Functional characterization of aryl hydrocarbon receptor (zfAHR2) localization and degradation in zebrafish (Danio...

The basic-helix-loop-helix/PAS (bHLH/PAS) family of proteins is a group of transcription factors that regulate key pathways during normal development and in the response to stress. The aryl hydrocarbon receptor (AHR) is a member of this family. Recently, Danio rerio (zebrafish) has become an important model system in the study of the signal transduction pathway and complements the results seen in mammalian models. However, studies of the AHR protein have been limited by the lack of antibody reagents and thus, little is known concerning the localization and degradation of the zebrafish AHR (zfAHR). In this report, we describe the production and characterization of specific polyclonal antibodies to the zfAHR2 protein and the analysis of AHR-mediated signal transduction in the zebrafish liver cell line (ZFL). The results show that the zfAHR2 is degraded via the 26S proteasome following exposure of cells to beta-naphthoflavone (BNF). Interestingly, the time course is slower and the magnitude of zfAHR2 degradation is not as great as seen for the mammalian AHR. Studies also show that the zfAHR2 is rapidly degraded in a ligand-independent manner by exposure of cells to geldanamycin (GA) to levels consistent with mammalian AHR. Finally, immunohistochemical staining of the ZFL cells suggest that the unliganded AHR resides in both the cytoplasm and nucleus and undergoes active nucleocytoplasmic shuttling in the absence of ligand. These results suggest that there is conservation of function between fish and mammals with respect to ligand-dependent and -independent degradation of the AHR and that the zfAHR2 is degraded via the 26S proteasome.