Serotonin 2A receptor disulfide bridge integrity is crucial for ligand binding to different signalling states but not for its homodimerization
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- dc.contributor.author Iglesias, Albaca
- dc.contributor.author Cimadevila, Martaca
- dc.contributor.author de la Fuente, Rocío Ailimca
- dc.contributor.author Martí Solano, Mariaca
- dc.contributor.author Cadavid, María Isabelca
- dc.contributor.author Castro, Mariánca
- dc.contributor.author Selent, Janaca
- dc.contributor.author Loza, María I.ca
- dc.contributor.author Brea, Joséca
- dc.date.accessioned 2018-05-23T07:28:25Z
- dc.date.issued 2017
- dc.description.abstract 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.
- dc.description.sponsorship 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).
- dc.format.mimetype application/pdf
- dc.identifier.citation Iglesias A, Cimadevila M, la Fuente RA, Martí-Solano M, Cadavid MI, Castro M et al. Serotonin 2A receptor disulfide bridge integrity is crucial for ligand binding to different signalling states but not for its homodimerization. Eur J Pharmacol. 2017 Nov 15;815:138-146. DOI: 10.1016/j.ejphar.2017.09.011
- dc.identifier.doi http://dx.doi.org/10.1016/j.ejphar.2017.09.011
- dc.identifier.issn 0014-2999
- dc.identifier.uri http://hdl.handle.net/10230/34704
- dc.language.iso eng
- dc.publisher Elsevierca
- dc.relation.ispartof European Journal of Pharmacology. 2017 Nov 15;815:138-46
- dc.relation.projectID info:eu-repo/grantAgreement/ES/3PN/SAF2009-13609-C04-01
- dc.relation.projectID info:eu-repo/grantAgreement/ES/1PE/SAF2014-57138-C2-2R
- dc.rights © Elsevier http://dx.doi.org/10.1016/j.ejphar.2017.09.011
- dc.rights.accessRights info:eu-repo/semantics/openAccess
- dc.subject.keyword Clozapine
- dc.subject.keyword DL-1,4-Dithiothreitol
- dc.subject.keyword Disulfide bridges
- dc.subject.keyword Dithiothreitol
- dc.subject.keyword Methysergide maleate
- dc.subject.keyword Phospholipase
- dc.subject.keyword Phospholipase C
- dc.subject.keyword Radioligand binding
- dc.subject.keyword Serotonin 2(A) receptor
- dc.subject.keyword Serotonin hydrochloride
- dc.subject.keyword Serotonina--Receptors
- dc.title Serotonin 2A receptor disulfide bridge integrity is crucial for ligand binding to different signalling states but not for its homodimerizationca
- dc.type info:eu-repo/semantics/article
- dc.type.version info:eu-repo/semantics/acceptedVersion