Elsevier

Biochemical Pharmacology

Volume 57, Issue 10, 15 May 1999, Pages 1177-1190
Biochemical Pharmacology

Gene Expression and Developments
Functional analysis of activation and repression domains of the rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNT) protein isoforms

https://doi.org/10.1016/S0006-2952(99)00036-2Get rights and content

Abstract

The aryl hydrocarbon receptor nuclear translocator (ARNT) protein is involved in many signaling pathways. Rainbow trout express isoforms of ARNT protein that are divergent in their C-terminal domains due to alternative RNA splicing. Rainbow trout ARNTb (rtARNTb) contains a C-terminal domain rich in glutamine and asparagine (QN), whereas the C-terminal domain of rtARNTa is rich in proline, serine, and threonine (PST). rtARNTb functions positively in AH receptor-mediated signaling, whereas rtARNTa functions negatively. Studies were performed to understand how changes in the C-terminal domains of the two rtARNT isoforms affect function. Deletion of the QN-rich C-terminal domain of rtARNTb did not affect function in aryl hydrocarbon receptor (AHR)-mediated signaling, whereas deletion of the PST-rich domain of rtARNTa restored function. Expression of the PST-rich domain on truncated rtARNTb or mouse ARNT (mARNT) reduced function of this protein by 50–80%. Gel shift assays revealed that the PST-rich domain affected AHR-mediated signaling by inhibiting DNA binding of the AHR•ARNT heterodimer. Gal4 transactivation assays revealed a potent transactivation domain in the QN-rich domain of rtARNTb. In contrast, Gal4 proteins containing the PST-rich domain of rtARNTa did not transactivate because the proteins did not bind to DNA. Secondary structure analysis of the PST-rich domain revealed hydrophilic and hydrophobic regions. Truncation of the hydrophobic domain that spanned the final 20–40 amino acids of the rtARNTa restored function to the protein, suggesting that repressor function was related to protein misfolding or masking of the basic DNA binding domain. Functional diversity within the C-terminal domain is consistent with other negatively acting transcription factors and illustrates a common biological theme.

Section snippets

Materials

Specific antibodies against mouse ARNT protein (R-1) and rainbow trout ARNT (rt-84) are identical to those described previously 13, 38. All antibodies are affinity-purified IgG fractions. Antibodies against Gal4 1–147 (anti-Gal4) were purchased from Santa Cruz Biotechnology. Specific antibodies against the FLAGTM epitope (M5 anti-FLAGTM) were purchased from the Kodak Co. For western blot analysis, goat anti-rabbit antibodies or goat anti-mouse antibodies conjugated to horseradish peroxidase

Ability of rtARNTb and mARNT to complement AHR-mediated signaling

The strategy of the studies in this report was to evaluate the ability of various rtARNT isoforms to complement AHR-mediated signal transduction in the type II Hepa-1 cell line. The choice of a mammalian cell line for analysis of rtARNT was due to (i) the fact that type II cells do not express high levels of mARNT, (ii) the ability to assay the expression of recombinant rtARNT protein in total cell lysates, (iii) the ability to evaluate complementation of AHR-mediated signaling by analysis of

Conclusions and implications

Recently, several bHLH/PAS proteins have been described that inhibit the functionality of bHLH/PAS heterodimers. One of the ARNT protein isoforms from rainbow trout, rtARNTa, can bind to mAHR and function as a dominant negative regulator of AHR-mediated signaling [13]. mSIM2, on the other hand, appears to bind to ARNT in a nonfunctional heterodimer 30, 31. In the case of rtARNTa, negative function appears to involve sequestration of liganded AHR in a complex that does not bind DNA, while mSIM2

Acknowledgements

This project was supported, in part, by grants to R. S. P. from the National Institute of Environmental Health Sciences (Grant ES 08980) and the South Carolina Seagrant Consortium (Grant R/ER-12) and a STAR Graduate Fellowship to B. N. from the Environmental Protection Agency (Fellowship 915218–01).

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