Characterization of the region of the aryl hydrocarbon receptor required for ligand dependency of transactivation using chimeric receptor between Drosophila and Mus musculus.
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor. Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is high affinity and toxic to many vertebrate animals, invertebrate AhRs including Drosophila melanogaster AhR (spineless) have no ability to bind exogenous chemicals as ligands. To analyze the ligand-binding domain (LBD) of AhR, we used chimeras between mouse and Drosophila AhR. The chimeric AhR revealed that the LBD determines constitutive transactivation in Drosophila AhR or ligand-dependent activation in mouse AhR. The LBD was further divided into three blocks that corresponded to amino acids 230-300, 301-361, and 361-420 of the mouse sequence. Six chimeric proteins clarified that amino acids 291-350 of the Drosophila LBD, i.e. the middle region, were required to keep the protein in the active form in the absence of ligand binding, whereas in the mouse AhR, this region was required to maintain the protein in the inactive form in the absence of ligand. Furthermore, Arg346 in the middle region of the mouse LBD, was identified as amino acids that were critical for AhR activation by site-directed mutagenesis.
Kudo
Kyoko
K
United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan.
Takeuchi
Takeshi
T
Murakami
Yusuke
Y
Ebina
Masayuki
M
Kikuchi
Hideaki
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000072317:Polychlorinated Dibenzodioxins / Q000493:pharmacokinetics; D017434:Protein Structure, Tertiary; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D015533:Transcriptional Activation
D017931:DNA Primers; D029721:Drosophila Proteins; D008024:Ligands; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011993:Recombinant Fusion Proteins; C112211:ss protein, Drosophila