Interactions of the crustacean nuclear receptors HR3 and E75 in the regulation of gene transcription.
Interactions of the crustacean nuclear receptors HR3 and E75 in the regulation of gene transcription.
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DOI：
10.1016/j.ygcen.2010.03.025
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发表时间：
2010-06-01
影响因子：
2.7
通讯作者：
LeBlanc, Gerald A.
中科院分区：
文献类型：
--
作者：
Hannas, Bethany R.;Wang, Ying H.;Baldwin, William S.;Li, Yangchun;Wallace, Andrew D.;LeBlanc, Gerald A.
内分泌信号转导通过级联发生，涉及配体依赖性和不依赖配体的核受体的作用。在昆虫中，两个这样的核受体HR3和E75相互作用，转导由表皮甾体引发的信号。我们从甲壳类动物水蚤中克隆了这些核受体，以评估它们在这一生态和经济上重要的生物群体中作为基因转录调节因子的功能。来自D. pulex的两个核受体DappuHR3 （NR1F组）和DappuE75 （NR1D组）与NR1F和NR1D组的其他成员具有高度的序列相似性，这表明它们与RORE（类视黄酮孤儿受体元件）具有单体结合。DappuE75具有血红素结合到配体结合域所需的关键氨基酸残基。接下来，我们开发了一种包含由RORE驱动的荧光素酶报告基因的基因转录报告试验。在这个系统中，DappuHR3而不是DappuE75激活了荧光素酶基因的转录。共转染实验显示，DappuE75以剂量依赖的方式抑制dappuhr3依赖性荧光素酶的转录。电泳迁移率转移实验证实DappuHR3与RORE结合。然而，我们没有发现DappuE75与响应元件类似地结合的证据。这些实验进一步证明了DappuE75阻止了DappuHR3与响应元件的结合。综上所述，DappuHR3是RORE调控基因的转录激活因子，而DappuE75是该活性的负调控因子。DappuE75不会通过占据应答元件来抑制DappuHR3的作用，但可能直接与DappuHR3蛋白相互作用。结合前人关于水蚤HR3受20-羟基蜕皮激素高度诱导的研究，这些结果支持了HR3在某些甲壳类动物中是蜕皮激素信号传导的主要成分，并受E75负调控的假设。
Endocrine signal transduction occurs through cascades that involve the action of both ligand-dependent and ligand-independent nuclear receptors. In insects, two such nuclear receptors are HR3 and E75 that interact to transduce signals initiated by ecdysteroids. We have cloned these nuclear receptors from the crustacean Daphnia pulex to assess their function as regulators of gene transcription in this ecologically and economically important group of organisms. Both nuclear receptors from D. pulex (DappuHR3 (group NR1F) and DappuE75 (group NR1D)) exhibit a high degree of sequence similarity to other NR1F and NR1D group members that is indicative of monomeric binding to the RORE (retinoid orphan receptor element). DappuE75 possesses key amino acid residues required for heme binding to the ligand binding domain. Next, we developed a gene transcription reporter assay containing a luciferase reporter gene driven by the RORE. DappuHR3, but not DappuE75, activated transcription of the luciferase gene in this system. Co-transfection experiments revealed that DappuE75 suppressed DappuHR3-dependent luciferase transcription in a dose-dependent manner. Electrophoretic mobility shift assays confirmed that DappuHR3 bound to the RORE. However, we found no evidence that DappuE75 similarly bound to the response element. These experiments further demonstrated that DappuE75 prevented DappuHR3 from binding to the response element. In conclusion, DappuHR3 functions as a transcriptional activator of genes regulated by the RORE and DappuE75 is a negative regulator of this activity. DappuE75 does not suppress the action of DappuHR3 by occupying the response element but presumably interacts directly with the DappuHR3 protein. Taken together with the previous demonstration that daphnid HR3 is highly induced by 20-hydroxyecdysone, these results support the premise that HR3 is a major component of ecdysteroid signaling in some crustaceans and is under the negative regulatory control of E75.
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