Essential role of the C148–C227 disulphide bridge in the human 5-HT2A homodimeric receptor

The 5-HT receptor is a homodimeric G protein-coupled receptor implied in multiple diseases, including schizophrenia. Recently, its co-crystallisation with the antipsychotic drugs zotepine and risperidone has revealed the importance of its extracellular domains in its pharmacology. Previous studies have shown that the non-specific disruption of extracellular disulphide bridges in the 5-HT receptor decreases ligand binding and receptor activation. There is enough evidence to hypothesize that this decrease may be due to a reduction of the disulphide bridge that links transmembrane domain 3 (TM-3) and extracellular loop 2 (ECL-2) of the 5-HT receptor via cysteine 148 (C148) and C227. Thus, to study the influence of the C148–C227 disulphide bridge on 5-HT receptor pharmacology, we substituted C148 and C227 in the human 5-HT receptor (WT) with alanines, to obtain two single mutants (C148A and C227A) and a double mutant (C148A/C227A), and the resultant DNA constructs were used to generate four stable cell lines. These substitutions reduced the binding of the 5-HT receptor to [H]lysergic acid diethylamide ([H]LSD) and impeded the 5-HT receptor-mediated activation of phospholipase C (PLC). Furthermore, bioluminescence resonance energy transfer (BRET) and western blotting analysis revealed that these mutations did not alter the homodimeric nature of the 5-HT receptor. However, fluorescence microscopy showed that these mutations hindered receptor trafficking to the cell membrane. These results illustrate the importance of the disulphide bridge between TM-3 and ECL-2 in maintaining the correct 5-HT receptor conformation to allow ligand binding and migration of the homodimeric receptor to the cell membrane.This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2014-57138-C2-1-R and SAF2017-85225-C3-1-R) and the European Regional Development Fund (ERDF). MC and LGG were supported by a grant from the Consellería de Cultura, Educación y Ordenación Universitaria, partially co-funded by the European Social Fund (ESF) program