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

Abstract

The serotonin 2 (5-HT) receptor is a G-protein coupled receptor (GPCR) with a conserved disulfide bridge formed by Cys (transmembrane helix 3, TM3) and Cys (extracellular loop 2, ECL-2). We hypothesized that disulfide bridges may determine serotonin 5-HT 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-HT 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-HT receptors.

A Biomarker to Differentiate between Primary and Cocaine-Induced Major Depression in Cocaine Use Disorder: The Role of Platelet IRAS/Nischarin (I1-Imidazoline Receptor)

“…Also, these biomarkers would improve the knowledge of neurobiological mechanisms of both types of depression associated to CUD and facilitate the appropriate treatment. In addition, comparable results (Trp depletion: 86–87%) have been reported in studies using a similar protocol in pure MDD ( 19 , 33 ).…”

“…The 5-HT 2A receptor antibody used [validated in brain tissue of 5-HT 2A receptor KO mice ( 30 )], immunodetected the platelet monomeric receptor as a ~60 kDa band and a ~120 kDa peptide which most probably represents (detected at twice the molecular mass) the homodimeric form of the 5-HT 2A receptor ( 31 , 32 ) resistant to the denaturalizing assay conditions (see Figure 1 ). In addition to the possibility of 5-HT 2A receptor heterodimerization ( 31 , 32 ), more recent studies have demonstrated that this serotonin receptor subtype can form functional homodimers resistant to the reducing agent dithiothreitol ( 33 , 34 ). The IRAS/nischarin antibody has also been validated in previous studies ( 15 , 16 ).…”

Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro

“…1, pEC 50 = 9.24 ± 0.17; t(9) = 2.56, p < 0.05). These data are consistent with previous reports showing a shift in the potency of the Ser23 allele relative to the Cys23 allele 27,29,43 . The 5-HT peak response in the Ser23 allele-expressing cells (Fig.…”

“…1; E max = 101.4 ± 7.82%; t(9) = 4.16, p < 0.05). As expression of the Ser23 allele results in the loss of the single cysteine residue in the N-terminus, the removal of the Cys-dependent disulfide bond formation may alter the conformation of the 5-HT 2C R and ultimately impact the potency of 5-HT to activate the Ser23 variant 43,44 .

Figure 1Serotonin-induced intracellular calcium release is lower in the Ser23-CHOp38 vs. Cys23-CHOp38 stably expressing cell lines. Average traces of a 5-HT induced concentration-dependent release in Cys23 and Ser23 5-HT 2C R-CHOp38 cells (normalized to the average 1 µM 5-HT response in the Cys23 5-HT 2C R-CHOp38 to give a percent response).

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Coupling of a viral K+-channel with a glutamate-binding-domain highlights the modular design of ionotropic glutamate-receptors

“…To test this hypothesis the two residues were substituted by cysteine. The resulting GluATCV* ΔNTD ΔM4 V152C/N407C construct and the GluATCV* and GluATCV* ΔNTD ΔM4 receptors were expressed in oocytes and the Glu-current response recorded in the absence and presence of dithiothreitol (DTT), which reduces disulfide bonds between cysteine residues 32 . The resulting Glu-dose–response curves in the absence of DTT were indistinguishable from those of the M4 containing GluATCV* receptors (GluATCV* and GluATCV* ΔNTD ).…”