CAR2 displays unique ligand binding and RXRalpha heterodimerization characteristics.
The constitutive androstane receptor (CAR; NR1I3) regulates the expression of genes involved in xenobiotic metabolism. Alternative splicing of the human CAR gene yields an array of mRNAs that encode structurally diverse proteins. One form of CAR, termed CAR2, contains an additional four amino acids (SPTV) that are predicted to reshape the ligand-binding pocket. The current studies show a marked, ligand-independent, CAR2-mediated transactivation of reporters containing optimal DR-3, DR-4, and DR-5 response elements, and reporters derived from the natural CYP2B6 and CYP3A4 gene promoters. Overexpression of the RXRalpha ligand binding domain was critical for achieving these effects. CAR2 interaction with SRC-1 was similarly dependent on the coexpression of RXRalpha. Mutagenesis of Ser233 (SPTV) to an alanine residue yielded a receptor possessing higher constitutive activity. Alternatively, mutating Ser233 to an aspartate residue drastically reduced the transactivation capacity of CAR2. The respective abilities of these mutagenized forms of CAR2 to transactivate a DR-4 x 3 reporter element correlated with their ability to interact with RxRalpha and to recruit SRC-1 in a ligand-regulated manner. Together, these results demonstrate a robust RXRalpha-dependent recruitment of coactivators and transactivation by CAR2. In addition, CAR2 displays novel dose responses to clotrimazole and androstanol compared with the reference form of the receptor while at the same time retaining the ability to bind CITCO. This result supports a hypothesis whereby the four-amino-acid insertion in CAR2 structurally modifies its ligand binding pocket, suggesting that CAR2 is regulated by a set of ligands distinct from those governing the activity of reference CAR.
Auerbach
Scott S
SS
Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary & Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802, USA.
Dekeyser
Joshua G
JG
Stoner
Matthew A
MA
Omiecinski
Curtis J
CJ
D000732:Androstanols / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D003022:Clotrimazole / Q000494:pharmacology; D000090702:Constitutive Androstane Receptor; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010091:Oximes / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D047490:Retinoid X Receptor alpha / Q000235:genetics / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology; D014157:Transcription Factors / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D000732:Androstanols; D000090702:Constitutive Androstane Receptor; D008024:Ligands; C000716327:NR1I3 protein, human; D010091:Oximes; D018160:Receptors, Cytoplasmic and Nuclear; D047490:Retinoid X Receptor alpha; D013844:Thiazoles; D014157:Transcription Factors; C000658:androstan-3-ol; C475093:6-(4-chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; D008156:Luciferases; D003022:Clotrimazole