Isolation and Expression of cDNAs from Rainbow Trout ( Oncorhynchus mykiss ) That Encode Two Novel

cDNAs encoding two distinct basic helix-loop-helix/ PER-ARNT-SIM (bHLH/PAS) proteins with similarity to the mammalian aryl hydrocarbon nuclear translocator (ARNT) protein were isolated from RTG-2 rainbow trout gonad cells. The deduced proteins, termed rtARNTa and rtARNTb, are identical over the first 533 amino acids and contain a basic helix-loop-helix domain that is 100% identical to human ARNT. rtARNTa and rtARNTb differ in their COOH-terminal domains due to the presence of an additional 373 base pairs of sequence that have the characteristics of an alternatively spliced exon. The presence of the 373-base pair region causes a shift in the reading frame. rtARNTa lacks the sequence and has a COOH-terminal domain of 104 residues rich in proline, serine, and threonine. rtARNTb contains the sequence and has a COOH-terminal domain of 190 residues rich in glutamine and asparagine. mRNAs for both rtARNT splice variants were detected in RTG-2 gonad cells, trout liver, and gonad tissue. rtARNTa and rtARNb protein were identified in cell lysates from RTG-2 cells. Transfection of rtARNT expression vectors into murine Hepa-1 cells that are defective in ARNT function (type II) result in rtARNT protein expression localized to the nucleus. Treatment of these cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a 20-fold greater induction of endogenous P4501A1 protein in cells expressing rtARNTb when compared with rtARNTa, even though both proteins effectively dimerize with the aryl hydrocarbon receptor. The decreased function of rtARNTa appears to be due to inefficient binding of rtARNTazAHR complexes to DNA. In addition, the presence of rtARNTa can reduce the aryl hydrocarbon receptor-dependent function of rtARNTb in vivo and in vitro. The aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins are constitutively expressed basic helix-loop-helix (bHLH) transcription factors that are thought to mediate many of the biological effects of halogenated aromatic hydrocarbons typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (1–3). These proteins are members of the bHLH/PAS family of transcription factors that also includes theDrosophila proteins SIM and PER (4–7). The common characteristics of these proteins are bHLH regions and a domain of approximately 300 amino acids termed PAS (PER, AHR ARNT, SIM) that is distal to the bHLH motif. Deletion and mutational analysis has identified the basic region of these proteins as critical for DNA binding, a finding consistent with other bHLH and bHLH leucine zipper proteins (8–12). The HLH and PAS domains appear to have multiple functions that include dimerization, hsp90 binding, and ligand binding in the AHR (8, 11, 13). Deletion analysis has defined regions in the COOH-terminal portion of both the AHR and ARNT as important in transactivation (8, 13–15). The hypoxia inducible factor 1a (Hif-1a) and a novel murine ARNT termed ARNT-2 have recently been added to the bHLH/PAS family (16, 17). The current model of AHR-mediated signal transduction in mammals hypothesizes that the AHR exists in association with hsp90 in a multiprotein complex primarily within the cytoplasmic compartment of cells (18–21). Following ligand binding, the AHR becomes localized within the nuclear compartment in a form that presumably has dissociated from hsp90 (20–21). Once in the nucleus, a fraction of the ligand-bound AHRs form heterodimers with the nuclear ARNT protein, whereas the majority of the AHRs are rapidly degraded (20, 22–25). The AHR•ARNT heterodimers are then capable of binding the consensus xenobiotic response element sequence 59-TNGCGTG-39 (XRE) to modulate gene expression (12, 26, 27). Presently, the complement of genes regulated by the AHR and ARNT is un* This work was supported in part by a grant from the South Carolina Seagrant Consortium and is part of the “Chemical-Biological Interactions in Aquatic Species” project funded by the National Seagrant Program. This work was also supported by the Medical University of South Carolina Institutional Research Funds of 1995–1996. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EBI Data Bank with accession number(s) U73840 and U73841. § To whom correspondence should be addressed: Dept. Biochemistry and Molecular Biology, MUSC, 171 Ashley Ave., Charleston, SC 29425. Tel. : 803-792-4321; Fax: 803-792-4322; E-mail: pollenzr@musc.edu. 1 The abbreviations used are: AHR, aryl hydrocarbon receptor; bHLH, basic helix-loop-helix; ARNT, aryl hydrocarbon nuclear translocator; hARNT, human ARNT; mARNT, mouse ARNT; TCDD, 2,3,7,8tetrachlorodibenzo-p-dioxin; PAS, PER-ARNT-SIM; GAR-TR, goat antirabbit Texas Red; Me2SO, dimethyl sulfoxide; XRE, xenobiotic response element; PCR, polymerase chain reaction; PAGE, polyacrylamide electrophoresis; bp, base pair(s); kb, kilobase pair(s); ECL, enhanced chemiluminescence; MOPS, 4-morpholinepropanesulfonic acid; RT, reversetranscriptase; EMSA, electrophoretic mobility shift assay; TNT, transcription and translation. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 48, Issue of November 29, pp. 30886–30896, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.