Bitra Kavita, Palli Subba Reddy
Department of Entomology, University of Kentucky, Lexington, KY 40506, USA.
Arch Insect Biochem Physiol. 2009 Feb;70(2):90-105. doi: 10.1002/arch.20281.
Ecdysteroids and juvenile hormones (JH) regulate a variety of developmental, physiological, behavioral, and metabolic processes. Ecdysteroids function through a heterodimeric complex of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP). An 85 kDa protein identified in Drosophila melanogaster methoprene-tolerant (Met) mutant binds to JH III with high affinity, and the mutant flies are resistant to juvenile hormone analog (JHA), methoprene. Reporter assays using the yeast two-hybrid system were performed in order to study the molecular interactions between EcR, USP and Met. As expected, EcR fused to the B42 activation domain and USP fused to the LexA DNA binding domain interacted with each other and supported induction of the reporter gene in the presence of stable ecdysteroid analog, RG-102240 or steroids, muristerone A and ponasterone A. The USP:USP homodimers supported expression of the reporter gene in the absence of ligand, and there was no significant increase in the reporter activity after addition of a JHA, methoprene. Similarly, Met:Met homodimers as well as Met:EcR and Met:USP heterodimers induced reporter activity in the absence of ligand and addition of ecdysteroid or JH analogs did not increase the reporter activity regulated by either homodimers or heterodimers of Met protein. Two-hybrid assays in insect cells and in vitro pull-down assays confirmed the interaction of Met with EcR and USP. These data suggest that the proteins that are involved in signal transduction of ecdysteroids (EcR and USP) and juvenile hormones (Met) interact to mediate cross-talk between these two important hormones. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.
蜕皮甾体和保幼激素（JH）调节多种发育、生理、行为和代谢过程。蜕皮甾体通过两种核受体——蜕皮激素受体（EcR）和超气门蛋白（USP）组成的异二聚体复合物发挥作用。在耐烯虫酯的黑腹果蝇（Met）突变体中鉴定出一种85 kDa的蛋白质，它与保幼激素III具有高亲和力，并且该突变体果蝇对保幼激素类似物（JHA）烯虫酯具有抗性。为了研究EcR、USP和Met之间的分子相互作用，使用酵母双杂交系统进行了报告基因检测。正如预期的那样，与B42激活结构域融合的EcR和与LexA DNA结合结构域融合的USP相互作用，并在存在稳定的蜕皮甾体类似物RG - 102240或甾体muristerone A和ponasterone A的情况下支持报告基因的诱导。USP:USP同二聚体在没有配体的情况下支持报告基因的表达，添加JHA烯虫酯后报告基因活性没有显著增加。同样，Met:Met同二聚体以及Met:EcR和Met:USP异二聚体在没有配体的情况下诱导报告基因活性，添加蜕皮甾体或JHA类似物不会增加由Met蛋白的同二聚体或异二聚体调节的报告基因活性。昆虫细胞中的双杂交检测和体外下拉检测证实了Met与EcR和USP的相互作用。这些数据表明，参与蜕皮甾体（EcR和USP）和保幼激素（Met）信号转导的蛋白质相互作用，以介导这两种重要激素之间的相互作用。《昆虫生物化学与生理学档案》。2008年。（c）2008威利 - 利斯公司。
