Regulated subcellular distribution of constitutive androstane receptor (CAR)

Nuclear translocation of constitutive androstane receptor (CAR) is a primary mechanism for the induction of cytochrome P450 genes by PB. Exogenous expression of the p160 coactivator glucocorticoid receptor interacting protein-1 (GRIP1) in hepatocytes can mediate PB-independent nuclear accumulation of murine CAR (mCAR). But the mechanism of nuclear translocation of CAR is largely unknown. The current study examined the mCAR structural determinants of its GRIP1-mediated nuclear localization. Through mutagenesis, in vivo gene delivery and confocal microscopy, I demonstrated that the xenobiotic response sequence (XRS), the activation function 2 motif and the DNA-binding domain (DBD) of CAR are all required for GRIP1-mediated nuclear accumulation of mCAR. In addition, my study indicated that the hinge domain and the ligand-binding domain (LBD) of CAR are both required for nuclear import of CAR. Further characterization of the XRS motif indicated that the XRS motif does not function simply as a novel nuclear localization signal (NLS). Instead, it may be involved in the interaction with an unknown protein which is critical for nuclear import of CAR. Also loss of the cytoplasmic retention of CAR is not sufficient for its maximal nuclear import.;The effects of three p160 coactivators, SRC-1, SRC-2/GRIP1 and SRC-3 on CAR localization and activity have been investigated and compared to determine if one of them is the preferred physiological coactivator for CAR. Our results show that the three members, while over-expressed, can mediate CAR localization and activity in a similar manner. Disruption of individual SRC genes in mice did not affect the PB-induced, CAR-targeted CYP2B10 gene transcription. Although the possibility that a certain SRC member is physiologically associated with CAR cannot be excluded, our results clearly suggest that the three SRC members compensate for each other in mediating CAR activity.;Overall, our studies are consistent with a model in which CAR is constantly shuttling between the cytoplasmic and the nucleus with the steady state strongly favoring cytoplasmic localization in untreated animals and strongly favoring nuclear localization in PB-treated animals. Regulation of the subcellular distribution of CAR is much more complicated than generally thought and might involve multiple regulatory factors and signal motifs.