title: Mass-spectrometric analysis of agonist-induced retinoic acid receptor gamma conformational change.
abstract: Apo and holo forms of retinoic acid receptors, and other nuclear receptors, display differential sensitivity to proteolytic digestion that likely reflects the distinct conformational states of the free and liganded forms of the receptor. We have developed a method for rapid peptide mapping of holo-retinoic acid receptor gamma that utilizes matrix-assisted laser-desorption-ionization time-of-flight MS to identify peptide fragments that are derived from the partially proteolysed holo-receptor. The peptide maps of retinoic acid receptor gamma bound by four different agonists were identical, suggesting that all four ligands induced a similar conformational change within the ligand-binding domain of the receptor. In all cases, this agonist-induced conformational change promoted the direct association of retinoic acid receptor gamma with the transcriptional co-activator p300 and inhibited interaction of the receptor with the nuclear receptor co-repressor. SR11253, a compound previously reported to exert mixed retinoic acid receptor gamma agonist/antagonist activities in cultured cells, was found to bind directly to, but only weakly altered the protease-sensitivity of, the receptor and failed to promote interaction of the receptor with p300 or induce dissociation of receptor-nuclear receptor co-repressor complexes. This technique should be generally applicable to other members of the nuclear receptor superfamily that undergo an induced structural alteration upon agonist or antagonist binding, DNA binding and/or protein-protein interaction.
authors.affiliation: Laboratory of Molecular Pharmacology, Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, U.S.A.
authors.forename: Valerie J
authors.initials: VJ
authors.lastname: Peterson
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Barofsky
authors.forename: Max L
authors.initials: ML
authors.lastname: Deinzer
authors.forename: Marcia I
authors.initials: MI
authors.lastname: Dawson
authors.forename: Kai-Chia
authors.initials: KC
authors.lastname: Feng
authors.forename: Xiao-kun
authors.initials: XK
authors.lastname: Zhang
authors.forename: Machender R
authors.initials: MR
authors.lastname: Madduru
authors.forename: Mark
authors.initials: M
authors.lastname: Leid
mesh_terms: D000595:Amino Acid Sequence; D006571:Heterocyclic Compounds / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D018168:Receptors, Retinoic Acid / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015534:Trans-Activators / Q000378:metabolism; D000097768:Retinoic Acid Receptor gamma
chemical_list: D006571:Heterocyclic Compounds; D009687:Nuclear Proteins; D018168:Receptors, Retinoic Acid; D015534:Trans-Activators; D000097768:Retinoic Acid Receptor gamma; C012947:1,3-dithiane