The AHR is a ligand dependent transcription factor, which belongs to the bHLH-PAS superfamily of proteins. Originally, the AHR was discovered for mediating the toxicity of environmental toxicants like TCDD and different PAHs. Advanced research showed that the AHR can be bound and activated by a wide range of agonists, including various exogenous and endogenous compounds. According to the classical description of the AHR pathway, the AHR is mainly located in the cytoplasm prior to ligandbinding. Most ligands own hydrophobic characteristics allowing them to enter the cell via diffusion.Ligand-binding to the AHR triggers a nuclear import followed by building a dimer with a protein called ARNT. This heterodimer recruits many co-factors, thereby getting access and subsequently binding to a special sequence in the DNA, the XRE. The AHR/ARNT complex bound to XRE induces the expression of xenobiotic metabolizing enzymes, including phase I and phase II enzymes, thus pointing to a key role of the AHR in xenobiotic metabolism. Examining the intracellular distribution of the AHR in living cells reveals that the AHR undergoes a shuttling between cytoplasm and nucleus. This shuttling occurs continuously and does not lead to any activation. Despite the huge number of studies examining the AHR, the localization remains scarcely investigated. In this thesis, the localization of the AHR was investigated thoroughly after transient transfection of EYFP-AHR leading to expression of a fluorescent fusion protein that can be detected in real-time in living cells. I validated this system by comparing the localization of the overexpressed AHR fusion protein with the endogenous AHR with and without the presence of ligands. First of all, in depth mutagenesis analyses on the crucial amino acids to dimerize with ARNT and bind to XRE were carried out. This aimed to characterize if these two significant steps might have an impact on maintaining the activated AHR in the nucleus after ligand-binding or generally affect its nucelo-cytoplasmic translocation in the basal state. Later, the regulatory motive responsible for intracellular distribution and nuclear import, namely NLS, was characterized using point mutations. Thereby, the intracellular distribution of the AHR variants that carry an impaired NLS was detected in the basal state or after ligand treatment. My results demonstrated that neither ARNT dimerization nor DNA binding have an impact on stabilizing the AHR in the nucleus prior or post ligand-binding. Moreover, the sequence of NLS was identified as (13RKRRK17) and (37KR-R40), whereby the first part represents the main regulator, while the second one has only a supportive and complementary role. Besides that, I examined both nuclear import processes of the AHR: basal and ligand-induced. Thereafter, the effect of novel import inhibitors IPZ and IVM was tested on the mentioned nuclear import of the AHR. I found that ligand induced import can occur when the basal import is diminished. At the same time, trying to block the nuclear import using import inhibitors showed comparable influence on both import pathways. My results proposed that ligand-induced and basal import happen autonomously and independent of each other, but they rely on the same particular mechanism. Taken together, this thesis expressed novel findings on the AHR’s sequence and the molecular components involved in its nucleo-cytoplasmic translocation using advanced methods.
