Received for publication August 31, 2006.
Revised December 14, 2006.
Accepted for publication December 27, 2006.

The human constitutive androstane receptor splice variant, CAR2, exhibits biologically distinct ligand binding and RxR heterodimerization character

Abstract

The constitutive androstane receptor (CAR, NR1I3) regulates the expression of genes involved in xenobiotic metabolism. Alternative splicing of the human CAR gene yields an array of mRNAs that encode structurally diverse proteins. One form of CAR, termed CAR2, contains an additional 4 amino acids (SPTV) that are predicted to reshape the ligand-binding pocket. The current studies show a marked, ligand-independent, CAR2-mediated transactivation of reporters containing optimal DR-3, DR-4 and DR-5 response elements, and reporters derived from the natural CYP2B6 and CYP3A4 gene promoters. Over expression of RXR's ligand binding domain was critical for achieving these effects. CAR2 interaction with SRC-1 was similarly dependent on the coexpression of RXR. Mutagenesis of S233 (SPTV) to an alanine residue yielded a receptor possessing higher constitutive activity. Alternatively, mutating S233 to an aspartate residue drastically reduced CAR2's transactivation capacity. The respective abilities of these mutagenized forms of CAR2 to transactivate a DR-4X3 reporter element correlated with their ability to interact with RxR, and to recruit SRC-1 in a ligand-regulated manner. Together, these results demonstrate a robust RXR-dependent recruitment of coactivators and transactivation by CAR2. In addition, CAR2 displays novel dose responses to clotrimazole and androstanol when compared to the reference form of the receptor while at the same time retaining the ability to bind CITCO. This result supports a hypothesis whereby the 4 amino acid insertion in CAR2 structurally modifies its ligand binding pocket, suggesting that CAR2 is regulated by a set of ligands distinct from those governing the activity of reference CAR.

Key words: CAR, enzyme induction, gene regulation, nuclear receptors, RXR