Solubilization of the Ecdysone Binding Protein from Anterior Silk Gland Cell Membranes of the Silkworm, Bombyx mori

Elmogy, Mohamed; Iwami, Masafumi; Sakurai, Shunsuke

doi:10.2108/zsj.24.971

Cited by 4 publications

(8 citation statements)

References 31 publications

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“…These membrane-bound molecules appear to be related to conventional EcR (Elmogy et al, 2004(Elmogy et al, , 2007.…”

Section: Introductionmentioning

confidence: 95%

Ecdysteroid receptor (EcR) is associated with microtubules and with mitochondria in the cytoplasm of prothoracic gland cells of Rhodnius prolixus (Hemiptera)

Vafopoulou¹

2009

Arch Insect Biochem Physiol

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We have shown previously that EcR in larval Rhodnius is present in the cytoplasm of various cell types and undergoes daily cycling in abundance in the cytoplasm (Vafopoulou and Steel, 2006. Cell Tissue Res 323:443-455). It is unknown which organelles are associated with EcR. Here, we report that cytoplasmic EcR in prothoracic gland cells is associated with both microtubules and mitochondria, and discuss the implications for both nuclear and non-genomic actions of EcR. EcR was localized immunohistochemically using several antibodies to EcR of Manduca and Drosophila and a confocal laser scanning microscope. Double labels were made to visualize EcR and (1) microtubules (using an antibody to tyrosylated alpha-tubulin) and (2) mitochondria (using a fluorescent MitoTracker probe), both after stabilization of microtubules with taxol. EcR co-localized with both tubulin and mitochondria. All the different EcR antibodies produced similar co-localization patterns. EcR was seen in the perinuclear aggregation of mitochondria, indicating that mitochondria are targets of ecdysone, which could influence mitochondrial gene transcription. EcR was also distributed throughout the microtubule network. Co-localization of EcR with tubulin or mitochondria was maintained after depolymerization of microtubules with colchicine. Treatment with taxol resulted in accumulation of EcR in the cytoplasm and simultaneous depletion of EcR from the nucleus, suggesting that microtubules may be involved in targeted intracellular transport of EcR to the nucleus (genomic action) or may play a role in rapid ecdysone signal transduction in the extranuclear compartment, i.e., in non-genomic actions of ecdysone. These findings align EcR more closely with steroid hormone receptors in vertebrates.

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“…These membrane-bound molecules appear to be related to conventional EcR (Elmogy et al, 2004(Elmogy et al, , 2007.…”

Section: Introductionmentioning

confidence: 95%

Ecdysteroid receptor (EcR) is associated with microtubules and with mitochondria in the cytoplasm of prothoracic gland cells of Rhodnius prolixus (Hemiptera)

Vafopoulou¹

2009

Arch Insect Biochem Physiol

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“…In addition, different Immuno-histochemical and biochemical localization techniques of Bombyx nuclear receptor BmEcR-B1 show that this protein is clearly indicated and is restricted in the cell nuclei and not translocated to cytosol or cell membrane during the prepupal period [19,20]. Also, the solubilisation scheme for the anterior silk gland membrane fractions preparations verified that the solubilized fraction using this technique retained 75% of its native ecdysone binding activity [21]. Accordingly, we used these fractions confidently for immunizing mice.…”

Section: Resultsmentioning

confidence: 96%

“…However, mEcR isolation still remains elusive [17,18,19,20,21]. A clear characterization and sequencing of the mEcR mediating the rapid nongenomic effects of ecdysone during the programmed cell death of B, mori anterior silk glands is still lacking.…”

Section: Resultsmentioning

confidence: 99%

“…Protein amounts were measured using a DC protein assay kit (Bio-Rad) with BSA as standard. The obtained membrane fractions were then solubilized according the solubilization scheme described in [21] using CHAPS and NaCl. The solubilized fractions retaining 75% of its native binding activity for PonA were used as antigen for immunization of mice.…”

Section: Methodsmentioning

confidence: 99%

“…Previous studies provided several biochemical and topological evidence for the presence of a putative membrane ecdysone receptor (mEcR) located in the plasma membranes of B. mori anterior silk glands (ASGs), which may act in concert with the conventional nuclear EcR [18,19,20]. Then after, we succeeded to identify, fully characterize and solubilize mEcR from ASGs [21]. Therefore, the present work aimed to raise monoclonal antibodies against mEcR.…”

Section: Introductionmentioning

confidence: 97%

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Production and characterization of monoclonal antibodies raised against the membrane ecdysone receptor from the anterior silk gland of the silkworm, Bombyx mori

Elmogy¹,

Elgendy²

2014

iosrphr

Self Cite

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Several evidences for nongenomic action of ecdysteroids through membrane receptor (mEcR) in programmed cell death (PCD) of the Bombyx mori anterior silk glands (ASGs) have been reported. The isolation, characterization and sequencing of mEcR still remain elusive. In the present work, monoclonal antibodies against mEcR has been raised, for the first time, and expected to facilitate any further molecular characterization of that novel receptor. The solubilized ASGs membrane fractions were used for immunizing mice. Three out of 4 injected mice showed immune-reactive signal, as revealed by dot blot assay. Out of the 3 mice, only one mouse sera showed to react with [ 3 H]PonA binding protein, as revealed by double antibody capture assay (DACA). Out of 117 primary hybridoma cell lines prepared only 43 were selected by using dot blot assay, while 7 out of the 43 were selected by using DACA. From the 7 selected clones, clone (13-2) of the highest bound dpm in DACA was used for limiting dilution assay. The pattern of immune-reactivity of 7 out of 8 of the diluted cultures were very similar and showed two signal bands of 65 and 42 kDa, as revealed by western blot analysis. From which, a single clone designated (13-2-23) of the highest bound dpm in DACA was reacted with a single protein band of 42 kDa, suggesting its probable specificity to the extracellular binding domain of mEcR. This work will pave the way for more understanding of the molecular mechanism of ecdysteroid rapid actions during PCD of ASGs in Bombyx mori.

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Ecdysone Receptors: From the Ashburner Model to Structural Biology

Hill

Billas

Bonneton

et al. 2013

Annu. Rev. Entomol.

136

101

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In 1974, Ashburner and colleagues postulated a model to explain the control of the puffing sequence on Drosophila polytene chromosomes initiated by the molting hormone 20-hydroxyecdysone. This model inspired a generation of molecular biologists to clone and characterize elements of the model, thereby providing insights into the control of gene networks by steroids, diatomic gases, and other small molecules. It led to the first cloning of the EcR subunit of the heterodimeric EcR-USP ecdysone receptor. X-ray diffraction studies of the ligand-binding domain of the receptor are elucidating the specificity of receptor-ecdysteroid interactions, the selectivity of some environmentally friendly insecticides, the evolution of the EcR-USP heterodimer, and indeed Ashburner's classical biochemical evidence for the central role of the ecdysone receptor in his model.

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Solubilization of the Ecdysone Binding Protein from Anterior Silk Gland Cell Membranes of the Silkworm, Bombyx mori

Cited by 4 publications

References 31 publications

Ecdysteroid receptor (EcR) is associated with microtubules and with mitochondria in the cytoplasm of prothoracic gland cells of Rhodnius prolixus (Hemiptera)

Ecdysteroid receptor (EcR) is associated with microtubules and with mitochondria in the cytoplasm of prothoracic gland cells of Rhodnius prolixus (Hemiptera)

Production and characterization of monoclonal antibodies raised against the membrane ecdysone receptor from the anterior silk gland of the silkworm, Bombyx mori

Ecdysone Receptors: From the Ashburner Model to Structural Biology

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