title: Amino acids important for ligand specificity of the human constitutive androstane receptor.
abstract: The human constitutive androstane receptor (CAR, NR1I3) is an important ligand-activated regulator of oxidative and conjugative enzymes and transport proteins. Because of the lack of a crystal structure of the ligand-binding domain (LBD), wide species differences in ligand specificity and the scarcity of well characterized ligands, the factors that determine CAR ligand specificity are not clear. To address this issue, we developed highly defined homology models of human CAR LBD to identify residues lining the ligand-binding pocket and to perform molecular dynamics simulations with known human CAR modulators. The roles of 22 LBD residues for basal activity, ligand selectivity, and interactions with co-regulators were studied using site-directed mutagenesis, mammalian co-transfection, and yeast two-hybrid assays. These studies identified several amino acids within helices 3 (Asn(165)), 5 (Val(199)), 11 (Tyr(326), Ile(330), and Gln(331)), and 12 (Leu(343) and Ile(346)) that contribute to the high basal activity of human CAR. Unique residues within helices 3 (Ile(164) and Asn(165)), 5 (Cys(202) and His(203)), and 7 (Phe(234) and Phe(238)) were found control the selectivity for CAR activators and inhibitors. A single residue in helix 7 (Phe(243)) appears to explain the human/mouse species difference in response of CAR to 17alpha-ethynyl-3,17beta-estradiol.
authors.affiliation: Department of Pharmaceutics, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Jyrkkärinne
authors.forename: Björn
authors.initials: B
authors.lastname: Windshügel
authors.forename: Janne
authors.initials: J
authors.lastname: Mäkinen
authors.forename: Markku
authors.initials: M
authors.lastname: Ylisirniö
authors.forename: Mikael
authors.initials: M
authors.lastname: Peräkylä
authors.forename: Antti
authors.initials: A
authors.lastname: Poso
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sippl
authors.forename: Paavo
authors.initials: P
authors.lastname: Honkakoski
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D000731:Androstanes / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D000090702:Constitutive Androstane Receptor; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013045:Species Specificity; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D000731:Androstanes; D000090702:Constitutive Androstane Receptor; D008024:Ligands; C000716327:NR1I3 protein, human; C000716328:Nr1i3 protein, mouse; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; C033936:androstane