Abstract
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 rtARNTa.AHR complexes to DNA. In addition, the presence of rtARNTa can reduce the aryl hydrocarbon receptor-dependent function of rtARNTb in vivo and in vitro.
Highlights
CDNAs encoding two distinct basic helix-loop-helix/ PER-ARNT-SIM proteins with similarity to the mammalian aryl hydrocarbon nuclear translocator (ARNT) protein were isolated from RTG-2 rainbow trout gonad cells
Based on the model of aryl hydrocarbon receptor (AHR)-mediated signal transduction in mammals, these results implied that a functional ARNT-like protein was expressed in RTG-2 cells
The amount of xenobiotic response element sequence 5Ј-TNGCGTG-3Ј (XRE) shifted by rtARNTa was 5 Ϯ 5% of the amount shifted by an identical amount of rtARNTb. These results suggest that rtARNTa does not complement AHR-mediated signal transduction due to reduced rtARNTaAHR binding at the XRE
Summary
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. These results provide strong evidence that the rtARNTa protein contains a COOH-terminal sequence that is highly divergent from mammalian ARNT proteins and is distinct from rtARNTb. Identification of rtARNTa and rtARNTb mRNAs and Protein in Rainbow Trout—To evaluate the expression of rtARNTa and rtARNTb in vivo, total RNA was isolated from RTG-2 cells, rainbow trout gonad tissue, and rainbow trout liver.
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