeczek F. Gabrion J. Bockaert J. Synaptogenesis of cultured striatal neurons in serum-free medium: a morphological and biochemical study.Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 2238-2242Crossref PubMed Scopus (172) Google Scholar). Briefly, dissociated cells from the cerebral cortex of 17 day-old Swiss mouse embryos were plated on 6- or 96-well plates coated successively with poly-l-ornithine (mol. Wt. = 40,000; 15 μg/ml) and 10% fetal calf serum + 1 μg/ml laminin. The culture medium included a 1:1 mixture of DMEM and F-12 nutrient supplemented with 33 mm glucose, 2 mm glutamine, 13 mm NaHCO3, 5 mm HEPES buffer, pH 7.4, 5 IU/ml (5 mg/ml) penicillin-streptomycin, and a mixture of salt and hormones containing 100 μg/ml transferin, 25 μg/ml insulin, 20 nm progesterone, 60 nm putrescine, and 30 nm Na2SeO3. Cultures were infected 5 days after seeding with the Sindbis virus expressing HA-tagged 5-HT2A receptor and were used 7 days after seeding. At this stage, they were shown to contain at least 95% of neurons (14.Weiss S. Pin J.P. Sebben M. Kemp D.E. Sladeczek F. Gabrion J. Bockaert J. Synaptogenesis of cultured striatal neurons in serum-free medium: a morphological and biochemical study.Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 2238-2242Crossref PubMed Scopus (172) Google Scholar). HEK-293 cells grown in SILAC media and transiently expressing 5-HT2A receptors were serum-starved for 4 h and challenged for 15 min with either vehicle (L), or lisuride (1 μm, M), or DOI (1 μm, H). Cells were lysed in 0.5 ml of ice-cold lysis buffer (50 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1% Triton X-100, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.27 m sucrose, 1 mm DTT). Cell lysates were clarified by centrifugation at 15,000 × g (20 min at 4 °C) and protein concentration was determined using the Bradford reagent. Equal amounts of proteins (3 mg) from each condition were mixed, reduced with 10 mm DTT, alkylated with 50 mm iodoacetamide and precipitated on ice with trichloroacetic acid (25%, 20 min) before their digestion with trypsin (1/200, w/w) in 2 m urea, 25 mm triethylammonium bicarbonate pH 7.8. Digests were acidified in 1% TFA, desalted on a 1-g Sep-Pak cartridge (Waters, Milford, MA), and subjected to hydrophilic interaction liquid chromatography (HILIC) using a 4.6 × 250-mm TSKgel Amide-80 5-μm particle column (Tosoh Biosciences) and an Alliance e2695 HPLC system (Waters), as previously described (15.McNulty D.E. Annan R.S. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.Mol. Cell. Proteomics. 2008; 7: 971-980Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Nine mg of peptides were loaded in 80% solvent B (100% acetonitrile with 0.1% TFA). Solvent A consisted of 0.1% TFA in water. Peptides were eluted with a gradient consisting of 80% B held for 5 min followed by 80% B to 60% B in 40 min and finally 0% B for 5 min. Fourteen fractions were collected throughout the gradient and further enriched in phosphorylated peptides by immobilized metal affinity chromatography (IMAC) (15.McNulty D.E. Annan R.S. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.Mol. Cell. Proteomics. 2008; 7: 971-980Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). HILIC fractions were dried and resuspended in 25% acetonitrile/0.1% TFA and incubated for 3 h with 8 μl of Phos-Select beads (Sigma Aldrich) under agitation. Beads were rinsed twice with 100 μl of 25% acetonitrile/0.1% TFA and loaded on a microC18 ZipTip (Millipore). Phosphorylated peptides were then eluted in two steps with 30 μl of 0.4 m NH4OH and then with 30 μl of 50% acetonitrile. They were analyzed by nano-flow HPLC-nanoelectrospray ionization using a LTQ Orbitrap Velos mass spectrometer coupled to an Ultimate 3000 HPLC (Thermo Fisher Scientific). Desalting and preconcentration of samples were performed on-line on a Pepmap® precolumn (0.3 mm × 10 mm, Thermo Fisher Scientific, Courtaboeuf, France). A gradient consisting of 2–40% buffer B (3–33 min), 40–80% B (33–34 min), 80–0% B (49–50 min), and equilibrated for 20 min in 0% B (50–70 min) was used to elute peptides at 300 nL/min from a Pepmap® capillary (0.075 mm × 150 mm) reversed-phase column (LC Packings). Mass spectra were acquired using a top-10 collision-induced dissociation (CID) data-dependent acquisition (DDA) method. The LTQ-Orbitrap was programmed to perform a Fourier transform (FT) full scan (60,000 resolution) on 400–1400 Th mass range with the top ten ions from each scan selected for LTQ-MS/MS with multistage activation on the neutral loss of 24.49, 32.66, and 48.99 Th. FT spectra were internally calibrated using a single lock mass (445.1200 Th). Target ion numbers were 500,000 for FT full scan on the Orbitrap and 10,000 MSn on the LTQ. The raw MS data were analyzed using the MaxQuant/Andromeda software (v. 1.2.2.5) (16.Cox J.R. