An in vitro assay for investigating factors which modulate formation of the nuclear aryl hydrocarbon (Ah) receptor complex was developed using 9S cytosolic Ah receptor isolated from wild-type Hepa 1c1c7 cells treated with 2, 3, 7, 8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) at 4°C and nuclei from Ah-responsive and -nonresponsive wild-type and mutant Hepa 1c1c7 cells. Incubation of the radiolabeled ([3H]TCDD) 9S cytosolic Ah receptor with nuclei from untreated wild-type Ah-responsive mouse Hepa 1c1c7 cells resulted in a time- and temperature-dependent formation of the nuclear Ah receptor complex as determined by either velocity sedimentation analysis or gel mobility shift assays using a consensus 32P-labeled dioxin-responsive element. Maximal levels of the nuclear Ah receptor formed within 30 min at 37°C and significantly lower levels were observed after incubation at 4, 15, or 25°C. Complementation studies using nuclei from untreated wild-type and Ah-nonresponsive class II mutant (translocation-deficient) cells and radiolabeled 9S cytosolic receptor (bound with [3H]TCDD) from both wild-type and mutant cell lines were also carried out. The results indicated that nuclear translocation was primarily inhibited using cytosol from mutant cells confirming the requirement for the aryl hydrocarbon receptor nuclear translocator protein for formation of the nuclear Ah receptor complex. The effects of a series of Ah receptor antagonists, ATP, vanadate apyrase, phosphatases, and lectin WGA, on formation of the nuclear Ah receptor complex were also investigated in the in vitro model using radiolabeled cytosolic Ah receptor and nuclei from untreated wild-type Hepa 1c1c7 cells. α-Naphthoflavone, ribonuclease, lectin WGA, vanadate, acid and alkaline phosphatase, and apyrase inhibited and ATP enhanced formation of the nuclear Ah receptor complex. The results also snow that TCDD-induced transformation of the Ah receptor was inhibited by α-naphthoflavone but not by ATP, lectin WGA, or apyrase. These data suggest that the cytosolic Ah receptor is phosphorylated prior to ligand binding and ATP is required for energy-dependent nuclear import of the Ah receptor.
ASJC Scopus subject areas
- Molecular Biology