Serotonin 2A receptor disulfide bridge integrity is crucial for ligand binding to different signalling states but not for its homodimerization.
Alba Iglesias, Marta Cimadevila, Rocío Ailim de La Fuente, María Martí-Solano, María Isabel Cadavid, Marián Castro, Jana Selent, María Isabel Loza, José Brea
European Journal of Pharmacology November 15, 2017 DOI: 10.1016/j.ejphar.2017.09.011 (opens in new tab)
Study at a glance
AI-extracted from the abstract| Characteristics | In vitro experimental study Peer reviewed |
|---|---|
| Intervention | Dithiothreitol (DTT) |
| Measures | Radioligand binding, bioluminescence resonance energy transfer (BRET), molecular dynamic simulations |
| Topics | Serotonin |
| Keywords | Clozapine pubchem cid Dl-1,4-dithiothreitol pubchem cid Disulfide bridges Phospholipase a2 Phospholipase c Radioligand binding Serotonin 2a receptor 3hlsd pubchem cid |
| Key findings | Reducing disulfide bridges with dithiothreitol decreased serotonin 5-HT2A receptor binding sites and the efficacy of both signaling pathways without changing affinity or potency, and did not affect homodimerization. Simulations suggest a broken cysteine bond makes extracellular loop 2 more flexible, sometimes collapsing the ligand binding pocket. The authors propose this disulfide bridge integrity is important for receptor function and ligand recognition. |
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.
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