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar) with a false discovery rate of less than 0.01 for peptides and phosphosites and a minimum peptide length of six amino acids. The mass accuracy of the precursor ions was improved by retention time-dependent mass recalibration. Andromeda was used to search the top eight per 100 Da peak lists against the human complete proteome set database (http://www.uniprot.org/uniprot/?query = organism:9606+keyword:1185) downloaded on February 22, 2012 (65,835 protein entries), combined with 248 frequently observed contaminants as well as reversed versions of all sequences. This version of the database contains both reviewed sequences from UniProtKB/Swiss-Prot and unreviewed sequences from UniProtKB/TrEMBL. Enzyme specificity was set to trypsin, additionally allowing cleavage N-terminal to proline and up to two missed cleavages. The search included cysteine carbamidomethylation as a fixed modification, protein N-terminal acetylation, oxidation of methionine and phosphorylation of Ser, Thr, and Tyr as variable modifications. Peptide identification was based on a search with a mass deviation of the precursor ion up to 7 ppm after recalibration, and the allowed fragment mass deviation was set to 0.5 Da. Identifications across different replicates and adjacent fractions was performed using the “match between runs” MaxQuant option with a 3 min time window. Quantification of SILAC triplex signals was performed by MaxQuant with standard settings. The phosphoSTY.txt file generated by MaxQuant was uploaded onto Perseus software (v. 1.2.0.17) to calculate B significance of phosphopeptide ratios in each of the three biological replicates (16.Cox J.R. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar). The new table was then uploaded onto the R environment in order to plot log H/M ratios against log M/L ratios and color-display the B significance count (p < 0.05) for each quantified phosphopeptide. HEK-293 cells transiently expressing HA-tagged 5-HT2A receptors were lysed in 50 mm Tris-HCl, pH 7.5, 1 mm EGTA, 1% Triton X-100, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 0.27 m sucrose, 1 mm DTT, and a protease inhibitor mixture (Roche). Samples were centrifuged at 15,000 × g for 30 min at 4 °C. Solubilized 5-HT2A receptors were immunoprecipitated with the agarose-conjugated anti-HA antibody (Sigma Aldrich). Immunoprecipitated HA-5-HT2A receptors were resolved by SDS-PAGE. Gel bands containing the receptor were excised and digested with trypsin (500 ng per condition). Peptides were analyzed by nano-LC-FT-MS/MS, top six per 30 Da windows peak lists were extracted using MSconvert 3.0 and searched with Mascot 2.4 against the same human Complete Proteome Set database, with phosphorylation of Ser, Thr, and Tyr as variable modifications, 7 ppm precursor mass tolerance, 0.5 Da fragment mass tolerance and trypsin/P digestion. MS2 spectra matching phosphorylated peptides with ion score over 15 were inspected using Prophossi software (17.Martin D.M. Nett I.R. Vandermoere F. Barber J.D. Morrice N.A. Ferguson M.A. Prophossi: automating expert validation of phosphopeptide-spectrum matches from tandem mass spectrometry.Bioinformatics. 2010; 26: 2153-2159Crossref PubMed Scopus (16) Google Scholar) for automatic annotation of unique transitions that pinpoint the position of phosphorylation sites. Ion signals corresponding to phosphorylated peptides were quantified from the maximal intensities measured in their ion chromatograms manually extracted using Qual browser v2.1 (Thermo Fisher Scientific) with a tolerance of 5 ppm for mass deviation, and normalized to signals of their nonphosphorylated counterparts. Ser280 phosphorylation of immunoprecipitated receptors was also analyzed by Western blotting using the phosphosite specific antibody. Proteins, resolved onto 10% polyacrylamide gels, were transferred to Hybond C nitrocellulose membranes (GE Healthcare). Membranes were immunoblotted with primary antibodies (anti phospho-Ser280 5-HT2A receptor, 1:300; anti phospho-Thr202/Tyr204-Erk1,2, 1:1000; anti Erk1,2, 1:1000; anti-HA, 1:1000; Anti-GFP, 1:1000; anti-RSK2, 1:1000) and then with either anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:3000, GE Healthcare). Immunoreactivity was detected with an enhanced chemiluminescence method (ECLTM plus detection reagent, GE Healthcare) and immunoreactive bands were quantified by densitometry using the ImageJ software. In protein phosphorylation analyses, the amount of each phosphoprotein was normalized to the amount of the corresponding total protein detected in the sample. Experiments were performed on wild type or 5-HT2A receptor-deficient mice (8.Gonzalez-Maeso J. Yuen T. Ebersole B.J. Wurmbach E. Lira A. Zhou M. Weisstaub N. Hen R. Gingrich J.A. Sealfon S.C. Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex.J. Neurosci. 