TY - JOUR T1 - Simultaneous Substitution of Phenylalanine-305 and Aspartate-318 of Rat Pregnane X Receptor with the Corresponding Human Residues Abolishes the Ability to Transactivate the CYP3A23 Promoter JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 571 LP - 582 DO - 10.1124/jpet.104.074971 VL - 312 IS - 2 AU - Xiulong Song AU - Yuxin Li AU - Jirong Liu AU - Madhu Mukundan AU - Bingfang Yan Y1 - 2005/02/01 UR - http://jpet.aspetjournals.org/content/312/2/571.abstract N2 - The pregnane X receptor (PXR) is a key regulator on the expression of genes involved in the elimination of chemicals. As one of the most divergent members in the nuclear receptor family, PXR is activated in a highly species-dependent manner by certain chemicals. Pregnenolone 16α-carbonitrile (PCN), a glucocorticoid antagonist, efficaciously activates rodent but not human PXR. This study was undertaken to investigate the structural basis for PCN-mediated activation of rat PXR. A series of rat-human chimeric PXRs were prepared to gradually replace the ligand-binding domain of human PXR with the corresponding rat sequence at an increasing length of 20 residues. Cotransfection experiments established that region306–326 acted as a transitional conjunction from none to full PCN responsive status. Site-directed mutagenesis study identified two residues (Phe-305 and Asp-318) that were critical in supporting PCN-mediated activation, and simultaneous substitution of both residues abolished the ability of rat PXR to transactivate the CYP3A23 promoter. In addition, substitutions on Phe-305, Asp-318, or both markedly reduced the basal transcriptional activity, and the reduction occurred with the CYP3A4 but not CYP3A23 promoter. Further study with CYP3A4 and CYP3A23 hybrid reporters demonstrated that the region harboring the distal PXR element in the CYP3A4 promoter mediated the repressive activity. PXR has been shown to interact with corepressors in the absence of ligand. The decreased responsiveness toward PCN and reduced basal transcriptional activity suggest that Phe-305 and Asp-318 are involved in both ligand-binding and corepressor interactions. ER -