Molecular Determinants of Differential Ligand Sensitivities of Insect Ecdysteroid Receptors
AUTOR(ES)
Wang, Sheng-Fu
FONTE
American Society for Microbiology
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=85723Documentos Relacionados
- Molecular identification of the insect adipokinetic hormone receptors
- Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors
- Ecdysteroid receptors in imaginal discs of Drosophila melanogaster.
- Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity
- Molecular Determinants of Snurportin 1 Ligand Affinity and Structural Response upon Binding