Expression of constitutive androstane receptor splice variants in rat liver and lung and their functional properties.
The mammalian constitutive androstane receptor (CAR) is a transcription factor that participates in controlling the expression of xenobiotic metabolizing and transporting genes in response to xenobiotics in an organ-specific manner. In addition to the wild-type CAR (CAR WT) mRNA, mRNAs for five splice variants (SVs) could be detected in the liver of 7-week-old male Wistar rats by RT-PCR using primer pairs covering a full-length mRNA derived from 9 exons; insertion of 18 bp at the 5'-end of intron 8 with or without deletion of 3 bp from the 5'-end of exon 7 (CAR SV1 or SV2), deletion of 4 bp from the 5'-end of exon 8 (CAR SV3), insertion of 195 bp intron 7 (CAR SV4), and insertion of 91 bp intron 6 (CAR SV5). In contrast, only CAR SV5 was detected in lung. Due to the introduction of novel stop codons, all the SVs were considered to code for premature proteins. The liver homogenate gave two protein bands in the vicinity of 37 kDa on Western blotting. They were attributable to CAR WT and SV-complex, respectively, based on their putative molecular weights in descending order. Upon cotransfection with the reporter plasmid, only the cells transfected with the CAR SV4-expression plasmid showed enhanced luciferase activity similar to the WT-transfected cells, for which the further splicing of the remaining intron 7 seemed to be responsible. The transactivation-defective SVs downregulated CAR WT-induced luciferase activity to some extent in the cotransfection experiments.
Kanno
Yuichiro
Y
Faculty of Pharmaceutical Sciences, Toho University, Japan.
Aoki
Sho
S
Mochizuki
Megumi
M
Mori
Emi
E
Nakahama
Takayuki
T
Inouye
Yoshio
Y
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D000090702:Constitutive Androstane Receptor; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D017930:Genes, Reporter; D008099:Liver / Q000378:metabolism*; D008168:Lung / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics
D000090702:Constitutive Androstane Receptor; D017931:DNA Primers; D018076:DNA, Complementary; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors