Use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin as a probe to determine the relative ligand affinity of human versus mouse aryl hydrocarbon receptor in cultured cells.
The aryl hydrocarbon receptor (AhR) is a ligand-induced transcription factor that is activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other related compounds, leading to toxicity. There is considerable variation in the response to TCDD among different species, and this may be correlated to differences in the AhR. Variations in the structure of the AhR could result in altered biochemical properties of the receptor, such as ligand affinity or transactivation potential. The difference between the mouse AhR b-1 allele (mAhR(b-1)) and human AhR (hAhR), in terms of their relative affinity for a photoaffinity ligand (2-azido-3-[(125)I]iodo-7,8-dibromodibenzo-p-dioxin), was assessed using both in vitro assays and assays performed directly in cell culture. Results revealed that the hAhR has a lower affinity for the photoaffinity ligand compared with mAhR(b-1). In contrast with a previous study, we found that a single amino acid (valine 381) in hAhR is responsible for the lower ligand affinity, and mutating this residue to alanine results in restoration of high ligand affinity in hAhR. In vitro ligand binding assays are limited by the low concentrations of protein in the assays, and it is not appropriate to compare ligand affinities of different receptors using this method without performing a competition assay or increasing the protein concentration in the assay. Because of the limitation of the in vitro assay, the relative ligand occupancy of mAhR(b-1) and hAhR was compared most effectively within cells, revealing that mAhR(b-1) has a 10-fold higher relative ligand affinity in cells, whereas mAhR(d) has a 2-fold higher relative ligand affinity than hAhR.
Ramadoss
Preeti
P
Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary Science, Graduate Program in Biochemistry, Microbiology and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
Perdew
Gary H
GH
D000409:Alanine / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002478:Cells, Cultured; D004147:Dioxins / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D051379:Mice; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D018336:Receptors, Aryl Hydrocarbon / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D014633:Valine / Q000235:genetics
D004147:Dioxins; D007457:Iodine Radioisotopes; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; C054916:2-azido-3-iodo-7,8-dibromodibenzo-1,4-dioxin; D014633:Valine; D000409:Alanine