title: Baculovirus expression of the Ah receptor and Ah receptor nuclear translocater. Evidence for additional dioxin responsive element-binding species and factors required for signaling.
abstract: In an effort to facilitate the structural and biochemical analyses of the Ah receptor (AHR) and the Ah receptor nuclear translocator (ARNT), a baculovirus system was developed to express microgram-milligram quantities of the human version of these proteins. To simplify purification, a polyhistidine tag was cloned at their C termini so that the recombinant proteins could be specifically adsorbed to nickel-nitriloacetic acid-Sepharose. Expression studies revealed that approximately 23% of the overexpressed AHR was recovered in cell extracts with the remaining 77% forming insoluble aggregates. ARNT was found to be more soluble, with 90% recovery from cell extracts and only 10% aggregation. Photoaffinity labeling and gel shift assays demonstrated that the recombinant proteins bound ligand, heterodimerized, and recognized their cognate "dioxin response element" (DRE) in a manner similar to their native counterparts. Coexpression of the AHR and ARNT in Sf9 cells resulted in the in vivo generation of heterodimers that bound the DRE in the absence of ligand. Studies with the nickel-nitriloacetic acid-purified recombinant proteins demonstrated that the AHR and ARNT could bind DRE only when reconstituted with a heat-sensitive factor(s) present in soluble extracts from a variety of cell types. Use of these proteins also demonstrated the existence of at least three AHR-dependent DRE-binding species, suggesting that the AHR can bind to DRE in at least three distinct conformations.
authors.affiliation: Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611.
authors.forename: W K
authors.initials: WK
authors.lastname: Chan
authors.forename: R
authors.initials: R
authors.lastname: Chu
authors.forename: S
authors.initials: S
authors.lastname: Jain
authors.forename: J K
authors.initials: JK
authors.lastname: Reddy
authors.forename: C A
authors.initials: CA
authors.lastname: Bradfield
mesh_terms: D000345:Affinity Labels; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004147:Dioxins / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis; D018336:Receptors, Aryl Hydrocarbon / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018411:Spodoptera; D014157:Transcription Factors
chemical_list: C497396:ARNT protein, human; D000345:Affinity Labels; D004268:DNA-Binding Proteins; D004147:Dioxins; D011506:Proteins; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator