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

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(b) lacks the sequence and has a COOH-terminal domain of 104 residues rich in proline, serine, and threonine. rtARNT, 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-tetrachlo rodibenzo-p-dioxin results in a 20-fold greater induction of endogenous P4501A1 protein in cells expressing rtARNT(a) when compared with rtARNT(b), 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.