This project is focused on identification of the mechanism by which steroid hormones, drugs, and polycyclic aromatic compounds induce cytochrome P450 in mammalian tissues and cultured cell lines. Intracellular protein receptors have been identified for glucocorticoids (the glucocorticoid receptor) and planar polycyclic aromatic compounds (the Ah receptor). These compounds bind to their respective receptors; the ligand-receptor complexes interact with DNA in the cell nucleus and apparently activate directly the transcription of specific genes. Monoclonal antibodies and cDNA clones for the glococorticoid receptor have been produced. A major goal of this project is to produce similar molecular probes for the Ah receptor. During the past year we have concentrated on the purification of the Ah receptor, and we have studied further the structural and functional homologies between the glucocorticoid and Ah receptors. We have found that certain covalently immobilized ligands (such as polycyclic aromatic dyes) can be used effectively for purification of both the glucocorticoid and Ah receptors. We have identified a human form of the Ah receptor in the hepatoblastoma HepG2 cell line, and we are carrying out experiments to """"""""rescue"""""""" the human Ah receptor gene by transfection of human genomic DNA into Ah receptor-deficient mutant mouse cells. Monoclonal antibodies and reverse phase HPLC have been used to isolate proteolytic fragments of the glucocorticoid receptor that represent the DNA and steroid-binding domains of this receptor. The HPLC methods developed initially for analysis of the glucocorticoid receptor have been useful particularly for purification and characterization of the Ah receptor.
