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

AuthorsCimadevila, M.; Gómez-García, L.; Martínez, A.L.; Iglesias, A.; López-Giménez, Juan F. ; Castro, M.; Cadavid, M.I.; Loza, María Isabel; Brea, J.
KeywordsSerotonin 2A receptor
Extracellular domains
Disulfide bridge
Ligand binding
Issue Date2020
CitationBiochemical Pharmacology 177 (2020)
AbstractThe 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.
Publisher version (URL)http://dx.doi.org/10.1016/j.bcp.2020.113985
Identifiersdoi: 10.1016/j.bcp.2020.113985
issn: 0006-2952
e-issn: 1873-2968
Appears in Collections:(IPBLN) Artículos
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