2024-03-29T08:56:24Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/2163102021-11-12T12:32:07Zcom_10261_64com_10261_1col_10261_317
Essential role of the C148–C227 disulphide bridge in the human 5-HT2A homodimeric receptor
Cimadevila, M.
Gómez-García, L.
Martínez, A.L.
Iglesias, A.
López-Giménez, Juan F.
Castro, M.
Cadavid, María Isabel
Loza, María Isabel
Brea, J.
Ministerio de Economía y Competitividad (España)
European Commission
Serotonin 2A receptor
GPCRs
Extracellular domains
Disulfide bridge
Ligand binding
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.
2020-07-09T07:50:12Z
2020-07-09T07:50:12Z
2020
2020-07-09T07:50:12Z
artículo
Biochemical Pharmacology 177 (2020)
http://hdl.handle.net/10261/216310
10.1016/j.bcp.2020.113985
http://dx.doi.org/10.13039/501100000780
http://dx.doi.org/10.13039/501100003329
http://dx.doi.org/10.1016/j.bcp.2020.113985
Sí
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/SAF2014-57138-C2-1-R
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/SAF2017-85225-C3-1-R
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