title: Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors.
abstract: The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.
authors.affiliation: Programs in Genetics, Cell & Molecular Biology, Department of Entomology, Michigan State University, East Lansing, Michigan 48824, USA.
authors.forename: S F
authors.initials: SF
authors.lastname: Wang
authors.forename: S
authors.initials: S
authors.lastname: Ayer
authors.forename: W A
authors.initials: WA
authors.lastname: Segraves
authors.forename: D R
authors.initials: DR
authors.lastname: Williams
authors.forename: A S
authors.initials: AS
authors.lastname: Raikhel
mesh_terms: D000330:Aedes; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002874:Chromosome Mapping; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D004440:Ecdysone / Q000378:metabolism* / Q000494:pharmacology; D004441:Ecdysterone / Q000378:metabolism* / Q000494:pharmacology; D008024:Ligands; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D008024:Ligands; D011987:Receptors, Steroid; D014157:Transcription Factors; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor; D004440:Ecdysone; D014443:Tyrosine; D010649:Phenylalanine; D004441:Ecdysterone; D004247:DNA