Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser 280 by Hallucinogenic versus Nonhallucinogenic Agonists Samah Karaki, Carine Becamel, Samy Murat, Clotilde Mannoury La Cour, Mark Millan, Laurent Prézeau, Joël Bockaert, Philippe Marin, Franck Vandermoere To cite this version: Samah Karaki, Carine Becamel, Samy Murat, Clotilde Mannoury La Cour, Mark Millan, et al.. Quan- titative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser 280 by Hallucinogenic versus Nonhallucinogenic Agonists. Molecular and Cellular Proteomics, American Soci- ety for Biochemistry and Molecular Biology, 2014, 13 (5), pp.1273-1285. 10.1074/mcp.M113.036558. hal-02366017 HAL Id: hal-02366017 https://hal.archives-ouvertes.fr/hal-02366017 Submitted on 15 Nov 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Research © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists*□S Samah Karaki‡§¶ʈ, Carine Becamel‡§¶ʈ, Samy Murat‡‡¶ʈ, Clotilde Mannoury la Cour**, Mark J. Millan**, Laurent Pre´ zeau‡§¶ʈ, Joe¨ l Bockaert‡§¶ʈ, Philippe Marin‡§¶ʈ§§, and Franck Vandermoere‡§¶ʈ‡‡§§ The serotonin 5-HT2A receptor is a primary target of psy- induced a less pronounced desensitization of receptor- chedelic hallucinogens such as lysergic acid diethyl- operated signaling in HEK-293 cells and neurons than did amine, mescaline, and psilocybin, which reproduce some nonhallucinogenic agonists. The mutation of Ser280 to as- of the core symptoms of schizophrenia. An incompletely partic acid (to mimic phosphorylation) reduced receptor resolved paradox is that only some 5-HT2A receptor ago- desensitization by nonhallucinogenic agonists, whereas nists exhibit hallucinogenic activity, whereas structurally its mutation to alanine increased the ability of hallucino- related agonists with comparable affinity and activity lack gens to desensitize the receptor. This study reveals a such a psychoactive activity. Using a strategy combining biased phosphorylation of the 5-HT2A receptor in re- stable isotope labeling by amino acids in cell culture with sponse to hallucinogenic versus nonhallucinogenic ago- enrichment in phosphorylated peptides by means of hy- nists, which underlies their distinct capacity to desensi- drophilic interaction liquid chromatography followed by tize the receptor. Molecular & Cellular Proteomics 13: immobilized metal affinity chromatography, we compared 10.1074/mcp.M113.036558, 1273–1285, 2014. the phosphoproteome in HEK-293 cells transiently express- ing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2- Among the G Protein-Coupled Receptors (GPCRs)1 acti- aminopropane (DOI) or the nonhallucinogenic 5-HT2A ago- nist lisuride. Among the 5995 identified phosphorylated vated by serotonin (5-hydroxytryptamine, 5-HT), the 5-HT2A peptides, 16 sites were differentially phosphorylated upon receptor continues to attract particular attention in view of its exposure of cells to DOI versus lisuride. These include a broad physiological role and implication in the actions of serine (Ser280) located in the third intracellular loop of the numerous psychotropic agents (1, 2). It is a primary target of 5-HT2A receptor, a region important for its desensitiza- widely used atypical antipsychotics such as clozapine, risperi- tion. The specific phosphorylation of Ser280 by hallucino- done, and olanzapine, which act as antagonists or inverse gens was further validated by quantitative mass spec- agonists (1, 3). The activation of 5-HT2A receptors expressed trometry analysis of immunopurified receptor digests and in the prefrontal cortex has also been implicated in the psy- by Western blotting using a phosphosite specific anti- cho-mimetic effects of psychedelic hallucinogens, such as body. The administration of DOI, but not of lisuride, to lysergic acid diethylamide (LSD), mescaline, and psilocybin, mice, enhanced the phosphorylation of 5-HT receptors 2A which are often used to model positive symptoms of schizo- at Ser280 in the prefrontal cortex. Moreover, hallucinogens phrenia (4–8). However, these psychoactive effects are not reproduced by structurally-related agonists, such as ergota- From the ‡CNRS, UMR-5203, Institut de Ge´ nomique Fonctionnelle, mine and the anti-Parkinson agent lisuride, despite the fact F-34094 Montpellier, France; §INSERM, U661, F-34094 Montpellier, that they exhibit comparable affinities and efficacies at 5-HT2A