cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt).

We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.

Hirose K ，Morita M ，Ema M ，Mimura J ，Hamada H ，Fujii H ，Saijo Y ，Gotoh O ，Sogawa K ，Fujii-Kuriyama Y ... -  《-》

Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT).

The activated aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT) bind DNA as a heterodimer. Both proteins represent a novel class of basic helix-loop-helix (bHLH)-containing transcription factors in that (i) activation of AHR requires the binding of ligand (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]), (ii) the xenobiotic responsive element (XRE) recognized by the AHR/ARNT heterodimer differs from the recognition sequence for nearly all other bHLH proteins, and (iii) both proteins contain a PAS homology region, which in the Drosophila PER and SIM proteins functions as a dimerization domain. A cDNA for mouse ARNT has been cloned, and potential functional domains of ARNT were investigated by deletion analysis. A mutant lacking all regions of ARNT other than the bHLH and PAS regions is unimpaired in TCDD-dependent dimerization and subsequent XRE binding and only modestly reduced in ability to complement an ARNT-deficient mutant cell line, c4, in vivo. Both the first and second alpha helices of the bHLH region are required for dimerization. The basic region is required for XRE binding but not for dimerization. Deletion of either the A or B segments of the PAS region slightly affects TCDD-induced heterodimerization, while deletion of the complete PAS region severely affects (but does not eliminate) dimerization. Thus, ARNT possesses multiple domains required for maximal heterodimerization. Mutants deleted for PAS A, PAS B, and the complete PAS region all retain some degree of XRE binding, yet none can rescue the c4 mutant. Therefore, both the PAS A and PAS B segments, besides contributing to dimerization, apparently fulfill additional, unknown functions required for biological activity of ARNT.

Reisz-Porszasz S ，Probst MR ，Fukunaga BN ，Hankinson O ... -  《-》

Functional diversity of vertebrate ARNT proteins: identification of ARNT2 as the predominant form of ARNT in the marine teleost, Fundulus heteroclitus.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of the bHLH/PAS protein superfamily. ARNT dimerizes with several PAS superfamily members, including the ligand-activated aryl hydrocarbon receptor (AHR), forming a complex that alters transcription by binding specific elements within the promoters of target genes. Two genes encode different forms of the protein in rodents: ARNT1, which is widely expressed, and ARNT2, which is limited to the brain and kidneys of adults and specific neural and branchial tissues of embryos. In an effort to characterize aryl hydrocarbon signaling mechanisms in Fundulus heteroclitus, a marine teleost that can develop heritable xenobiotic resistance, we have isolated a liver cDNA encoding an ARNT homolog. The protein exhibits AHR-dependent DNA binding capability typical of other vertebrate ARNTs. Unexpectedly, phylogenetic analysis reveals that the cDNA encodes an ARNT2. This is the only detectable ARNT sequence in Fundulus liver, gill, ovary, and brain, suggesting that ARNT2 is the predominant form of ARNT in this species. Also surprising is the relative lack of sequence identity with another fish ARNT protein, rainbow trout ARNTb, which we show forms a distinct branch outside the ARNT1 and ARNT2 clades in phylogenetic analyses. Functional diversity of ARNT proteins in fish may have important implications for the assessment of aryl hydrocarbon effects on natural populations. The increasing use of fish models in developmental and toxicological studies underscores the importance of identifying taxon-specific roles of ARNT proteins and their potential dimeric partners in the PAS superfamily.

Powell WH ，Karchner SI ，Bright R ，Hahn ME ... -  《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》

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 mRN

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.

Pollenz RS ，Sullivan HR ，Holmes J ，Necela B ，Peterson RE ... -  《JOURNAL OF BIOLOGICAL CHEMISTRY》

Functional analysis of aryl hydrocarbon receptor nuclear translocator interactions with aryl hydrocarbon receptor in the yeast two-hybrid system.

The aryl hydrocarbon receptor (AHR) mediates dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced transcriptional activation of a battery of genes by interaction with a cofactor, called aryl hydrocarbon receptor nuclear translocator (ARNT) protein. Both AHR and ARNT belong to a family of proteins that includes the Drosophila circadian-rhythm protein and "single-minded" protein. These proteins share a domain called the PAS domain. In addition to the PAS domain, both AHR and ARNT contain basic helix-loop-helix (bHLH) and glutamine (Q)-rich domains. The roles of these domains in the receptor-mediated transcriptional activation are not understood completely. By using the yeast two-hybrid system with the N-terminal half of AHR as a probe, which contains the bHLH and PAS regions, to screen cDNA libraries prepared from human lymphocytes and C57BL mouse liver for clones encoding proteins capable of binding to these regions, we isolated a partial ARNT cDNA clone. These results demonstrated that the N-terminal half of AHR is capable of interacting with ARNT in yeast (probably through the bHLH motif). A fusion protein containing the GAL4 DNA binding domain (DB) linked to the full-length AHR was not capable of activating expression of a reporter gene containing the GAL4 DNA binding site, suggesting that ligand-free AHR alone has no transactivating properties in yeast. However, the C-terminal portion (amino acid residues 580-797) of the AHR, including the Q-rich domain, could confer transactivation of the reporter gene expression in the same system, suggesting that the N-terminal portion of the AHR contains transcription repression properties. In contrast, GAL4(DB)-ARNT fusion protein was able to activate expression of the same reporter gene. Deletion analysis of ARNT revealed that the C-terminal 75 amino acids, including the Q-rich domain, exhibited full transactivation function in yeast and mammalian cells. These results revealed different structural organizations for the transactivation properties between AHR and ARNT, although both contained transactivation domains at the C-termini.

Yamaguchi Y ，Kuo MT 《BIOCHEMICAL PHARMACOLOGY》