Abstract
The serotonin 5-HT 2A receptor is a primary target of psy-chedelic hallucinogens such as lysergic acid diethyl-amine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT 2A receptor ago-nists 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 hy-drophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT 2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT 2A ago-nist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser 280) located in the third intracellular loop of the 5-HT 2A receptor, a region important for its desensitiza-tion. The specific phosphorylation of Ser 280 by hallucino-gens was further validated by quantitative mass spec-trometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific anti-body. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT 2A receptors at Ser 280 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 Ser 280 to as-partic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucino-gens to desensitize the receptor. This study reveals a biased phosphorylation of the 5-HT 2A receptor in response to hallucinogenic versus nonhallucinogenic ago-nists, which underlies their distinct capacity to desensi-tize the receptor. Molecular & Cellular Proteomics 13: 10.1074/mcp.M113.036558, 1273-1285, 2014. Among the G Protein-Coupled Receptors (GPCRs) 1 activated by serotonin (5-hydroxytryptamine, 5-HT), the 5-HT 2A receptor continues to attract particular attention in view of its broad physiological role and implication in the actions of numerous psychotropic agents (1, 2). It is a primary target of widely used atypical antipsychotics such as clozapine, risperi-done, and olanzapine, which act as antagonists or inverse agonists (1, 3). The activation of 5-HT 2A receptors expressed in the prefrontal cortex has also been implicated in the psy-cho-mimetic effects of psychedelic hallucinogens, such as lysergic acid diethylamide (LSD), mescaline, and psilocybin, which are often used to model positive symptoms of schizo-phrenia (4-8). However, these psychoactive effects are not reproduced by structurally-related agonists, such as ergota-mine and the anti-Parkinson agent lisuride, despite the fact that they exhibit comparable affinities and efficacies at 5-HT 2A receptors (7, 9). This paradox was partially resolved by the demonstration that hallucinogens induce a specific transcrip-From the ‡CNRS,