Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori
Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori
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DOI：
10.1111/j.1432-1033.2004.04249.x
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发表时间：
2004-08-01
期刊：
影响因子：
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通讯作者：
Sakurai, S
中科院分区：
文献类型：
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作者：
Elmogy, M;Iwami, M;Sakurai, S
非基因组作用的昆虫类固醇激素，20-羟基蜕皮激素（20 E），已牵连在几个20 E依赖的事件，包括程序性细胞死亡的前丝腺（ASG），但没有信息是可用的作用模式。我们提供了一个假定的膜受体位于质膜的ASGs的证据。从ASG制备的膜组分显示出对[H-3]ponasterone A（PonA）的高结合活性。膜级分不含常规的蜕皮激素受体，如通过使用抗人蜕皮激素受体A（EcR-A）的抗体的Western印迹分析所揭示的。结合活性不被IM NaCl或0.05%（w/v）MEGA-8溶解，表明结合位点位于膜中。在Triton X-114中的差异增溶和温度诱导相分离表明，结合位点可能是整合的膜蛋白。这些结果表明，结合位点位于质膜蛋白，我们将其称为膜蜕皮激素受体（mEcR）。mEcR表现出对[H-3]PonA的饱和结合（Kd = 17.3nm，B-max = 0.82 pmol.mg（-1）蛋白）。结合和解离动力学表明，[H-3]PonA与mEcR在几分钟内结合并解离。综合结果支持质膜蜕皮激素受体的存在，这可能与传统的EcR在各种20 E依赖的发展事件中的一致行动。
Nongenomic action of an insect steroid hormone, 20-hydroxyecdysone (20E), has been implicated in several 20E-dependent events including the programmed cell death of Bombyx anterior silk glands (ASGs), but no information is available for the mode of the action. We provide evidence for a putative membrane receptor located in the plasma membrane of the ASGs. Membrane fractions prepared from the ASGs exhibit high binding activity to [H-3]ponasterone A (PonA). The membrane fractions did not contain conventional ecdysone receptor as revealed by Western blot analysis using antibody raised against Bombyx ecdysone receptor A (EcR-A). The binding activity was not solubilized with 1 M NaCl or 0.05% (w/v) MEGA-8, indicating that the binding sites were localized in the membrane. Differential solubilization and temperature-induced phase separation in Triton X-114 showed that the binding sites might be integrated membrane proteins. These results indicated that the binding sites are located in plasma membrane proteins, which we putatively referred to as membrane ecdysone receptor (mEcR). The mEcR exhibited saturable binding for [H-3]PonA (K-d = 17.3 nm, B-max = 0.82 pmol.mg(-1) protein). Association and dissociation kinetics revealed that [H-3]PonA associated with and dissociated from mEcR within minutes. The combined results support the existence of a plasmalemmal ecdysteroid receptor, which may act in concert with the conventional EcR in various 20E-dependent developmental events.