The unliganded AhR is located in the cytosol as part of a multimeric complex. After AhR binds ligand it dissociates from the complex and then dimerizes with the ARNT protein. The AHR/ARNT dimer binds to xenobiotic responsive element (XRE) sequences located in the 5 flanking region of the CYP1A1 gene and certain other genes and activates their transcription. Transcriptional activation may be a means whereby AhR mediates carcinogenesis by its ligands. Both AhR and ARNT contain basic helix-loop-helix (bHLH) domains. However, the AhR/ARNT heterodimer differs from previously described bHLH-containing transcription factors in at least four ways: (1) AhR is unique among these factors in that its activity depends upon binding of ligand. (2) The XRE sequence is different from the E-box sequence, the recognition sequence for nearly all other bHLH proteins. (3) The DNA binding region of AhR is different from that of other bHLH proteins. (4) Dimerization between AhR and ARNT requires approximately 300 amino acid PAS homology regions unique to these proteins, as well as their bHLH domains. Site-directed mutagenesis will be performed in order to characterize the DNA binding regions of AhR and ARNT in detail. Constructs containing the DNA binding regions of AhR and ARNT that are small in size but nevertheless capable of dimerizing and binding DNA will be constructed. The structure of these constructs bound to the XRE sequence will be analyzed by X-ray crystallography. The mouse Sim-1 protein can dimerize with ARNT. The DNA binding sequence recognized by the ARNT/Sim-1 dimer (if any) will be identified. Experiments will address whether the ARNT/Sim-1 dimer acts as a transcriptional activator or repressor, whether Sim-1 inhibits activity of the AhR/ARNT dimer, and whether the endogenous ARNT and Sim-1 proteins interact within cells in vivo. Experiments will also address whether ARNT interacts with the mouse Sim-2 protein. Additional dimerization partners for ARNT, AhR, Sim-1, and Sim-2 will be isolated. The dimers so identified will be analyzed in the same fashion as for the ARNT/Sim-1 dimer. The developmental and tissue-specific expression of these novel proteins and their mRNAs will also be determined. Alternative dimerization partners for ARNT may be receptors for other xenobiotics. Alternative dimerization partners for AhR may dictate binding to a DNA sequence different from the XRE, thus potentially providing a novel route through which ligands for AhR regulate gene expression. Mutants of the mouse Hepa-1 cell line defective in the B gene appear to be affected in a factor required for transcription of the AhR gene. This factor will be identified or cloned. The D mutant of Hepa-1 cells is most probably defective in post-translational modification of the AhR protein. The modification defect of the mutant will be determined. The factors defective in the B and D mutants are likely to play important roles in the regulation of AhR activity.
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