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

作者：Sheng Fu Wang, Ken Miura, Richard J. Miksicek, William A. Segraves, Alexander S. Raikhel · 发表于：Journal of Biological Chemistry · 年份：1998 · DOI：10.1074/jbc.273.42.27531 · 被引用次数：83 · 研究领域：Neurobiology and Insect Physiology Research、Animal Behavior and Reproduction、Insect and Pesticide Research

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 AaEcR.AaUSP 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 AaEcR.AaUSP exhibits a broad binding specificity, forming complexes with inverted (IR) and direct (DR) repeats of the nuclear receptor response element half-site consensus sequence AGGTCA separated by spacers of variable length. A single nucleotide spacer was optimal for both imperfect (IRhsp-1) and perfect (IRper-1) inverted repeats; adding or removing 1 base pair in an IRhsp-1 spacer practically abolished binding. However, changing the half-site to the consensus sequence AGGTCA (IRper-1) increased binding of AaEcR.AaUSP 10-fold over IRhsp-1 and, at the same time, reduced the stringency of the spacer length requirement, with IRper-0 to IRper-5 showing detectable binding. Spacer length was less important in DRs of AGGTCA (DR-0 to DR-5); although 4 bp ...