Isolation and Expression of cDNAs from Rainbow Trout (Oncorhynchus mykiss) That Encode Two Novel Basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) Proteins with Distinct Functions in the Presence of the Aryl Hydrocarbon Receptor

doi:10.1074/jbc.271.48.30886

Isolation and Expression of cDNAs from Rainbow Trout (Oncorhynchus mykiss) That Encode Two Novel Basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) Proteins with Distinct Functions in the Presence of the Aryl Hydrocarbon Receptor

EVIDENCE FOR ALTERNATIVE mRNA SPLICING AND DOMINANT NEGATIVE ACTIVITY IN THE bHLH/PAS FAMILY*

From the ^‡ Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425 and the
^¶ Environmental Toxicology Center and School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53706

^§ 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{at}musc.edu

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 rtARNT_a and rtARNT_b, are identical over the first 533 amino acids and contain a basic helix-loop-helix domain that is 100% identical to human ARNT. rtARNT_a and rtARNT_b 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. rtARNT_a lacks the sequence and has a COOH-terminal domain of 104 residues rich in proline, serine, and threonine. rtARNT_b 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. rtARNT_a and rtARN_b 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 rtARNT_b when compared with rtARNT_a, even though both proteins effectively dimerize with the aryl hydrocarbon receptor. The decreased function of rtARNT_a appears to be due to inefficient binding of rtARNT_a·;AHR complexes to DNA. In addition, the presence of rtARNT_a can reduce the aryl hydrocarbon receptor-dependent function of rtARNT_b in vivo and in vitro.

Footnotes

↵* 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. The 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™/EMBL Data Bank with accession number(s) U73840 and U73841.
↵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

2,3,7,8-tetrachlorodibenzo-p-dioxin

PAS

PER-ARNT-SIM

GAR-TR

goat anti-rabbit 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

reverse-transcriptase

EMSA

electrophoretic mobility shift assay

TNT

transcription and translation.
↵² R. S. Pollenz, unpublished observations.
↵³ The band between the 1183 and 810 fragments appears to be a PCR artifact containing a duplex of the 1183 and 810 bands. RT-PCR from the alternatively spliced plasma membrane calcium ATPase has been reported to result in the appearance of similar artifact bands (48). The 960-bp band was never isolated during the screening of 300 colonies that resulted in the isolation of pcrG41 and pcrG79.
↵⁴ RTG-2 cells stained with rt84 show strong nuclear reactivity (R. S. Pollenz, manuscript in preparation).
↵⁵ Preliminary deletion studies with mARNT, suggest that removal of the RXXKRR domain results in a protein with a predominantly cytoplasmic location in type II cells (R. S. Pollenz, manuscript in preparation).
↵⁶ Very high levels of basal luciferase activity were detected in type II cells co-transfected with pMVrtARNT_a or pMVrtARNT_b and an XRE-driven luciferase reporter plasmid. However, in all experiments the level of luciferase activity associated with cells expressing rtARNT_b was 3-10-fold higher than the luciferase activity associated with cells expressing rtARNT_a. (H. L. Root and R. S. Pollenz, unpublished observations.)
↵⁷ In one transfection experiment, the pSV-β-galactosidase expression vector was also included. When the P4501A1 protein concentration in each sample was normalized to the β-galactosidase activity of that sample, the mean ± S.D. for cells expressing rtARNT_b was 51 ± 22 compared with 22 ± 2 for cells expressing both rtARNT_b and rtARNT_a.
- Received July 12, 1996.
- Revision received September 17, 1996.