2003; 23: 8836-8843Crossref PubMed Google Scholar) and conformed to European ethics standards (86/609-EEC) and to decrees of the French National Ethics Committee (N° 87/848) for the care and use of laboratory animals. Mice (∼30 g) were injected intraperitoneal with either vehicle (5% DMSO/5% Tween 80) or DOI or lisuride (10 mg/kg each). Thirty min after the onset of the treatment, mice were anesthetized with pentobarbital (100 mg/kg intraperitoneal, Ceva SA) and rapidly perfused transcardiacally with fixative solution containing 4% w/v paraformaldehyde in 0.1 m sodium phosphate buffer (pH 7.5) containing NaF (100 mm) and sodium orthovanadate (1 mm). Brains were post-fixed for 48 h in the same solution and stored at 4 °C. Fifty micrometers-thick sections were cut with a vibratome (Leica), permeabilized with 0.2% Triton X-100 in Tris buffer saline (TBS) for 20 min, saturated for 1 h with 10% goat serum in TBS containing 0.03% Triton X-100 and incubated for 48 h at 4 °C with primary antibodies (anti phospho-Ser280 5HT2A receptor, 1:100 or anti 5-HT2A receptor, 1:500) in TBS. After four washes, they were incubated for 1 h with an Alexa Fluor® 488-conjugated anti-rabbit antibody (1:1000, Invitrogen) in TBS. Immunofluorescent staining was observed with a Zeiss Axioimager Z1 microscope equipped with apotome. Images were acquired using the Axiovision 4.8 software driving an AxioCam MRm CCD camera (Carl Zeiss Microimaging). Inositol phosphate production was analyzed as previously described (18.Chanrion B. Mannoury la Cour C. Gavarini S. Seimandi M. Vincent L. Pujol J.F. Bockaert J. Marin P. Millan M.J. Inverse agonist and neutral antagonist actions of antidepressants at recombinant and native 5-hydroxytryptamine2C receptors: differential modulation of cell surface expression and signal transduction.Mol. Pharmacol. 2008; 73: 748-757Crossref PubMed Scopus (90) Google Scholar). HEK-293 cells transiently expressing HA-tagged 5-HT2A receptors and grown on glass cover-slips were incubated with the rabbit anti HA antibody (1/500, 30 min at 10 °C) and then with drugs for 1 h at 37 °C. Cells were fixed in 4% (w/v) paraformaldehyde, 4% sucrose in PBS for 20 min, quenched 4 times 10 min with PBS containing 4% sucrose and 0.1 m glycine, and incubated for 60 min at 4 °C with an Alexa Fluor® 594-coupled anti-rabbit antibody (1:1000 in PBS supplemented with 2% goat serum, Invitrogen) to label cell surface receptors. They were then rinsed three times PBS containing 2% goat serum, permeabilized with 0.2% (w/v) Triton X-100 in PBS containing 2% goat serum for 15 min and incubated for 30 min at 4 °C with the Alexa Fluor® 488-coupled anti-rabbit antibody (1:1000 in PBS supplemented with 2% goat serum, 0.05% Triton X-100) to label internalized receptors. After three washes, coverslips were mounted on glass slides in Mowiol® 4.88 (Calbiochem). Series of optical sections were collected with a Zeiss Axioimager Z1 microscope equipped with apotome. Images were acquired using the Axiovision 4.8 software driving an AxioCam MRm CCD camera (Carl Zeiss Microimaging). Quantification of receptor cell surface expression was performed by ELISA under nonpermeabilized conditions as previously described (18.Chanrion B. Mannoury la Cour C. Gavarini S. Seimandi M. Vincent L. Pujol J.F. Bockaert J. Marin P. Millan M.J. Inverse agonist and neutral antagonist actions of antidepressants at recombinant and native 5-hydroxytryptamine2C receptors: differential modulation of cell surface expression and signal transduction.Mol. Pharmacol. 2008; 73: 748-757Crossref PubMed Scopus (90) Google Scholar). To directly compare the phosphorylation patterns generated upon 5-HT2A receptor stimulation by a hallucinogenic (DOI) and by a nonhallucinogenic (lisuride) agonist, we used the SILAC technology under three experimental conditions: light condition (vehicle-treated cells), semiheavy label (lisuride-treated cells), and heavy label (DOI-treated cells). As cultured neurons do not divide in vitro, a complete SILAC labeling prerequisite for unbiased quantification could not be achieved in these cultures. Therefore, we performed our phosphoproteomics screen in HEK-293 cells transiently expressing 5-HT2A receptors. We first examined whether this model recapitulates the biased signaling at 5-HT2A receptors initially described in neurons, i.e. specific activation of Gi/o signaling by hallucinogens (7.Gonzalez-Maeso J. Weisstaub N.V. Zhou M. Chan P. Ivic L. Ang R. Lira A. Bradley-Moore M. Ge Y. Zhou Q. Sealfon S.C. Gingrich J.A. Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior.Neuron. 2007; 53: 439-452Abstract Full Text Full Text PDF PubMed Scopus (550) Google Scholar). Exposure of cells to D" @default.
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• W2116715079 date "2014-05-01" @default.
• W2116715079 modified "2023-10-14" @default.
• W2116715079 title "Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists" @default.
• W2116715079 cites W1503765703 @default.
• W2116715079 cites W1625702540 @default.
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