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J. Biol. Chem., Vol. 280, Issue 7, 5960-5971, February 18, 2005

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Amino Acids Important for Ligand Specificity of the Human Constitutive Androstane Receptor^*

Johanna Jyrkkärinne, Björn Windshügel¶, Janne Mäkinen, Markku Ylisirniö||, Mikael Peräkylä||, Antti Poso**, Wolfgang Sippl¶, and Paavo Honkakoski

From the Departments of Pharmaceutics, ^||Chemistry, and ^**Pharmaceutical Chemistry, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland and the ^¶Department of Pharmaceutical Chemistry, Martin-Luther-University, Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle (Saale), Germany

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 (Asn¹⁶⁵), 5 (Val¹⁹⁹), 11 (Tyr³²⁶, Ile³³⁰, and Gln³³¹), and 12 (Leu³⁴³ and Ile³⁴⁶) that contribute to the high basal activity of human CAR. Unique residues within helices 3 (Ile¹⁶⁴ and Asn¹⁶⁵), 5 (Cys²⁰² and His²⁰³), and 7 (Phe²³⁴ and Phe²³⁸) were found control the selectivity for CAR activators and inhibitors. A single residue in helix 7 (Phe²⁴³) appears to explain the human/mouse species difference in response of CAR to 17-ethynyl-3,17-estradiol.

Received for publication, October 1, 2004 , and in revised form, November 12, 2004.

^* This work was supported by Academy of Finland Grants 44040 and 51610 (to P. H.) and 104622 (to M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

To whom correspondence should be addressed: Dept. of Pharmaceutics, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland. Tel.: 358-17-162490; Fax: 358-17-162252; E-mail: paavo.honkakoski{at}uku.fi.

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