Functional Selectivity in Serotonin Receptor 2A (5-HT_2A) Endocytosis, Recycling, and Phosphorylation

Abstract

G protein-coupled receptor (GPCR) signaling is modulated by endocytosis and endosomal sorting of receptors between degradation and recycling. Differential regulation of these processes by endogenous ligands and synthetic drugs is a poorly understood area of GPCR signaling. Here, we describe remarkable diversity in the regulation of trafficking of GPCR induced by multiple ligands. We show that the serotonin receptor 2A (5-HT 2A ), a prototypical GPCR in the study of functional selectivity at a signaling receptor, is functionally selective in endocytosis and recycling in response to five ligands tested: endogenous agonists serotonin (5-HT) and dopamine (DA), synthetic agonist 1-(2,5-dimethoxy-4-iodophenyl)-aminopropane (DOI), antagonist ketanserin, and inverse agonist and antipsychotic drug clozapine. Only four ligands (5-HT, DA, DOI, and clozapine) bring about receptor endocytosis. As we have earlier described with 5-HT and DA, there is ligand-specific requirement for protein kinase C (PKC) in endocytosis. We now show 5-HT 2A phosphorylation by PKC is necessary for 5-HT-mediated and DOI-mediated receptor endocytosis, but DA-mediated and clozapine-mediated internalization is not affected if PKC is inhibited. Internalized receptors are recycled to the cell surface, but there is variability in the time course of recycling. 5-HT-and DA-internalized receptors are recycled in 2.5 hours while agonist DOI and antagonist clozapine bring about recycling in 7.5 hours. Recycling in response to those ligands that require PKC activation to effect receptor endocytosis is dependent on receptor dephosphorylation by protein phosphatase 2A (PP2A). Thus, internalization and phosphorylation/dephosphorylation cycles may play a significant role in the regulation of 5-HT 2A by functionally and therapeutically important ligands.

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

“…To our knowledge, no study has so far compared the ability of hallucinogenic and nonhallucinogenic compounds to desensitize 5-HT 2A receptors. Nonetheless, the present findings are consistent with a recent study, which demonstrated that DOI was less efficient than 5-HT to internalize eGFP-tagged 5-HT 2A receptors stably expressed in HEK-293 cells (27). They also provide convergent evidence indicating that the different effects of hallucinogenic versus non hallucinogenic agonists upon receptor desensitization reflect their differential capacity to promote Ser 280 phosphorylation: (1) mutating Ser 280 into alanine or aspartate abolished the difference in the agonist effects upon receptor desensitization; (2) hallucinogens were able to desensitize S 280 A receptor to an extent comparable to that induced by nonhallucinogenic agonists in cells expressing wild type receptor and (3) nonhallucinogenic agonists did not promote desensitization of the Ser 280 D receptor mutant.…”

“…Given the role of PKC in 5-HT 2A receptor desensitization and internalization and the importance of the 5-HT 2A receptor i3 loop in the regulation of receptor responsiveness (26–29), we next examined whether hallucinogenic and nonhallucinogenic agonists differentially modulate receptor desensitization. Pretreatment of HEK-293 cells with either lisuride or ergotamine for 1 h, followed by extensive drug washout, inhibited inositol phosphate production induced by a further exposure of cells to 5-HT, whereas pretreating cells with DOI or LSD did not significantly affect the 5-HT response (Figs.…”

A High-ResolutionIn VivoAtlas of the Human Brain's Serotonin System

“…In addition, the 5-HT 2A R mRNA levels are almost twice as high compared with the other investigated targets. We speculate that high mRNA levels enable the system to regulate synaptic 5-HT 2A R levels quickly, consistent with the ligand-induced endocytosis and recycling of 5-HT 2A R (Raote et al, 2013). We observed a moderate correlation association between 5-HT 4 R mRNA and 5-HT 4 R protein density (Fig.…”

Psychedelics

“…For example, PKC phosphorylation of serine residue S291 in the 5-HT 2A receptor is also thought to play a role in modulation of receptor signaling. Using five different ligands that activate the 5-HT 2A receptor, Raote et al (2013) showed that different biochemical pathways are involved in receptor trafficking. These investigators used the fluorescence of rat 5-HT 2A enhanced green fluorescent protein (EGFP) to follow receptor internalization.…”