Abstract

The constitutive androstane receptor (CAR) mediates the hepatic induction of various xenobiotic metabolizing enzymes and transporters after specific chemical exposures. Recent reports have established the existence of several human CAR mRNA splice variants, including a prominently expressed form termed CAR3, a receptor that possesses a 5 amino acid insertion within its ligand binding domain. In this study, we demonstrate that, in contrast to the constitutively active reference form of the receptor, CAR3 is ligand-activated, transactivating an optimized DR-4 × 3 reporter in response to the human CAR ligand 6-(4-chlorophenyl)imidazo[2,1-b]thiazole-5-carbaldehyde O-(3, 4-dichlorobenzyl)oxime (CITCO). The transactivation response requires the DNA binding domain and AF-2 motif of CAR3 and is markedly enhanced by retinoid X receptor-α (RXR) cotransfection. The stimulatory effects of RXR involve a unique mechanism, because they were completely dependent on the RXR AF-2 function but independent of both the RXR A/B domain and its C domain/heterodimerization region. Mammalian two-hybrid results demonstrated that RXR enhanced CITCO-dependent interaction of CAR3 with the receptor interaction domain of SRC-1, indicating that RXR augments CAR3 activity by facilitating coactivator recruitment. It is noteworthy that clotrimazole also functions as a ligand activator of CAR3, in contrast to the inverse agonist activity exhibited by this agent on the reference form of the receptor. Furthermore, results of transfection assays reveal that CAR3 is capable of transactivating the natural CYP2B6 and CYP3A4 gene enhancers, exhibiting both ligand- and RXR-dependence. These results demonstrate that CAR3, unlike CAR1, is a ligand-activated receptor and that CAR3 may regulate gene expression in vivo in a manner distinct from the reference form of the receptor.