In primary cultures of hepatocytes or in vivo, CAR resides in the cytoplasm forming a complex with heat shock protein 90, cytoplasmic CAR retention protein, and membrane-associated subunit of protein phosphatase 1 (PPP1R16A) (Kobayashi et al., 2003; Yoshinari et al., http://www.selleckchem.com/products/Sorafenib-Tosylate.html 2003; Sueyoshi et al., 2008). CAR translocates into the nucleus and turns on its target gene expression only after exposure to chemical activators such as 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime (CITCO) a human-specific CAR agonist, and phenobarbital (PB), a universal indirect activator of CAR (Kawamoto et al., 1999; Maglich et al., 2003). In contrast to the observations in primary hepatocytes, expression of CAR in immortalized cell lines, such as HepG2 cells results in spontaneous nuclear accumulation and constitutive activation of this receptor independent of xenobiotic activation (Baes et al.

, 1994; Zelko et al., 2001; Li et al., 2009). The lack of cell lines that maintain CAR distribution and activation in a physiologically relevant manner has become a major obstacle in investigating the mechanisms of xenobiotic-mediated CAR activation. To date, although several chaperone molecules involving CAR cytoplasmic retention such as cytoplasmic CAR retention protein and PPP1R16A have been identified, the role of CAR protein variants in the constitutive versus chemical-mediated CAR activation remains unclear. Many naturally occurring alternative splicing variants of hCAR have recently been identified and functionally characterized by several groups (Arnold et al.

, 2004; Jinno et al., 2004; Auerbach et al., 2005). Among these spliced hCAR transcripts, the hCAR3, which contains an in-frame insertion of five amino acids (APYLT) in the highly conserved region of the ligand-binding domain (LBD), exhibited minimal basal, but potent ligand-induced activities in cell-based reporter assays (Auerbach et al., 2005; Faucette et al., 2007). It is intriguing that CITCO treatment was incapable of facilitating hCAR3 nuclear translocation in COS1 cells or rat primary hepatocytes (Jinno et al., 2004; Auerbach et al., 2005). Given the complexities encountered in studying hCAR activation in vitro, these initial observations of hCAR3 make it an attractive target for illustrating the mechanisms of hCAR activation.

To define the contribution of the five-amino-acid insertion on the functional transformation of hCAR3, we have generated a series of chimeric constructs containing various residues of the five-amino-acid insertion and evaluated their function in response to prototypical hCAR activators. The current studies demonstrate that retention of the alanine residue alone (hCAR1+A) seems sufficient to shift the constitutively activated hCAR1 to Brefeldin_A the xenobiotic-sensitive hCAR3. The chemical specificities of hCAR1+A activation closely resemble that of the reference hCAR1.