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Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
Published on: December 19, 2018
CAR2 displays unique ligand binding and RXRalpha heterodimerization characteristics
Scott S Auerbach1, Joshua G Dekeyser, Matthew A Stoner
1Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary & Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802, USA.
Drug Metabolism and Disposition: the Biological Fate of Chemicals
|December 30, 2006
Summary
A novel variant of the constitutive androstane receptor (CAR2) exhibits ligand-independent activity, suggesting altered regulation by distinct ligands compared to the reference CAR. This CAR2 variant demonstrates RXRalpha-dependent coactivator recruitment and transactivation.
Area of Science:
- Pharmacology
- Molecular Biology
- Genetics
Background:
- The constitutive androstane receptor (CAR; NR1I3) is a key regulator of xenobiotic metabolism.
- Alternative splicing of the human CAR gene generates diverse protein isoforms, including CAR2.
- CAR2 possesses a unique four-amino-acid insertion (SPTV) predicted to alter its ligand-binding pocket.
Purpose of the Study:
- To investigate the functional consequences of the CAR2 variant.
- To elucidate the role of RXRalpha and coactivators in CAR2-mediated transactivation.
- To compare the ligand-dependent regulation of CAR2 with the reference CAR.
Main Methods:
- Reporter gene assays to measure transactivation.
- Co-immunoprecipitation to assess protein interactions.
- Site-directed mutagenesis to probe the role of specific amino acids (Ser233).
Main Results:
- CAR2 demonstrated significant ligand-independent transactivation of reporter genes and natural promoters (CYP2B6, CYP3A4).
- RXRalpha overexpression was essential for CAR2 activity and coactivator (SRC-1) recruitment.
- Mutagenesis of Ser233 affected CAR2's constitutive activity, RXRalpha interaction, and SRC-1 recruitment.
- CAR2 exhibited distinct dose-response profiles to clotrimazole and androstanol compared to reference CAR.
Conclusions:
- CAR2 exhibits robust, RXRalpha-dependent transactivation and coactivator recruitment.
- The SPTV insertion in CAR2 modifies its ligand-binding pocket, leading to altered ligand sensitivity and potentially distinct regulatory mechanisms.
- CAR2 represents a functionally unique CAR isoform with implications for drug metabolism and response.
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