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Use of [ 125 I]4′‐iodoflavone as a tool to characterize ligand‐dependent differences in Ah receptor behavior
Author(s) -
Swanson Hollie I.,
Whitelaw Murray L.,
Petrulis John R.,
Perdew Gary H.
Publication year - 2002
Publication title -
journal of biochemical and molecular toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.526
H-Index - 58
eISSN - 1099-0461
pISSN - 1095-6670
DOI - 10.1002/jbt.10053
Subject(s) - radioligand , ligand (biochemistry) , hela , chemistry , receptor , nuclear receptor , cytosol , ligand binding assay , biophysics , biochemistry , cell , biology , transcription factor , gene , enzyme
We have synthesized [ 125 I]4′‐iodoflavone to study Ah receptor (AhR)–ligand interactions by a class of AhR ligands distinct from the prototypic ligand 2,3,7,8‐tetrachlorodibenzo‐ p ‐dioxin (TCDD). This radioligand allows the comparison of AhR–ligand interactions using a ligand that differs in AhR affinity, and yet has the same radiospecific activity as [ 125 I]2‐iodo‐7,8‐dibromodibenzo‐ p ‐dioxin. Specific binding of [ 125 I]4′‐iodoflavone with the AhR was detected as a single radioactive peak (∼9.7 S) following density sucrose gradient analysis. Cytosolic extracts from both Hepa 1 and HeLa cells were used as the source of mouse and human AhR, respectively. A ∼6.7 S form of radioligand‐bound Ah receptor was detected in the high salt nuclear extracts of both cell lines. In HeLa cells approximately twofold more [ 125 I]4′‐iodoflavone–AhR 6 S complex, compared with [ 125 I]2‐iodo‐7,8‐dibromodibenzo‐ p ‐dioxin, was recovered in nuclear extracts. A comparison of the ability of 4′‐iodoflavone and TCDD to cause time‐dependent translocation of AhR‐yellow fluorescent protein revealed that 4′‐iodoflavone was more efficient at enhancing nuclear accumulation of the receptor. These results suggest that [ 125 I]4′‐iodoflavone is a particularly useful and easily synthesized ligand for studying the AhR. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:298–310, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10053