Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists

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.

Psychedelics Recruit Multiple Cellular Types and Produce Complex Transcriptional Responses Within the Brain

“…Our results indicate that a large number of 5-HT 2A -expressing cells undergo dramatic receptor redistribution to the soma following ( R )-DOI in all frontal areas that normally express 5-HT 2A receptors, and we have also observed this phenomena following LSD (unpublished observations). These results are consistent with agonist-induced receptor internalization, which has been observed previously for the 5-HT 2A receptor in vitro ( Berry et al, 1996 , Karaki et al, 2014 ). Interestingly, many neurons that internalize 5-HT 2A receptors upon ( R )-DOI administration do not express cFos.…”

What Have Mass Spectrometry-Based Proteomics and Metabolomics (Not) Taught Us about Psychiatric Disorders?

“…An example of the potential that the study of PTMs holds for elucidating the regulation of molecular events involved in psychiatric phenotypes includes a global quantitative phosphoproteomics analysis based on stable isotope labeling in cells which revealed differences in serotonin receptor phosphorylation when exposed to hallucinogenic versus non-hallucinogenic agonists. These results were also confirmed in vivo in mice [52]. Since hallucinations are a core symptom of schizophrenia, this work may pave the way for more effective treatments of positive symptoms of this disorder.…”

PGC-1α/ERRα-Sirt3 Pathway Regulates DAergic Neuronal Death by Directly Deacetylating SOD2 and ATP Synthase β

“…The primary neuron cultures were derived from the midbrain or cortex of E16 ICR mice or Sprague-Dawley (SD) rats (Vitalriver, Beijing, China) as described previously (25,57,59).…”