title: Mutational analysis reveals that all-trans-retinoic acid, 9-cis-retinoic acid, and antagonist interact with distinct binding determinants of RARalpha.
abstract: Retinoids exert their pleiotropic effects on cell differentiation and proliferation through specific nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Two biologically highly active natural retinoids have been identified, all-trans-retinoic acid (t-RA) and 9-cis-retinoic acid (9-cis-RA). The RXRs exclusively bind 9-cis-RA, whereas the RARs bind both isomers of RA with comparable affinity. Recently published results suggest that RARs have the same binding site for t-RA and 9-cis-RA but with different determinants (1-3). Antagonist binding on RARalpha has been suggested to induce distinct conformational changes in comparison with agonist binding. To elucidate the region minimally required for efficient binding of agonist (t-RA and 9-cis-RA) and antagonist Ro 41-5253 to the RARalpha, we generated N- and C-terminally truncated mutants of the receptor. Characterization of these deletion mutant proteins using protease mapping and ligand binding experiments revealed that different parts of the ligand-binding domain are necessary for t-RA, 9-cis-RA, and antagonist binding. Three distinct regions of the ligand-binding domain of the human retinoic acid receptor-alpha are required for binding of t-RA (RARalpha187-402), 9-cis-RA (RARalpha188-409), and the antagonist Ro 41-5253 (RARalpha226-414).
authors.affiliation: Preclinical Research, Department of Infectious Diseases, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Keidel
authors.forename: F P
authors.initials: FP
authors.lastname: Lamour
authors.forename: C M
authors.initials: CM
authors.lastname: Apfel
mesh_terms: D000077556:Alitretinoin; D001565:Benzoates / Q000378:metabolism*; D001665:Binding Sites; D002839:Chromans / Q000378:metabolism*; D004252:DNA Mutational Analysis; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D018168:Receptors, Retinoic Acid / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D000072482:Retinoic Acid Receptor alpha; D012176:Retinoids / Q000378:metabolism; D017384:Sequence Deletion; D014212:Tretinoin / Q000378:metabolism*
chemical_list: D001565:Benzoates; D002839:Chromans; D017931:DNA Primers; D010446:Peptide Fragments; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D000072482:Retinoic Acid Receptor alpha; D012176:Retinoids; C084904:Ro 41-5253; D000077556:Alitretinoin; D014212:Tretinoin