Essential role of the C148–C227 disulphide bridge in the human 5-HT2A homodimeric receptor
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...
| Autores: | , , , , , , , , |
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| Tipo de recurso: | artículo |
| Fecha de publicación: | 2020 |
| País: | España |
| Institución: | Universidad de Santiago de Compostela (USC) |
| Repositorio: | Minerva. Repositorio Institucional de la Universidad de Santiago de Compostela |
| Idioma: | inglés |
| OAI Identifier: | oai:minerva.usc.gal:10347/45302 |
| Acceso en línea: | https://hdl.handle.net/10347/45302 |
| Access Level: | acceso abierto |
| Palabra clave: | Serotonin 2A receptor GPCRs Extracellular domains Disulfide bridge Ligand binding |
| Sumario: | 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. |
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