tailieunhanh - Báo cáo khoa học: Monitoring ligand-mediated nuclear receptor–coregulator interactions by noncovalent mass spectrometry

Retinoid receptors are ligand-dependent transcription factors belonging to the nuclear receptor superfamily. Retinoic acid (RARa, b, c) and retinoid X (RXRa, b, c) 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 com-plexes at target gene promoters. It has been shown that the interaction of corepressors with nuclear receptors in-volves conserved LXXI/HIXXXI/L consensus sequences termed corepressor nuclear receptor (CoRNR) boxes | Eur. J. Biochem. 271 4958-4967 2004 FEBS 2004 doi Monitoring ligand-mediated nuclear receptor-coregulator interactions by noncovalent mass spectrometry Sarah Sanalier1 William Bourauet2 Pierre Germain2 Virainie Chavant2. Dino Moras2 Hinrich Gronemeyer2 Noelle Potier1 and Alain Van Dorsselaer1 1 Laboratoire de Spectrométrie de Masse Bio-Organique CNRS UMR 7509 ECPM Strasbourg France 2Institut de Génétique et de Biologie Moleculaire et Cellulaire CNRS INSERM ULP College de France Illkirch France Retinoid receptors are ligand-dependent transcription factors belonging to the nuclear receptor superfamily. Retinoic acid RARa b c and retinoid X RXRa b c 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 RARa-RXRa hetero dimer. The stability of the RARa-RXRa-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. Keywords ESI-MS noncovalent mass spectrometry nuclear receptors protein-protein interactions protein-ligand .