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-HT and glutamate mGlu receptors continue to attract particular attention, given their implication in psychosis associated with schizophrenia and the mechanism of action of atypical antipsychotics and a new class of antipsychotics, respectively. A large body of evidence indicates a functional crosstalk between both receptors in the brain, but the underlying mechanisms are not entirely elucidated. Here, we have explored the influence of 5-HT receptor upon the phosphorylation pattern of mGlu receptor in light of the importance of specific phosphorylation events in regulating G protein-coupled receptor signaling and physiological outcomes. Among the five mGlu receptor-phosphorylated residues identified in HEK-293 cells, the phosphorylation of Ser was enhanced upon mGlu receptor stimulation by the orthosteric agonist LY379268 only in cells co-expressing the 5-HT receptor. Likewise, administration of LY379268 increased mGlu receptor phosphorylation at Ser in prefrontal cortex of wild-type mice but not 5-HT mice. Exposure of HEK-293 cells co-expressing mGlu and 5-HT receptors to 5-HT also increased Ser phosphorylation state to a magnitude similar to that measured in LY379268-treated cells. In both HEK-293 cells and prefrontal cortex, Ser phosphorylation elicited by 5-HT receptor stimulation was prevented by the mGlu receptor antagonist LY341495, while the LY379268-induced effect was abolished by the 5-HT receptor antagonist M100907. Mutation of Ser into alanine strongly reduced G signaling elicited by mGlu or 5-HT receptor stimulation in cells co-expressing both receptors. Collectively, these findings identify mGlu receptor phosphorylation at Ser as a key molecular event that underlies the functional crosstalk between both receptors.

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