Essential role of the C148–C227 disulphide bridge in the human 5-HT2A homodimeric receptor
| Autor: | Juan F. López-Giménez, María Isabel Cadavid, José Brea, María Isabel Loza, Marián Castro, Antón L. Martínez, L. Gómez-García, Marta Cimadevila, Alba Iglesias |
|---|---|
| Přispěvatelé: | Ministerio de Economía y Competitividad (España), European Commission |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Pharmacology Phospholipase C Chemistry Mutant Biochemistry GPCRs Cell membrane Serotonin 2A receptor 03 medical and health sciences Transmembrane domain 030104 developmental biology 0302 clinical medicine medicine.anatomical_structure 030220 oncology & carcinogenesis Extracellular Biophysics medicine Disulfide bridge Extracellular domains Receptor Ligand binding Cysteine G protein-coupled receptor |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 2014-5713 |
| Popis: | 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. 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. |
| Databáze: | OpenAIRE |
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