Abstract
The constitutive androstane receptor (CAR) NR1I3 is a transcription factor that upon activation by xenobiotics induces transcription of drug-metabolizing and drug transporter genes. Our goal was to identify whether alternative splicing of CAR makes an important contribution to the generation of novel CAR proteins. The wild-type CAR mRNA (CAR.1) and splice variants (SVs) were amplified from human liver cDNAs and in a panel of cDNAs from 36 human tissues, using exon 1- and 3′-untranslated region primers, cloned and sequenced. Twenty-two unique hCAR splice variants (CAR-SVs) containing different combinations of splicing (deletion of exons 2, 4, 5, 7, partial deletion of exon 9, or insertion of 12 or 15 base pairs from introns 6 or 7) were identified. CAR mRNAs were expressed in small intestine, kidney, testis, adrenal, and brain caudate nucleus. Intestine expressed only CAR.1 mRNA, whereas spleen, heart, and prostate expressed only CAR-SVs. In vitro transcription and translation of CAR-SVs lacking exon 2 (missing ATG start site) generated CAR proteins that differed in Mr from CAR.1. These CAR-SVs used a translation initiation site in exon 1, generating CAR with a unique amino-terminal sequence. Transcripts lacking part of exon 9 altered the CAR reading frame generating CAR proteins with a unique carboxy-terminal end. CAR-SVs demonstrated compromised binding to CYP2B6 NR elements and transcriptional activation of a CYP2B6 luciferase reporter. The expression of CAR in additional human cell types increases the range of tissue specific transcriptional responses regulated by this receptor, suggesting additional biological roles for CAR and CAR-SV proteins in these tissues.
Footnotes
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Normal human liver (processed through Dr. Mary Relling's laboratory at St. Jude Children's Research Hospital) and hepatocytes were obtained through the Liver Tissue Procurement and Distribution System (Pittsburgh, PA), which was funded by National Institutes of Health Contract N01-DK-9-2310 and by the Cooperative Human Tissue Network. This study was supported in part by National Institutes of Health Grant GM60346, by the National Institutes of Health/National Institute of General Medical Sciences Pharmacogenetics Research Network and Database (U01GM61374, http://pharmgkb.org) under Grant U01 GM61393, by National Institutes of Health Grant P30 CA21765 Cancer Center Support Grant, and by the American Lebanese Syrian Associated Charities (ALSAC).
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doi:10.1124/jpet.104.069310.
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ABBREVIATIONS: CAR, constitutive androstane receptor; CITCO, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; bp, base pair(s); SV, splice variant; PCR, polymerase chain reaction; TNT, transcription and translation; EMSA, electrophoretic mobility shift assay; DBD, DNA binding domain.
- Received March 31, 2004.
- Accepted June 9, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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