BACULOVIRUS EXPRESSION OF THE AH RECEPTOR AND AH RECEPTOR NUCLEAR TRANSLOCATOR - EVIDENCE FOR ADDITIONAL DIOXIN-RESPONSIVE ELEMENT-BINDING SPECIES AND FACTORS REQUIRED FOR SIGNALING

In an effort to facilitate the structural and biochemical cal analyses of the Ah receptor (AKR) and the Ah receptor nuclear translocator (AR NT), a baculovirus system was developed to express microgram-milligram quantities of the human version of these proteins. To simplify purifi cation, a polyhistidine tag was cloned at their C termini so that the recombinant proteins could be specifically adsorbed to nickel-nitriloa cetic acid-Sepharose. Expression studies revealed that approximately 2 3% of the overexpressed AHR was recovered in cell extracts with the re maining 77% forming insoluble aggregates. ARNT was found to be more so luble, with 90% recovery from cell extracts and only 10% aggregation. Photoaffinity labeling and gel shift assays demonstrated that the reco mbinant proteins bound ligand, heterodimerized, and recognized their c ognate ''dioxin response element'' (DRE) in a manner similar to their native counterparts. Coexpression of the AHR and ARNT in Sf9 cells res ulted in the in vivo generation of heterodimers that bound the DRE in the absence of ligand. Studies with the nickel-nitriloacetic acid-puri fied recombinant proteins demonstrated that the AHR and ARNT could bin d DRE only when reconstituted with a heat-sensitive factor(s) present in soluble extracts from a variety of cell types. Use of these protein s also demonstrated the existence of at least three AHR-dependent DRE- binding species, suggesting that the AHR can bind to DRE in at least t hree distinct conformations.