Identification of novel splice variants of ARNT and ARNT2 in the rat.
Most of the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the bHLH/PAS protein AH receptor (AHR). For regulation of gene activities, AHR dimerizes with another member of the bHLH/PAS protein family, AHR nuclear translocator (ARNT). A substrain of Wistar rats, Han/Wistar (Kuopio) (H/W), is about 1000-fold more resistant to the acute lethality of TCDD than other strains, exemplified by Long-Evans (Turku/AB) (L-E); the LD50 values for these two strains are >9600 and 10-20 microg/kg, respectively. Previous studies have demonstrated that the major reason for the exceptional TCDD resistance of H/W rats lies in their AHR, which is remodeled at its C-terminal transactivation domain, but there appears to be another contributing gene product. The present study set out to compare the primary structure of ARNT and the closely related ARNT2 proteins in H/W and L-E rats by cDNA cloning. To our surprise, we found several isoforms of these proteins only one of which has previously been reported in rats. All of the isoforms appeared to arise from alternative splicing. For ARNT, isoforms with deletions at exon 5, 3(') end of exon 6 or 5(') end of exon 11, or with an insertion at 5(') end of exon 20 were discovered. There was also interindividual variation in the number of glutamine-encoding codons at 5(') end of exon 16. The most exciting new variant was revealed for ARNT2, because the insertion found at 5(') end of exon 19 disrupts the functionally critical transactivation domain in the protein, implying a dominant negative role for this isoform. The relative expression levels of the variants did not differ in the two rat strains, nor did TCDD modify the ratios, suggesting that the variants do not contribute to TCDD resistance. However, the regulation of ARNT and ARNT2 activities may be more intricate than previously assumed.
Korkalainen
Merja
M
National Public Health Institute, Laboratory of Toxicology, Department of Environmental Health, P.O. Box 95, Neulaniementie 4, FIN-70701 Kuopio, Finland. Merja.Korkalainen@ktl.fi
Tuomisto
Jouko
J
Pohjanvirta
Raimo
R
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D014644:Genetic Variation; D007031:Hypothalamus / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D020318:Rats, Long-Evans; D017208:Rats, Wistar; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D020033:Protein Isoforms; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; C497398:ARNT protein, rat; C490991:Arnt2 protein, rat