The Q-rich subdomain of the human Ah receptor transactivation domain is required for dioxin-mediated transcriptional activity

The aryl hydrocarbon receptor (AhR), a basic helix-loop-helix/Per-Arnt-Sim transcription factor, mediates many of the toxic and biological effects of the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin, which i nclude the transcriptional activation of dioxin-responsive genes such as CY P1A1. Many aspects of this process are known; however, the mechanism of tra nscriptional activation and the proteins that are key to this process remai n to be determined. The hAhR has a complex transactivation domain, composed of three potentially distinct subdomains. Deletional analysis of the hAhR transactivation domain indicates that removal of the P/S/T-rich subdomain e nhances transcriptional activity, whereas the Q-rich subdomain is critical for hAhR transactivation potential, and the acidic subdomain by itself fail s to activate a dioxin response element-driven reporter gene. Deletional an alysis of the Q-rich subdomain identified a critical stretch of 23 amino ac ids between residues 666 and 688 of the hAhR, which are required for transa ctivation potential. Alanine scanning mutagenesis of this region identified a leucine residue (Leu-678), which is required for hAhR activity. Function al analysis of this point mutant revealed that it is capable of binding lig and, heterodimerization, and subsequent binding to dioxin response elements . Further, when bAhR/L678A and hAhR containing only the acidic subdomain we re overexpressed they acted as dominant negative receptors and repressed wi ld-type hAhR activity. In addition, the hAhR/L678A failed to activate CYP1A 1 gene transcription in transfected BP-8 cells and exhibited reduced bindin g to RIP140 in vitro. Thus, Leu-678 appears to be critical for efficient tr ansactivation activity of the hAhR and appears to disrupt recruitment of co -regulators.