The Ah receptor is a soluble protein complex of about 280 kD. The receptor binds, and mediates carcinogenesis by, polycyclic aromatic hydrocarbons (PAHs), which are found in cigarette smoke and smog, heterocyclic amines, which constitute the principal carcinogens in cooked meat, and chlorinated aromatic compounds, such as, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The Ah receptor translocates from the cytoplasm to the nucleus on binding ligand. The best understood activity of the receptor concerns its role in the induction of cytochrome P450IA1. Induction occurs as the result of binding of the Ah receptor-ligand complex to xenobiotic responsive transcriptional enhancer elements (XREs) located in the upstream region of the P450IAl gene. The Ah receptor nuclear translocator protein, Arnt, is required for the Ah receptor to translocate to the nucleus; is a structural component of the nuclear form of the Ah receptor; and most probably also forms part of the complex (also containing the ligand-binding subunit of the Ah receptor) that binds and activates the XRE. Experiments will investigate whether Arnt i) is also a structural component of the cytosolic form of the Ah receptor, ii) itself translocates to the nucleus after the Ah receptor binds ligand, iii) binds the XRE directly, or indirectly via its association with the ligand-binding subunit, and iv) if it binds directly, to which nucleotides in the XRE Arnt and the ligand-binding subunit each bind. An experiment will be performed to ascertain whether Arnt binds to other proteins, besides the ligand-binding subunit of the Ah receptor. The protein-binding capacity of Arnt will be utilized to clone cDNAs for those proteins that interact with Arnt, including the ligand-binding subunit. The role of these proteins in Ah receptor action will be investigated by inhibiting protein activity, using antisense oligonucleotides or antisense cDNAs. In vitro mutagenesis experiments will be performed on Arnt in order to identify putative functional domains, including domains for nuclear translocation, for binding the ligand-binding subunit, for binding the XRE, and for transcriptional activation. The amt gene mutations in the two C- mutants of Hepa-1 cells will be sought in order to confirm that the putative mutations responsible for loss of Ah receptor translocation in these mutants reside in the amt gene. The promoter region of the human amt gene will be cloned and sequenced, in order to identify potential regulatory effectors of the amt gene, and to provide the means for identifying the start site of transcription.
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