Serotonin 2A receptor disulfide bridge integrity is crucial for ligand binding to different signalling states but not for its homodimerization

The serotonin 2A (5-HT2A) receptor is a G-protein coupled receptor (GPCR) with a conserved disulfide bridge formed by Cys148 (transmembrane helix 3, TM3) and Cys227 (extracellular loop 2, ECL-2). We hypothesized that disulfide bridges may determine serotonin 5-HT2A receptor functions such as receptor activation, functional selectivity and ligand recognition. We used the reducing agent dithiothreitol (DTT) to determine how the reduction of disulfide bridges affects radioligand binding, second messenger mobilization and receptor dimerization. A DTT-induced decrease in the number of binding sites (1190 ± 63.55 fmol/mg protein for control cells compared with 921.2 ± 60.84 fmol/mg protein for DTT-treated cells) as well as in the efficacy of both signalling pathways characterized was observed, although the affinity and potency were unchanged. Bioluminiscence resonance energy transfer (BRET) assays revealed the DTT treatment did not modify the homodimeric nature of serotonin 5-HT2A receptors. In molecular dynamic simulations, the ECL-2 of the receptor with a broken cysteine bond adopts a wider variety of conformations, some of which protrude deeper into the receptor orthosteric binding pocket leading to collapse of the pocket. A shrunken binding pocket would be incapable of accommodating lysergic acid diethylamide (LSD). Our findings suggest that the decrease of efficacy may be due to disruption of disulfide bridge between TM3 and ECL-2. This reveals the integrity of the ECL-2 epitope, which should be explored in the development of novel ligands acting as allosteric modulators of serotonin 5-HT2A receptors.his work was supported by Ministerio de Ciencia e Innovacion (SAF2009-13609-C04-01), Ministerio de Economia y Competitividad (SAF2014-57138-C2-2R), and Innopharma project (PI12/00742; Ministerio de Economía y Competitividad -FEDER). A.I. was recipient of a FPI fellowship from Ministerio de Ciencia e Innovacion. M. Cimadevila is recipient of a financial support from the Xunta de Galicia (ED481A-2016/096) and the European Union (European Social Fund - ESF)

Serotonin 2A receptor disulfide bridge integrity is crucial for ligand binding to different signalling states but not for its homodimerization

The serotonin 2A (5-HT2A) receptor is a G-protein coupled receptor (GPCR) with a conserved disulfide bridge formed by Cys148 (transmembrane helix 3, TM3) and Cys227 (extracellular loop 2, ECL-2). We hypothesized that disulfide bridges may determine serotonin 5-HT2A receptor functions such as receptor activation, functional selectivity and ligand recognition. We used the reducing agent dithiothreitol (DTT) to determine how the reduction of disulfide bridges affects radioligand binding, second messenger mobilization and receptor dimerization. A DTT-induced decrease in the number of binding sites (1190 ± 63.55 fmol/mg protein for control cells compared with 921.2 ± 60.84 fmol/mg protein for DTT-treated cells) as well as in the efficacy of both signalling pathways characterized was observed, although the affinity and potency were unchanged. Bioluminiscence resonance energy transfer (BRET) assays revealed the DTT treatment did not modify the homodimeric nature of serotonin 5-HT2A receptors. In molecular dynamic simulations, the ECL-2 of the receptor with a broken cysteine bond adopts a wider variety of conformations, some of which protrude deeper into the receptor orthosteric binding pocket leading to collapse of the pocket. A shrunken binding pocket would be incapable of accommodating lysergic acid diethylamide (LSD). Our findings suggest that the decrease of efficacy may be due to disruption of disulfide bridge between TM3 and ECL-2. This reveals the integrity of the ECL-2 epitope, which should be explored in the development of novel ligands acting as allosteric modulators of serotonin 5-HT2A receptors.his work was supported by Ministerio de Ciencia e Innovacion (SAF2009-13609-C04-01), Ministerio de Economia y Competitividad (SAF2014-57138-C2-2R), and Innopharma project (PI12/00742; Ministerio de Economía y Competitividad -FEDER). A.I. was recipient of a FPI fellowship from Ministerio de Ciencia e Innovacion. M. Cimadevila is recipient of a financial support from the Xunta de Galicia (ED481A-2016/096) and the European Union (European Social Fund - ESF)

Development of fluorescent probes that target serotonin 5-HT2B receptors

Some 5-HT2B fluorescent probes were obtained by tagging 1-(2,5-dimethoxy-4-iodophenyl)-propan-2-amine (DOI) with a subset of fluorescent amines. Some of the resulting fluorescent ligands showed excellent affinity and selectivity profiles at the 5-HT2B receptors (e.g. 12b), while retain the agonistic functional behaviour of the model ligand (DOI). The study highlighted the most salient features of the structure-activity relationship in this series and these were substantiated by a molecular modelling study based on a receptor-driven docking model constructed on the basis of the crystal structure of the human 5-HT2B receptor. One of the fluorescent ligands developed in this work, compound 12i, specifically labelled CHO-K1 cells expressing 5-HT2B receptors and not parental CHO-K1 cells in a concentration-dependent manner. 12i enables imaging and quantification of specific 5-HT2B receptor labelling in live cells by automated fluorescence microscopy as well as quantification by measurements of fluorescence intensity using a fluorescence plate reader.This research was carried out within the framework of the Cost Action GLISTEN and financially supported by the Spanish Government (grant numbers SAF2009-13609-C04-03 and GPC2014/003 (PS09/63) to E.S. and SAF2014-57138-C2-1-R to M.C. and M.I.L.). Authors also thanks financial support from Consellería de Cultura, Educación e Ordenación Universitaria of the Galician Government: (grant: GPC2014/03), Centro Singular de Investigación de Galicia accreditation 2016-2019 (ED431G/09). J. S. acknowledges financial support from Instituto de Salud Carlos III FEDER (CP12/03139 and PI15/00460)