Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors
Autor(es): Wang, SF; Ayer, S; Segraves, WA; Williams, DR; Raikhel, AS
Resumo: 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.
Palavras-Chave: Retinoid-X-Receptor; Drosophila Ecdysone Receptor; Thyroid-Hormone Receptor; Manduca-Sexta; Nuclear Receptors; Messenger-Rna; Developmental Expression; Prothoracic Glands; Crystal-Structure; Tobacco Hornworm
Imprenta: Molecular and Cellular Biology, v. 20, n. 11, p. 3870-3879, 2000
Identificador do objeto digital: 10.1128/MCB.20.11.3870-3879.2000
Descritores: Aedes aegypti - Cell ; Aedes aegypti - DNA ; Aedes aegypti - Proteins ; Aedes aegypti - RNA
Data de publicação: 2000
