This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The constitutive androstane receptor (CAR) is unique among nuclear receptors because it is expressed almost exclusively in hepatocytes, remains in an active conformation in the cytoplasm, and is activated by many chemicals with which it does not directly interact. A CAR activator is the sedative phenobarbital (PB), even though PB does not bind directly to CAR. Primary hepatocyte model systems are often used to demonstrate that PB and PB-like chemicals induce the expression of CAR target genes such as cytochrome P450 2B6 (CYP2B6) and other metabolizing enzymes and transport proteins implicated in acetaminophen toxicity, bilirubin clearance, and obesity-related disorders. Besides PB, multiple neuroactive chemicals including other barbiturates, benzodiazepines, and steroids are transcriptional activators of CYP2B6 gene expression in the liver.
The Aims of this project are to: 1) Define the hepatocyte membrane chemical sensor upstream of CAR activation, and 2) Identify key regulators of CAR subcellular distribution (nuclear import/export). Human hepatocytes and transfected cells will be treated with various neuroactive chemicals, and experiments will analyze subsequent intracellular compartmentalization of CAR, which is controlled by protein phosphorylation, a nuclear localization signal in the DNA binding domain and a xenobiotic response sequence (XRS) in the ligand binding domain;other experiments will focus on nuclear export of CAR by adapter calreticulin. Lentiviral infections of siRNAs and chimeric CAR-green fluorescent proteins in human hepatocytes and mouse livers will be used to gain a clearer understanding of CAR cytoplasmic sequestration, nuclear import and export, and unanticipated involvement of CAR in metabolism of neuroactive pharmacological agents.
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