France; ¶Universite´ Montpellier 1, F-34094 Montpellier, France; ʈUni- receptors (7, 9). This paradox was partially resolved by the versite´ Montpellier 2, F-34094 Montpellier, France; **Institut de Re- demonstration that hallucinogens induce a specific transcrip- cherches Servier, Croissy-sur-Seine, France Received November 27, 2013, and in revised form, February 19, 2014 1 The abbreviations used are: GPCR, G protein-coupled receptor; Published, MCP Papers in Press, March 17, 2014, DOI 10.1074/ 5-HT, 5-hydroxytryptamine, serotonin; DOI, 1-[2,5-dimethoxy-4-iodo- mcp.M113.036558 phenyl]-2-aminopropane; HILIC, hydrophilic interaction liquid chro- Author contributions: C.M., M.J.M., J.B., P.M., and F.V. designed matography; LSD, lysergic acid diethylamine; PKB, Protein Kinase B; research; S.K., S.M., P.M., and F.V. performed research; C.B. and PKC, Protein Kinase C; PLC, phospholipase C; PTX, Pertussis toxin; L.P. contributed new reagents or analytic tools; S.K. and F.V. ana- RSK, Ribosomal S6 kinase; SILAC, stable isotope labeling by amino lyzed data; M.J.M., J.B., P.M., and F.V. wrote the paper. acids in cell culture. Molecular & Cellular Proteomics 13.5 1273 Biased 5-HT2A Receptor Phosphorylation tomic signature because of the specific engagement of a and arginine, supplemented with 10% dialyzed serum and either 2 Pertussis toxin-sensitive G /Src signaling pathway which is L-lysine/L-arginine for light label (K0R0, L) or L-Lysine-2HCl ( H4, i/o 13 96–98%)/L-Arginine-HCl ( C6, 99%) for semi-heavy label (K4R6, M) not activated by nonhallucinogenic agonists (7, 8). These 13 15 13 or L-Lysine-2HCl ( C6, 99%; N2, 99%)/L-Arginine-HCl ( C6, 99%; findings suggest that hallucinogenic and nonhallucinogenic 15 N4, 99%) for heavy label (K8R10, H) (percentages represent the agonists induce different conformational states of the 5-HT2A isotopic purity of the labeled amino acids). Under these conditions, receptor, and represent a striking example of functional se- analysis of semi-heavy amino acid incorporation at the protein level lectivity that translates into contrasting pattern of mice behav- indicated a median ratio of 93% (first quartile at 88%, third quartile at ior: induction of head-twitches by hallucinogenic but not by 95%). A similar distribution was observed for the incorporation of the heavy amino acids. nonhallucinogenic agonists (9). Primary cultures of cortical neurons were prepared as described The differential influence of hallucinogenic versus nonhallu- previously (14). Briefly, dissociated cells from the cerebral cortex of 17 cinogenic agonists on signaling pathways suggests that they day-old Swiss mouse embryos were plated on 6- or 96-well plates trigger contrasting patterns of protein phosphorylation. To coated successively with poly-l-ornithine (mol. Wt. ϭ 40,000; 15 ␮ ϩ ␮ address this issue, we employed a quantitative phosphopro- g/ml) and 10% fetal calf serum 1 g/ml laminin. The culture medium included a 1:1 mixture of DMEM and F-12 nutrient supple- teomics strategy to directly compare the phosphoproteomes mented with 33 mM glucose, 2 mM glutamine, 13 mM NaHCO3,5mM generated in HEK-293 cells by the synthetic hallucinogen HEPES buffer, pH 7.4, 5 IU/ml (5 mg/ml) penicillin-streptomycin, and 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) and a mixture of salt and hormones containing 100 ␮g/ml transferin, 25 ␮ the nonhallucinogenic 5-HT2A agonist lisuride. We found that g/ml insulin, 20 nM progesterone, 60 nM putrescine, and 30 nM DOI, but not lisuride, induced the phosphorylation of a serine Na2SeO3. Cultures were infected 5 days after seeding with the Sind- bis virus expressing HA-tagged 5-HT receptor and were used 7 residue (Ser280) located in the third intracellular loop of the 2A days after seeding. At this stage, they were shown to contain at least receptor itself. The hallucinogen-specific phosphorylation of 95% of neurons (14). this residue was further validated in vitro and in vivo by using Global Quantitative Phosphoproteomics Analyses—HEK-293 cells a phosphosite-specific antibody. These findings were fol- grown in SILAC media and transiently expressing 5-HT2A receptors lowed by a series of experiments to determine the impact of were serum-starved for 4 h and challenged for 15 min with either ␮ ␮ Ser280 phosphorylation upon receptor desensitization and vehicle (L), or lisuride