title: P23 enhances the formation of the aryl hydrocarbon receptor-DNA complex.
abstract: The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that requires heterodimerization with its partner, the Ah receptor nuclear translocator (Arnt), for activation of transcription. The heterodimer specifically recognizes the dioxin response element (DRE), which contains a core sequence (5'-TNGCGTG-3'). This AhR/Arnt/DRE complex has been well characterized and can be observed readily by the gel shift assay. Human AhR and Arnt with a C-terminal histidine tag have been expressed functionally using a baculovirus expression system. However, after purification of these proteins using the metal resin, they are not able to bind the response element in a ligand-dependent manner unless crude extracts, such as the rabbit reticulocyte lysate (RRL), are reconstituted with these proteins. Proteins in the RRL are responsible for this restoration of the gel shift complex because the activity is sensitive to both heat and proteolytic treatments. We have examined whether hsp90 and p23 are among the protein factors in the RRL that are responsible for this activity. By performing fractionation studies using filtration devices and immunodepletion studies, we have selectively fractionated these proteins. Among all the fractions, the centricon-10 retentate, which contains 100% of p23 but no hsp90, possessed the most enriched activity. Purified bacterial-expressed p23 restored the gel shift complex; the mechanism was mediated at the heterodimerization step and was hsp90-dependent.
authors.affiliation: Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Premnath V
authors.initials: PV
authors.lastname: Shetty
authors.forename: Bhagyashree Y
authors.initials: BY
authors.lastname: Bhagwat
authors.forename: William K
authors.initials: WK
authors.lastname: Chan
mesh_terms: D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D000071876:Prostaglandin-E Synthases; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C497396:ARNT protein, human; D004268:DNA-Binding Proteins; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D010750:Phosphoproteins; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D004247:DNA; C490849:PTGES3 protein, human; D000071876:Prostaglandin-E Synthases