title: Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer.
abstract: Several G protein-coupled receptors have been shown to exist as homo-and hetero-oligomeric complexes in living cells. However, the link between ligand-induced receptor activation and its oligomerization state as well as the proportion of the total receptor population that can engage in oligomeric complexes remain open questions. Here, the closely related human MT1 and MT2 melatonin receptors (MT1R, MT2R) were used to address these issues. Bioluminescence resonance energy transfer (BRET) experiments in living HEK 293 cells revealed that these receptors form homo- and hetero-oligomers. Constitutive energy transfer was observed for all receptor combinations at physiological expression levels and could be detected in single cell BRET experiments. Inhibition of the energy transfer by dilution of the BRET partners identified MT1R and MT2R dimers as the predominant receptor species, and this oligomerization state did not change upon agonist and antagonist binding. Agonists, neutral antagonists, and inverse agonists all promoted increases in BRET values for MT2R but not for MT1R homodimers in living cells and isolated plasma membranes. This indicates that no correlation could be inferred between the receptor activation state and the dimerization state of the receptor. This also suggests that ligand-promoted BRET increases represent specific ligand-induced conformational changes of pre-existing dimers rather then increased dimerization. The observation that ligands favored the energy transfer within the hetero-oligomer from MT1R to MT2R but not in the reverse orientation, from MT2R to MT1R, supports this view.
authors.affiliation: Department of Cell Biology, Institut Cochin, INSERM U 567, CNRS UMR 8104, 22 rue Méchain, 75014 Paris, France.
authors.forename: Mohammed A
authors.initials: MA
authors.lastname: Ayoub
authors.forename: Cyril
authors.initials: C
authors.lastname: Couturier
authors.forename: Estelle
authors.initials: E
authors.lastname: Lucas-Meunier
authors.forename: Stephane
authors.initials: S
authors.lastname: Angers
authors.forename: Philippe
authors.initials: P
authors.lastname: Fossier
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
authors.forename: Ralf
authors.initials: R
authors.lastname: Jockers
mesh_terms: D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D017353:Gene Deletion; D006801:Humans; D008024:Ligands; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000378:metabolism; D044094:Receptors, Melatonin; D013050:Spectrometry, Fluorescence; D014162:Transfection
chemical_list: D008024:Ligands; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D044094:Receptors, Melatonin; D000242:Cyclic AMP