title: Stability of GABAB receptor oligomers revealed by dual TR-FRET and drug-induced cell surface targeting.
abstract: The function of cell surface proteins likely involves the formation and dissociation of oligomeric complexes. However, the dynamics of this process are unknown. Here we examined this process for the GABA(B) receptors that assemble into oligomers of heterodimers through the association of their GABA(B1) subunit. We report a method to study oligomer dynamics based on a drug-controlled cell surface targeting of intracellularly retained receptors and a parallel measurement of two FRET signals in HEK293 cells. GABA(B1) subunits at the cell surface (4.0 ± 0.6 a.u.) are labeled with a pair of fluorophores (donor and red acceptor). New receptors are then targeted to the cell surface during 3h treatment with AP21967 such that the number of receptors is doubled (9.1 ± 0.7 a.u.). After labeling these new receptors with a second acceptor (green), the red FRET remained unchanged (5189 ± 36 vs. 4783 ± 32 cps), supporting the stability of the preformed oligomers. However, new oligomers are detected by the green FRET signal indicating both receptor populations are in the same microdomains. As a control, we confirmed the strict stability of the GABA(B) heterodimer itself. Herein, using a novel method to monitor the dynamics of cell surface complexes, we provide evidence for the stability of GABA(B) oligomers.
authors.affiliation: Institut de Génomique Fonctionnelle, Centre National de Recherche Scientifique (CNRS) UnitéMixte de Recherche -5203, Institut National de Santé et de Recherche Médicale (INSERM) U661, Montpellier, France.
authors.forename: Laëtitia
authors.initials: L
authors.lastname: Comps-Agrar
authors.forename: Julie
authors.initials: J
authors.lastname: Kniazeff
authors.forename: Carsten
authors.initials: C
authors.lastname: Brock
authors.forename: Eric
authors.initials: E
authors.lastname: Trinquet
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pin
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D055503:Protein Multimerization / Q000187:drug effects; D055550:Protein Stability; D021122:Protein Subunits / Q000378:metabolism; D018080:Receptors, GABA-B / Q000737:chemistry* / Q000187:drug effects; D020123:Sirolimus / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: C539629:AP 21967; D021122:Protein Subunits; D018080:Receptors, GABA-B; D020123:Sirolimus