Transcriptional Activation by the Mouse Ah Receptor.: INTERPLAY BETWEEN MULTIPLE STIMULATORY AND INHIBITORY FUNCTIONS

Qiang Ma,Liqun Dong,James P Whitlock

doi:10.1074/jbc.270.21.12697

Abstract

The aromatic hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates cellular responses to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We cloned AhR cDNA from C57BL/6 mouse liver and verified by transfection that it encodes a functional protein. Analyses of deletion mutants indicate that the carboxyl half of AhR contains several types of transactivation domain, which function independently of domains that mediate TCDD recognition, DNA binding, and heterodimerization with the Ah receptor nuclear translocator (Arnt) protein. The transactivation domains function independently of each other, display different levels of activity, and act synergistically when linked. In addition, AhR contains an 82-amino acid domain that inhibits transactivation. The inhibitory domain displays specificity, in that it blocks the transactivating functions of AhR and Arnt, but not that of the herpes simplex protein VP16. The inhibitory activity depends upon the cell type in which AhR is expressed, implying that a cell-specific protein mediates the effect.

Highlights

The aromatic hydrocarbon receptor (AhR) is a liganddependent transcription factor that mediates cellular responses to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
As a prelude to analyzing the biological function of AhR, we cloned its cDNA from mouse liver using a PCR-based approach
The findings in this report indicate that AhR, like other transcription factors, contains domains that function independently

Summary

EXPERIMENTAL PROCEDURES

Materials-Vent polymerase was from New England Biolabs (Beverly, MA). The plasmids pRc/CMV and pCHllO were from Invitrogen Corp. (San Diego, CAl and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ), respectively. The forward primers for the NH 2-terminal deletions were as follows: N2: primer F2, 5' -TGTCGTCTAGAATGAGCTTCTTTGATGTTGCATT3', which contains an XbaI site and nucleotides 238-257 of AhR coding sequence (underlined). NC2: forward primer F8 and reverse primer R4, 5' -TGCCGGGCCCTCACTGCACACTCTTGGAAT-3', containing an ApaI site, a stop codon, and nucleotides 2138-2154 of the AhR noncoding strand (underlined). NC7: forward primer F2 and reverse primer R5: 5' -TGCCTCTAGATTCTCCAGTCTTAATCATGC-3', which contains an XbaI site and nucleotides 998-1017 of the AhR noncoding strand (underlined). Each cDNA construct was used as a template to direct protein synthesis in an in vitro transcription/translation system, as described below These studies verified that the full-length AhR, its deletion mutants, and the GaI4:AhR/Arnt fusion proteins had the expected molecular weights and were expressed at similar levels.

A Report er ORE

A AhR G4:N6 G4:NC4:N6 G4:N8 G4:NC4:N8 G4:NC5 G4:NC6 G4 B Arnt

Findings

DISCUSSION