Abstract

The constitutive androstane receptor (CAR) is constitutively activated in immortalized cell lines independent of xenobiotic stimuli. This feature of CAR has limited its use as a sensor for xenobiotic-induced expression of drug-metabolizing enzymes. Recent reports, however, reveal that a splicing variant of human CAR (hCAR3), which contains an insertion of 5 amino acids (APYLT), exhibits low basal but xenobiotic inducible activities in cell-based reporter assays. Nonetheless, the underlying mechanisms of this functional shift are not well understood. We have now generated chimeric constructs containing various residues of the 5 amino acids of hCAR3 and examined their response to typical hCAR activators. Our results showed that the retention of alanine (hCAR1+A) alone is sufficient to confer the constitutively activated hCAR1 to the xenobiotic sensitive hCAR3. Notably, hCAR1+A was significantly activated by a series of known hCAR activators, and displayed activation superior to that of hCAR3. Moreover, intracellular localization assays revealed that hCAR1+A exhibits nuclear accumulation upon CITCO treatment in COS1 cells, which differs from the spontaneous nuclear distribution of hCAR1 and the non-translocatable hCAR3. Mammalian two-hybrid and GST pull-down assays further demonstrated that hCAR1+A interacts with the co-activator SRC-1 and GRIP-1 at low level prior to activation, while at significantly enhanced level in the presence of CITCO. Thus, the alanine residue in the insertion of hCAR3 seems in charge of the xenobiotic response of hCAR3 through direct and indirect mechanisms. Activation of hCAR1+A may represent a sensitive avenue for the identification of hCAR activators.