Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis.

Détails

ID Serval
serval:BIB_1D30A40BE443
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis.
Périodique
Journal of Biological Chemistry
Auteur⸱e⸱s
Ilegems E., Pick H.M., Deluz C., Kellenberger S., Vogel H.
ISSN
0021-9258[print], 0021-9258[linking]
Statut éditorial
Publié
Date de publication
2004
Volume
279
Numéro
51
Pages
53346-53352
Langue
anglais
Résumé
Sequential stages in the life cycle of the ionotropic 5-HT(3) receptor (5-HT(3)R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT(3)R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT(3)R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT(3) R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT(3) receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT(3) receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.
Mots-clé
Actins/metabolism, Binding Sites, Cell Line, Cell Membrane/metabolism, DNA/metabolism, Electrophysiology, Endocytosis, Endoplasmic Reticulum/metabolism, Golgi Apparatus/metabolism, Green Fluorescent Proteins/metabolism, Humans, Hydrogen-Ion Concentration, Kinetics, Lasers, Ligands, Microscopy, Confocal, Microscopy, Fluorescence, Protein Binding, Protein Transport, Receptors, Serotonin, 5-HT3/metabolism, Time Factors, Transfection
Pubmed
Web of science
Open Access
Oui
Création de la notice
24/01/2008 12:45
Dernière modification de la notice
20/08/2019 12:53
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