Expression of the Human Aryl Hydrocarbon Receptor Complex in Yeast: ACTIVATION OF TRANSCRIPTION BY INDOLE COMPOUNDS

Charles A Miller

doi:10.1074/jbc.272.52.32824

Abstract

The human aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator protein (ARNT) were coexpressed in the yeast Saccharomyces cerevisiae to create a system for the study of this heterodimeric transcription factor. Specific transcriptional activation mediated by AHR/ARNT heterodimer, which is a functional indicator of receptor expression, was assessed by beta-galactosidase activity produced from a reporter plasmid. Yeast expressing AHR and ARNT displayed constitutive transcriptional activity that was not augmented by addition of AHR agonists in strains that required exogenous tryptophan for viability. In contrast, strains with an intact pathway for tryptophan biosynthesis responded to AHR agonists and had lower levels of background beta-galactosidase activity. Hexachlorobenzene, benzo(a)pyrene, and beta-naphthoflavone were effective AHR agonists in the yeast system, and had EC50 values of 200, 40, and 20 nM, respectively, for beta-galactosidase activity induction. Tryptophan, indole, indole acetic acid, and tryptamine activated transcription in yeast coexpressing AHR and ARNT (EC50 values approximately 300 microM). Indole-3-carbinol was an exceptionally potent AHR agonist (EC50 approximately 10 microM) in yeast. This yeast system is useful for the study of AHR/ARNT protein complexes, and may be generally applicable to the investigation of other multiprotein complexes.

Highlights

The human aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator protein (ARNT) were coexpressed in the yeast Saccharomyces cerevisiae to create a system for the study of this heterodimeric transcription factor
Dose-dependent responses to ␣- and ␤-naphthoflavones showed that intact AHR functions in yeast when coexpressed with intact ARNT protein, the signal from the DRE-directed reporter plasmid was relatively weak
The observation that the human AHR and ARNT genes function in yeast is remarkable when one considers the many complex processes that must be conserved for human aryl hydrocarbon receptor complex (AHRC) to function as a transcription factor when expressed in yeast

Summary

ACTIVATION OF TRANSCRIPTION BY INDOLE COMPOUNDS*

AHR is activated by exposure to exogenous aromatic ligands, several investigations have demonstrated constitutive AHRC activation in mammalian cell lines that were not exposed to known agonists. Genetic evidence suggesting endogenous AHR activation in vivo in the absence of exogenously added ligands is demonstrated by the 10-fold reduction in the AHRC-responsive hepatic CYP1A2 mRNA in mice which lack AHR [12]. This result is consistent with the presence of an endogenous AHR agonist in vivo and, along with the results above, supports the hypothesis of an endogenous AHR agonist(s). Dose-dependent responses to ␣- and ␤-naphthoflavones showed that intact AHR functions in yeast when coexpressed with intact ARNT protein, the signal from the DRE-directed reporter plasmid was relatively weak. The data shown below indicate that considerable AHR activation occurs in yeast and are consistent with hypothesis that an endogenous AHR agonist is present, even though yeast express no known homologues of AHR and ARNT

MATERIALS AND METHODS

RESULTS

TABLE I Plasmids used in this study

DISCUSSION