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