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 ecdy...
详细信息
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.
Nuclear receptors contain a conserved hydrophobic ligand binding pocket that is particularly amenable to structure-based protein engineering. Thus, site-directed mutagenesis of the ligand binding pocket has resulted i...
详细信息
Nuclear receptors contain a conserved hydrophobic ligand binding pocket that is particularly amenable to structure-based protein engineering. Thus, site-directed mutagenesis of the ligand binding pocket has resulted in the creation of nuclear receptors with novel ligand specificities. Such proteins are now being used to control gene expression in vivo in a ligand-dependent manner.
Specific binding of the insect steroid hormone 2-hydroxyecdysone to imaginal discs of D. melanogaster was investigated. Evidence was presented showing that most of the specific binding was located in the nuclear fract...
详细信息
Specific binding of the insect steroid hormone 2-hydroxyecdysone to imaginal discs of D. melanogaster was investigated. Evidence was presented showing that most of the specific binding was located in the nuclear fraction at the time changes in gene function were observed. Nuclear binding is high affinity, analog specific, apparently saturable and unaffected by inhibitors of RNA and protein synthesis. The association kinetics of nuclear binding are very similar to the specific binding in whole cells. Specific binding to whole discs and to disc nuclei is temperature-dependent, but equal levels of nuclear binding are achieved after 1 h at ***. C and 8 h at ***. C. There is little or no lag in the nuclear location of specific binding at either temperature. The biochemical properties of the specific nuclear binding are consistent with the involvement of the sites in the hormone detection and response system mediating imaginal disc morphogenesis.
On the basis of chromatographic evidence, it appears that the major circulating molting hormone is β-ecdysone in the crab, Pachygrapsus crassipes. This circulating molting hormone does not appear to be bound to any c...
详细信息
暂无评论