A Lysate-Based Microscale Thermophoresis Assay for the Analysis of Ligand-Dependent Aryl Hydrocarbon Receptor Responses

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in xenobiotic metabolism, immune regulation, and cellular homeostasis. AhR-ligand interactions have traditionally been investigated using radioligand binding assays and, more recently, by microscale thermophoresis (MST) applied to purified recombinant AhR-aryl hydrocarbon receptor nuclear translocator (AhR-ARNT) complexes. Although these approaches enable characterization of ligand binding, they require either radiolabeled ligands or purified recombinant receptor systems and therefore do not fully reproduce the molecular complexity surrounding the receptor in cells. Here we develop a lysate-based MST assay to characterize ligand-dependent AhR responses within a cell-derived molecular environment without requiring receptor purification. An AhR-green fluorescent protein (AhR-GFP) rescue cell line was generated in an AHR knockout background, allowing the fluorescently tagged AhR to serve simultaneously as the target protein and intrinsic fluorescent tracer. Functional validation confirmed ligand-induced nuclear translocation of AhR-GFP and cytochrome P450 family 1 subfamily A member 1 (CYP1A1) induction with sensitivity comparable to that of wild-type cells. MST measurements performed directly in cellular lysates revealed concentration-dependent responses to the AhR agonists 6-formylindolo[3,2-b]carbazole (FICZ), 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), β-naphthoflavone, and 1-hydroxyphenazine, as well as the antagonists CH223191 and BAY 2416964, enabling determination of apparent half-maximal effective concentration (EC50app). Collectively, these findings establish a purification-free MST assay for probing ligand-dependent AhR responses within a complex cell-derived molecular environment.