Serotonin 2A receptor disulfide bridge integrity is crucial for ligand binding to different signalling states but not for its homodimerization
| Autor: | María Isabel Loza, María Isabel Cadavid, Jana Selent, Marta Cimadevila, Marián Castro, Maria Marti-Solano, José Brea, Rocio A. de la Fuente, Alba Iglesias |
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| Rok vydání: | 2017 |
| Předmět: |
Models
Molecular 0301 basic medicine Allosteric regulation CHO Cells Ligands Dithiothreitol 03 medical and health sciences chemistry.chemical_compound Cricetulus 0302 clinical medicine Radioligand binding Phospholipase C Cricetinae Functional selectivity Animals Humans Receptor Serotonin 5-HT2A Amino Acid Sequence Disulfides Phospholipase Serotonina--Receptors Binding site Protein Structure Quaternary Receptor Clozapine Serotonin hydrochloride G protein-coupled receptor Pharmacology Methysergide maleate Binding Sites Chemistry Serotonin 2(A) receptor Nuclear Proteins Disulfide bridges Ligand (biochemistry) 030104 developmental biology Biochemistry DL-1 4-Dithiothreitol Biophysics Protein Multimerization 030217 neurology & neurosurgery Protein Binding Signal Transduction Cysteine |
| Zdroj: | Recercat. Dipósit de la Recerca de Catalunya instname |
| ISSN: | 0014-2999 |
| Popis: | 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). |
| Databáze: | OpenAIRE |
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