Abstract
The dioxin receptor is a cytoplasmic basic helix-loop-helix/Per-Arnt-Sim homology (bHLH/PAS) protein known to bind planar polycyclic ligands including polycyclic aromatic hydrocarbons, benzoflavones, heterocyclic amines, and halogenated aromatic hydrocarbons, e.g. dioxins. Ligand-induced activation of the dioxin receptor initiates a process whereby the receptor is transformed into a nuclear transcription factor complex with a specific bHLH/PAS partner protein, Arnt. In analogy to the glucocorticoid receptor, the latent dioxin receptor is found associated with the molecular chaperone hsp90. We have defined and isolated a minimal ligand binding domain of the dioxin receptor from the central PAS region, comprising of amino acids 230 to 421, and found this domain to interact with hsp90 in vitro. Expression of the minimal ligand binding domain in wheat germ lysates or bacteria, systems which harbor hsp90 homologs unable to interact with the glucocorticoid or dioxin receptors, resulted in non-ligand binding forms of this minimal 230 to 421 fragment. Importantly, affinity of the minimal ligand binding domain for dioxin was similar to the affinity inherent in the full-length dioxin receptor, and a profile of ligand structures which specifically bound the minimal ligand binding domain was found to be conserved between this domain and the native receptor. These experiments show that the minimal ligand binding domain maintains the quantitative and qualitative aspects of ligand binding exhibited by the full-length receptor, implying that the central ligand binding pocket may exist to accommodate all classes of specific dioxin receptor ligands, and that this pocket is critically dependent upon hsp90 for its ligand binding conformation.
Highlights
Signal transduction by dioxins, related halogenated hydrocarbons, and polycyclic aromatic hydrocarbons is mediated by the dioxin receptor, an intracellular bHLH/PAS1 protein which, in response to
The dioxin receptor and Arnt are distinguished from other members of the bHLH family of transcription factors by virtue of PAS (Per-Arnt-Sim) homology regions, segments of 250 to 300 amino acids juxtaposed to the bHLH motifs which share similarity with the bHLH/PAS Drosophila transcription factor Sim (Nambu et al, 1991), and Drosophila PAS circadian oscillator Per (Huang et al, 1993)
A second chimera, DBD/DR280 – 421 (Fig. 1), consisting of a 50 amino acid truncation from the N-terminal end of the core 230 – 421 dioxin receptor domain, produced a fusion protein with greatly reduced dioxin binding capability (Fig. 2B), as did a C terminally truncated chimera DBD/DR230 – 400. These results indicate that the region of the dioxin receptor between amino acids 230 and 421 provides an accurate demarcation of the core ligand binding domain and that this domain, exhibiting a ligand binding capability similar to that of DBD/DR83– 805, very likely maintains most if not all of the ligand binding activity shown by the native dioxin receptor
Summary
Plasmid Constructs—Plasmids pDBD/GEM, pDBD/DR83– 805/ GEM, and pDBD/DR230 – 421/GEM have been previously described (Whitelaw et al, 1993a). HisHaLBD/pET19b was generated by subcloning into pET19b (Qiagen) a fragment encompasssing tagged ligand binding domain (LBD) codons. This DNA fragment was generated by PCR using an upstream primer containing an NdeI restriction site and bases coding for the hemagglutinin epitope (Kolodziej and Young, 1991) of the monoclonal antibody 12CA5 (Babco) (5Ј-GCTGAGGCATATGTATCTTTATGATGTTCTTGATTATGCAATGAATTTCCAAGGGAGG-3Ј) and a downstream primer (5Ј-GCTTTTGGATCCTTATGGTAGGGGATCCATTATGGGA-3Ј). The HisHaLBD protein which does not contain the additional pET19b 20 amino acids in the C terminus but encompasses only the His tag, the hemagglutinin epitope, and the minimal ligand binding domain (amino acids 230 – 421) of the dioxin receptor was obtained by subcloning the NcoI/BglII fragment from HisHaLBD/pET19b and the BglII/SphI fragment from pLBD/Gem7Zf into pSP72 (Promega), providing pHisHaLBD/SP72. Immunoprecipitations were performed with monoclonal anti-hsp IgM antibody 3G3 (Affinity Bioreagents), or an equal concentration of control mouse IgM antibody TEPC 183 (Sigma) as described previously (McGuire et al, 1994; Perdew and Whitelaw, 1991)
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