DNA Binding and Transactivation Characteristics of the Mosquito Ecdysone Receptor-Ultraspiracle Complex

Abstract

The steroid hormone 20-hydroxyecdysone is a key regulatory factor, controlling blood-meal triggered egg maturation in mosquitoes. To elucidate the ecdysone hierarchy governing this event, we cloned and characterized the ecdysone receptor (AaEcR) and the nuclear receptor Ultraspiracle (AaUSP), a retinoid X receptor homologue, from the mosquito, Aedes aegypti, which form a functional complex capable of ligand and DNA binding. Here we analyzed the DNA-binding properties of the AaEcR⅐AaUSP heterodimer with respect to the effects of nucleotide sequence, orientation, and spacing between half-sites in natural Drosophila and synthetic ecdysone response element (EcREs). By using an electrophoretic gel mobility shift assay, we showed that AaEcR⅐AaUSP exhibits a broad binding specificity,

Distinct roles of Broad isoforms in regulation of the 20-hydroxyecdysone effector gene, Vitellogenin, in the mosquito Aedes aegypti

“…The coding region sequences of the four BR-C isoforms were inserted into the pAc5.1/ V5/HisA (Invitrogen) vector and fused to a V5 and a His tag (Chen et al, 2004). The construction of plasmids pVg2.1-luc, pAc5-AaEcR and pAc5-AaUSP has been described elsewhere (Wang et al, 1998).…”

Cyclicity of mosquito vitellogenic ecdysteroid-mediated signaling is modulated by alternative dimerization of the RXR homologue Ultraspiracle

“…The green African monkey kidney CV-1 cell line (American Type Culture Collection) was used for the mammalian two-hybrid assay and transfected as described elsewhere (16). In brief, 3 ϫ 10 5 cells were plated on 35-mm dishes for 18-24 h before transfection.…”

“…Two EcR isoforms (AaEcR-A and AaEcR-B) and two USP isoforms (AaUSP-A and AaUSP-B) are expressed with unique patterns during pre-and vitellogenic periods (12)(13)(14)(15). The mosquito EcR-USP heterodimer is capable of binding to various EcREs to modulate ecdysone regulation of target genes (16). However, regulatory regions of two YPP genes have similar EcREs, which are direct repeats with a single spacer (DR-1) (ref.…”

Identification of Glucocorticoid Receptor Domains Involved in Transrepression of Transforming Growth Factor-β Action

“…p6RMR and p6RMR604.GR (44) were digested with KpnI and XbaI, whereas p6RGR524.MR and p6RGR438.MR, were digested with XbaI; the resulting fragments containing the nuclear receptor or chimeras were subcloned into the corresponding sites in pcDNA3.1/Zeo(ϩ). Ultraspiracle expressed with pcDNA3.1/Zeo(ϩ) is as described before (45).…”

“…9A, lanes 1-3). As a negative control, we tested binding of Smad3C to the mosquito Ultraspiracle protein, the insect homolog of retinoid X receptor (45), which displayed virtually no binding to either GST-mad3C or GST (Fig. 9A, lanes 4 -6).…”