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首页> 外文期刊>Molecular and Cellular Biology >Molecular Determinants of Differential Ligand Sensitivities of Insect Ecdysteroid Receptors
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Molecular Determinants of Differential Ligand Sensitivities of Insect Ecdysteroid Receptors

机译：昆虫蜕皮甾体受体的不同配体敏感性的分子决定因素。

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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 theDrosophila 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 ofAedes 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 AedesEcR, 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.
机译：昆虫蜕皮甾体激素的功能性受体是异二聚体，由两个核激素受体，蜕皮甾体受体（EcR）和类维生素X受体同源物Ultraspiracle（USP）组成。尽管通常认为蜕皮激素是一种激素前体和生理活性类固醇20-羟基蜕皮激素（20E），但关于蜕皮类固醇在各种节肢动物中的相对活性知之甚少。作为表征潜在的差异配体识别的步骤，我们已经使用Schneider-2（S2）细胞中的凝胶迁移率迁移测定和转染测定分析了各种蜕皮类固醇的活性。蜕皮激素几乎没有激活果蝇受体复合物（DmEcR-USP）。相比之下，这种类固醇作为蚊子埃及伊蚊受体复合物（AaEcR-USP）的有效配体，可以显着增强DNA结合并激活S2细胞中的报告基因。蚊子受体还显示出比果蝇受体更高的激素非依赖性DNA结合活性。亚单位交换实验表明，EcR蛋白而非USP蛋白是配体特异性的原因。使用域交换技术，我们制作了一系列的 Aedes 和 Drosophila EcR嵌合构建体。差异配体响应性被映射到配体结合域的C末端附近，在先前与核受体的二聚体特异性有关的身份框中。通过与从其他核受体获得的可用晶体结构比较确定，该区域包括螺旋9和10。定点诱变表明，伊蚊 EcR中的Phe529与果蝇 EcR中的Tyr611对应，对配体特异性和激素非依赖性DNA结合活性至关重要。这些结果表明，蜕皮激素可以以物种特异性的方式充当善意配体。

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