AH RECEPTOR PHOSPHORYLATION - LOCALIZATION OF PHOSPHORYLATION SITES TO THE C-TERMINAL HALF OF THE PROTEIN

The aryl hydrocarbon receptor (AhR) is a transcriptional enhancer acti vated by the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related xenobiotics. Ligand binding initiates a series of poorly unde rstood molecular events which confers recognition of cis-acting elemen ts located in regulatory regions of particular structural genes, such as CYP1A1. Several studies have suggested that AhR phosphorylation may be instrumental in activating the AhR to a DNA-binding state. In agre ement with previous investigations, treatment of the AhR with acid pho sphatase resulted in the loss of DNA-binding activity. To further eval uate the functional role of AhR phosphorylation we determined whether TCDD binding altered total AhR phosphorylation, and identified phospho rylated regions by the examination of chemical cleavage patterns. The AhR was isolated by immunoprecipitation from [P-32]-orthophosphate-lab eled Hepa 1 cells grown in the presence or absence of TCDD. Examinatio n of the amount of P-32 associated with the AhR indicated that the tot al level of AhR phosphorylation was not affected by ligand binding. Ch emical cleavage with hydroxylamine and cyanogen bromide also revealed a similar pattern for liganded and unliganded AhR. The shortest region s of overlap determined by the chemical cleavage patterns localized ph osphorylation sites to two regions in the C-terminal half of the AhR, One region is centrally located between amino acids 368 and 605 and wi thin or adjacent to a DNA binding repressor domain. The other region i s located at the glutamine-rich carboxyl terminus between amino acids 636 and 759. These data coupled with previous observations imply that total AhR phosphorylation is not altered by the ligand-elicited transf ormation to a DNA-binding form, but that phosphorylation nevertheless plays an important role in the ability of an active AhR-Arnt complex t o associate with cis-acting regulatory elements. (C) 1995 Academic Pre ss,Inc.