RT Journal Article
T1 Essential role of the C148–C227 disulphide bridge in the human 5-HT2A homodimeric receptor
A1 Cimadevila Fondevila, Marta
A1 Gómez-García, L.
A1 Martínez Rodríguez, Antón Leandro
A1 Iglesias, Alba
A1 López-Giménez, J.
A1 Castro Pérez, María de los Ángeles
A1 Loza García, María Isabel
A1 Cadavid Torres, Isabel
A1 Brea Floriani, José Manuel
K1 Serotonin 2A receptor
K1 GPCRs
K1 Extracellular domains
K1 Disulfide bridge
K1 Ligand binding
AB 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.
PB Elsevier
YR 2020
FD 2020-07
LK https://hdl.handle.net/10347/45302
UL https://hdl.handle.net/10347/45302
LA eng
NO Biochemical Pharmacology Volume 177, July 2020, 113985
NO 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.
DS Minerva
RD 29 abr 2026