| Together, the studies presented here have focused on characterizing the functional impact of identified genetic and structural alterations within key regulatory pathways that modify responsiveness to xenobiotics in the human liver.; The constitutive androstane receptor mediates the transcriptional activation of a variety of drug metabolizing enzymes following exposure to the prototypical inducer phenobarbital. In the first chapter, 4 splice variants of the human CAR transcript are described. One of the variants results from a 12 nucleotide insertion between exon 6 and exon 7, generated through the use of an alternative splice acceptor site in intron 6 (CAR2). The CAR2 protein contains a 4 amino acid insertion (SPTV) within the ligand binding domain. CAR2 possesses a compromised constitutive activity that is a byproduct of poor DNA binding and a reduced ability of the receptor to recruit coactivators. Studies of RXRalpha's influence on CAR2 revealed that deficiencies in receptor's activity can be rescued by RXRalpha expression. Further analysis of RXRalpha's impact on CAR2 function suggest that the primary means of CAR2 rescue occurs through an RXRalpha-dependent recruitment of coactivators to the variant receptor.; Another variant CAR transcript (CAR3) retains 15 nucleotides due to an alternative splice acceptor site in intron 7. CAR3 contains a 5 amino acid insertion (APYLT) that alters the heterodimerization interface. Unlike CAR1, CAR3 is inactive in the absence of ligand. Detailed studies of CAR3 transcriptional activation revealed that cotransfection of an RXRalpha expression plasmid enhanced the activity of the variant receptor. Paradoxically, the enhancement of CAR3 activity by RXRalpha is independent of heterodimeration, and instead may involve the interaction of shared coregulatory factors.; Also described in this thesis an N-terminal truncated PXR form that is generated from the use of an internal translation start site, P261L; a naturally occurring genetically polymorphic form of RXRalpha; and, PGC-1alpha*, a truncated form of the PGC-1alpha coactivator that is produced by alternative splicing.; Overall this thesis research has defined multiple molecular variables that impact not only the activity of CAR, but many other hepatic and non-hepatic nuclear receptor signaling pathways that mediate the disposition of drugs and other environmental chemicals. |
