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.