title: Monitoring ligand-mediated nuclear receptor-coregulator interactions by noncovalent mass spectrometry.
abstract: Retinoid receptors are ligand-dependent transcription factors belonging to the nuclear receptor superfamily. Retinoic acid (RARalpha, beta, gamma) and retinoid X (RXRalpha, beta, gamma) receptors mediate the retinoid/rexinoid signal to the transcriptional machineries by interacting at the first level with coactivators or corepressors, which leads to the recruitment of enzymatically active noncovalent complexes at target gene promoters. It has been shown that the interaction of corepressors with nuclear receptors involves conserved LXXI/HIXXXI/L consensus sequences termed corepressor nuclear receptor (CoRNR) boxes. Here we describe the use of nondenaturing electrospray ionization mass spectrometry (ESI-MS) to determine the characteristics of CoRNR box peptide binding to the ligand binding domains of the RARalpha-RXRalpha heterodimer. The stability of the RARalpha-RXRalpha-CoRNR ternary complexes was monitored in the presence of different types of agonists or antagonists for the two receptors, including inverse agonists. These results show unambiguously the differential impact of distinct retinoids on corepressor binding. We show that ESI-MS is a powerful technique that complements classical methods and allows one to: (a) obtain direct evidence for the formation of noncovalent NR complexes; (b) determine ligand binding stoichiometries and (c) monitor ligand effects on these complexes.
authors.affiliation: Laboratoire de Spectrométrie de Masse Bio-Organique, CNRS UMR 7509, ECPM, Strasbourg, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Sanglier
authors.forename: William
authors.initials: W
authors.lastname: Bourguet
authors.forename: Pierre
authors.initials: P
authors.lastname: Germain
authors.forename: Virginie
authors.initials: V
authors.lastname: Chavant
authors.forename: Dino
authors.initials: D
authors.lastname: Moras
authors.forename: Hinrich
authors.initials: H
authors.lastname: Gronemeyer
authors.forename: Noelle
authors.initials: N
authors.lastname: Potier
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
mesh_terms: D000595:Amino Acid Sequence; D019281:Dimerization; D008024:Ligands; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D008024:Ligands; D018168:Receptors, Retinoic Acid