Wang S F, Miura K, Miksicek R J, Segraves W A, Raikhel A S
Programs in Genetics, Cell & Molecular Biology, Departments of Entomology, Michigan State University, East Lansing, Michigan 48824, USA.
J Biol Chem. 1998 Oct 16;273(42):27531-40. doi: 10.1074/jbc.273.42.27531.
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 was optimal, DR-3 and DR-5 bound AaEcR.AaUSP almost as efficiently as DR-4. Furthermore, AaEcR. AaUSP also bound DRs separated by 11-13 nucleotide spacers. Competition experiments and direct estimation of binding affinity (Kd) indicated that, given identical consensus half-sites and an optimal spacer, the AaEcR.AaUSP heterodimer bound an IR with higher affinity than a DR. Co-transfection assays utilizing CV-1 cells demonstrated that the mosquito EcR.USP heterodimer is capable of transactivating reporter constructs containing either IR-1 or DR-4. The levels of transactivation are correlated with the respective binding affinities of the response elements (IRper-1 > DR-4 > IRhsp-1). Taken together, these analyses predict broad variability in the EcREs of mosquito ecdysone-responsive genes.
类固醇激素20-羟基蜕皮酮是一个关键调控因子，控制着蚊子中血餐引发的卵子成熟过程。为了阐明调控这一过程的蜕皮激素层级关系，我们从埃及伊蚊中克隆并鉴定了蜕皮激素受体（AaEcR）和核受体超气门蛋白（AaUSP，一种类视黄醇X受体同源物），它们形成了一个能够结合配体和DNA的功能复合物。在此，我们分析了AaEcR.AaUSP异源二聚体的DNA结合特性，涉及天然果蝇和合成蜕皮激素反应元件（EcREs）中半位点之间的核苷酸序列、方向和间距的影响。通过使用电泳凝胶迁移率变动分析，我们表明AaEcR.AaUSP表现出广泛的结合特异性，与由可变长度间隔区隔开的核受体反应元件半位点共有序列AGGTCA的反向（IR）和正向（DR）重复序列形成复合物。单个核苷酸间隔区对于不完全（IRhsp - 1）和完全（IRper - 1）反向重复序列都是最佳的；在IRhsp - 1间隔区中添加或去除1个碱基对实际上会消除结合。然而，将半位点改变为共有序列AGGTCA（IRper - 1）使AaEcR.AaUSP的结合比IRhsp - 1增加了10倍，同时降低了间隔区长度要求的严格性，IRper - 0至IRper - 5显示出可检测的结合。在AGGTCA的DRs（DR - 0至DR - 5）中，间隔区长度的重要性较低；尽管4个碱基对是最佳的，但DR - 3和DR - 5与AaEcR.AaUSP的结合效率几乎与DR - 4相同。此外，AaEcR.AaUSP还能结合由11 - 13个核苷酸间隔区隔开的DRs。竞争实验和结合亲和力（Kd）的直接估计表明，在共有半位点相同且间隔区最佳的情况下，AaEcR.AaUSP异源二聚体与IR的结合亲和力高于DR。利用CV - 1细胞进行的共转染实验表明，蚊子EcR.USP异源二聚体能够反式激活含有IR - 1或DR - 4的报告基因构建体。反式激活水平与反应元件各自的结合亲和力相关（IRper - 1 > DR - 4 > IRhsp - 1）。综上所述，这些分析预测蚊子蜕皮激素反应基因的EcREs存在广泛的变异性。
