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.
Highlights
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]-2aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride
Exposure of cells to DOI or lysergic acid diethylamide (LSD) induced comparable stimulation of inositol phosphate production and Extracellular-regulated kinase (Erk)1,2 phosphorylation to those elicited by two nonhallucinogenic agonists, lisuride and ergotamine, though activation of PLC by DOI and Erk1,2 phosphorylation elicited by LSD were slightly more pronounced
Pre-treating cells with Pertussis toxin (PTX) decreased PLC activation induced by DOI and LSD and abolished Erk1,2 phosphorylation induced by both hallucinogens, whereas PTX treatment did not affect the lisuride and ergotamine responses
Summary
Materials—Human Embryonic Kidney-293 (HEK-293) cells were from the European Collection of Cell Cultures, culture media from Invitrogen (Carlsbad, CA). Peptides were analyzed by nanoLC-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. 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. ELISA—Quantification of receptor cell surface expression was performed by ELISA under nonpermeabilized conditions as previously described (18)
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