The Zebrafish (Danio rerio) Aryl Hydrocarbon Receptor Type 1 Is a Novel Vertebrate Receptor

Abstract

Fish are known to have two distinct classes of aryl hydrocarbon receptors, and their roles in mediating xenobiotic toxicity remain unclear. In this study, we have identified and characterized a cDNA tentatively named zebrafish AHR1 (zfAHR1). Analysis of the deduced amino acid sequence reveals that the protein is distinct from zfAHR2 and is more closely related to the mammalian aryl hydrocarbon receptor (AHR). zfAHR1 and zfAHR2 share 40% amino acid identity overall and 58% in the N-terminal half. The zfAHR1 gene maps to linkage group 16 in a region that shares conserved synteny with human chromosome 7 containing the human AHR, suggesting that the zfAHR1 is the ortholog of the human AHR. zfAHR2 maps to a separate linkage group (LG22). Both zfAHR mRNAs are expressed in early development, but they are differentially expressed in adult tissues. zfAHR2 can dimerize with zfARNT2b and binds with specificity to dioxin-responsive elements (DREs). Under identical conditions, zfAHR1/zfARNT2b/DRE complexes are formed; however, the interactions are considerably weaker. In COS-7 cells expressing zfARNT2b and zfAHR2, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure leads to a significant induction of dioxin-responsive reporter genes. In identical experiments, TCDD exposure fails to induce the reporter gene in zfAHR1-expressing cells. Ligand-binding experiments suggested that the differential zfAHR activities are attributable to differences in TCDD binding because only zfAHR2 exhibits high-affinity binding to [ 3 H]TCDD or ␤-naphthoflavone. Finally, using chimeric zfAHR1/zfAHR2 constructs, the lack of TCDDmediated transcriptional activity was localized to the ligandbinding and C-terminal domains of zfAHR1.The aryl hydrocarbon receptor (AHR) is a member of the basic helix-loop-helix PAS family of proteins. Members of this family include the aryl hydrocarbon receptor nuclear translocators (ARNT, ARNT2, and ARNT3), hypoxia-inducible factor 1␣, Drosophila melanogaster single-minded, D. melanogaster period, Clock, and others. These proteins are involved in mediating responses to environmental contaminants, low oxygen tension and glucose, circadian rhythm, and various other cues (for review, see Gu et al., 2000). The basic components of the AHR signal transduction pathway are well understood (for review, see Schmidt and Bradfield, 1996). The cytosolic AHR is complexed with at least three chaperone proteins, two molecules of HSP90 and the aryl hydrocarbon interacting protein (AIP, also known as ARA9 or XAP2), enabling proper conformation for ligand binding. Once bound by ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the cytosolic AHR translocates to the nucleus, dissociates from the chaperone proteins, and dimerizes with ARNT. This heterodimeric AHR/ARNT complex associates with specific DNA sequences termed dioxin-response elements (DRE, alternatively known as xenobiotic response el-

Functional properties of the four Atlantic salmon (Salmo salar) aryl hydrocarbon receptor type 2 (AHR2) isoforms

“…This is consistent with other studies indicating that AHR2 is the predominant form in fish (Abnet et al 1999a;Karchner et al 1999;Roy and Wirgin 1997;Tanguay et al 1999). Also, in zebrafish (Danio rerio) the AHR1 forms are either inactive (AHR1a; (Andreasen et al 2002a)) or have reduced TCDD sensitivity as compared with AHR2 (AHR1b ). Furthermore, in zebrafish the single AHR2 has been shown to mediate CYP1A induction and developmental toxicity (Prasch et al 2003).…”

Identification of Zebrafish ARNT1 Homologs: 2,3,7,8-Tetrachlorodibenzo-p-dioxin Toxicity in the Developing Zebrafish Requires ARNT1

“…Instead, this suggests that there are likely to be structural and functional differences between the proteins that affect their ability to interact with zfAHR2 and XRE sequences. As has been reported previously (Tanguay et al, 2000;Andreasen et al, 2002a), this complex migrates as two distinct shifted bands, although the nature of this doublet is still unclear.…”

“…To use zebrafish as a model to study TCDD developmental toxicity, it was essential to identify and characterize the zebrafish AHR signaling pathway, and much is now known about its components. Three forms of the AHR (zfAHR1, zfAHR1B, and zfAHR2) have been identified in zebrafish (Tanguay et al, 1999;Andreasen et al, 2002a;Karchner et al, 2005), and morpholino knockdown of zfAHR2 has identified it as the receptor that binds TCDD, leading to downstream effects (Prasch et al, 2003;Teraoka et al, 2003;Dong et al, 2004). In untreated zebrafish liver cells, zfAHR2 has been shown to shuttle between the cytoplasm and nucleus.…”

Photobiological effects of UVA and UVB light in zebrafish embryos: Evidence for a competent photorepair system

“…The frequency of hatching and mortality are not reciprocal, as there were larvae that hatched but died prior to 5 d. Similar representation of zebrafish hatch and mortality rates is seen in toxicological studies [30,31]. The time of hatching in zebrafish embryos can vary within the third day after fertilization and thus is not a precise staging index for development [1].…”