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

• Published in: Biochemical pharmacology 2020-07, Vol.177, p.113985-113985, Article 113985
• Main Authors: Cimadevila, M., Gómez-García, L., Martínez, A.L., Iglesias, A., López-Giménez, J., Castro, M., Cadavid, M.I., Loza, M.I., Brea, J.
• Format: Article
• Language: English
• Subjects: Disulfide bridge; Extracellular domains; GPCRs; Ligand binding; Serotonin 2A receptor
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2020.113985

[Display omitted] The 5-HT2A 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-HT2A 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-HT2A receptor via cysteine 148 (C148) and C227. Thus, to study the influence of the C148–C227 disulphide bridge on 5-HT2A receptor pharmacology, we substituted C148 and C227 in the human 5-HT2A 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-HT2A receptor to [3H]lysergic acid diethylamide ([3H]LSD) and impeded the 5-HT2A 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-HT2A 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-HT2A receptor conformation to allow ligand binding and migration of the homodimeric receptor to the cell membrane.