title: Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions.
abstract: In order to further establish zebrafish as a vertebrate model for studying the mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity it is necessary to characterize the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AhR/ARNT) signaling pathways in this species. In this study, three zfARNT2 cDNAs were isolated, expressed, and characterized and named zfARNT2b, zfARNT2c, and zfARNT2a. zfARNT2b, zfARNT2c, and zfARNT2a encode proteins with theoretical molecular weights of 81, 79, and 45 kDa, respectively. zfARNT2b and zfARNT2a proteins are identical over the first 403 amino acids but differ in their C-terminal domains as a result of alternative mRNA splicing. zfARNT2c is nearly identical to zfARNT2b, with the exception of an in frame 15 amino acid deletion adjacent to the basic region of zfARNT2c. Using quantitative RT-PCR methods the tissue distribution of each zfARNT2 isoform was determined. In COS-7 cells expressing zfARNT2b and zfAhR2, 10 nM TCDD causes a nine-fold induction of a dioxin responsive reporter gene. In COS-7 cells expressing zfARNT2a or zfARNT2c, TCDD does not induce reporter gene expression. In contrast, all three zfARNT2 proteins induce reporter gene activity under control of hypoxia responsive elements when cotransfected with the zebrafish endothelial specific PAS protein 1. DNA gel shift analysis suggests that the decreased function of zfARNT2a is due to inefficient binding of zfARNT2a/zfAhR2 complexes to dioxin responsive elements. These results also indicate that alternative mRNA splicing results in formation of ARNT proteins with distinct functional properties.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262, USA. robert.tanguay@uchsc.edu
authors.forename: R L
authors.initials: RL
authors.lastname: Tanguay
authors.forename: E
authors.initials: E
authors.lastname: Andreasen
authors.forename: W
authors.initials: W
authors.lastname: Heideman
authors.forename: R E
authors.initials: RE
authors.lastname: Peterson
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D019556:COS Cells; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004147:Dioxins / Q000494:pharmacology; D020869:Gene Expression Profiling; D017930:Genes, Reporter / Q000235:genetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010100:Oxygen / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology / Q000633:toxicity; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D016415:Sequence Alignment; D015398:Signal Transduction / Q000187:drug effects; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D004147:Dioxins; D010100:Oxygen; D000072317:Polychlorinated Dibenzodioxins; D020033:Protein Isoforms; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D015534:Trans-Activators; D014157:Transcription Factors; D029961:Zebrafish Proteins; D000099162:Endothelial PAS Domain-Containing Protein 1; C490990:arnt2 protein, zebrafish