Activation of genes in response to extracellular stimuli, pathogens, pharmaceutical administration, or environmental agents, requires a highly integrated signal transduction process that directs the transcriptional machinery to modulate the appropriate network of genes. An array of cytochrome P450 (CYP) genes, as well as other biotransformation functions, are targets of these signals. Like the CYPs, the nuclear hormone receptors (NHRs) are encoded by a superfamily of genes and largely orchestrate the responses triggered by chemical and hormonal effectors. This research proposal will focus in particular on the human constitutive androstane receptor (CAR) and the RXRalpha receptor (RXRa), NHRs that have been characterized recently to mediate the transcriptional activity of 'phenobarbital-like' inducer compounds that include a variety of pharmaceuticals and xenobiotic agents. The primary hypothesis that will be tested with this program is that the CAR and RXRa nuclear receptors are genetically polymorphic and structurally variant in human populations. Furthermore, we will test the hypothesis that the respective variant receptors dictate differential biological responses. A series of 3 experimental aims are advanced to facilitate the progression of the research program through initial discovery phases to in vitro and in vivo approaches to enable biological characterization of CAR and RXRa structural variants. Investigations will include the deployment of novel knockout mouse models that enable expression of the human NHRs, and structural variants thereof, in primary hepatocytes and in liver tissues that otherwise possess null receptor backgrounds. The """"""""humanized"""""""" mice will be analyzed for altered biological activities associated with expression of the receptor isoforms, including perturbations in response to drug exposures, aberrant receptor interactions using scanning fluorescence microscopy, and changes in the repertoire of gene expression using DNA microarray hybridizations. The presence of variant NHR receptor variants in the population likely dictates substantial interindividual differences in drug response and toxicities resulting from chemical exposures. In addition, the existence of structurally variant receptors may contribute to individual differences in the risk for human diseases, including certain cancers. The data obtained from these investigations will generate important new genetic and biological information regarding the structural diversity of prominent members of the NHR superfamily.
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