Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer co nsisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and t he retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), th e physiologically active steroid, little is known about the relative activi ty of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activit ies of various ecdysteroids using gel mobility shift assays and transfectio n assays in Schneider-2 (S2) cells. Ecdysone showed little activation of th e Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this s teroid functioned as a potent ligand for the mosquito Aedes aegypti recepto r complex (AaEcR-USP), significantly enhancing DNA binding and transactivat ing a reporter gene in S2 cells. The mosquito receptor also displayed highe r hormone-independent DNA binding activity than the Drosophila receptor. Su bunit-swapping experiments indicated that the EcR protein, not the USP prot ein, was responsible for ligand specificity. Using domain-swapping techniqu es, we made a series of Aedes and Drosophila EcR chimeric constructs. Diffe rential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimeri zation 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 Phe5 29 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critic al for ligand specificity and hormone-independent DNA binding activity. The se results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.