Properties of nuclear and cytosolic ecdysteroid receptors from an epithelial cell line from Chironomus tentans

Turberg, Andreas; Spindler, Klaus-Dieter

doi:10.1016/0022-1910(92)90036-d

Cited by 30 publications

(12 citation statements)

References 33 publications

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“…Ligand binding was determined with [ 3 H]-ponasterone A (specific activity 2.5 TBq/mmol) using a filter assay as already described previously (Turberg and Spindler, 1992). The receptor concentration of yeast extracts was quantified by western blots (Rauch et al, 1998) and a constant ratio of EcR/USP was used in all experiments (Grebe et al, 2003).…”

Section: Ligand Binding Assaysmentioning

confidence: 99%

Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor

Grebe

Fauth

Spindler-Barth

2004

Insect Biochemistry and Molecular Biology

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Section: Ligand Binding Assaysmentioning

confidence: 99%

Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor

Grebe

Fauth

Spindler-Barth

2004

Insect Biochemistry and Molecular Biology

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“…CHO cells were transfected as described above and cell extracts prepared as described previously (Azoitei & Spindler‐Barth, 2009). Ligand binding was determined with 3 [H]‐Ponasterone A (specific activity 7.9 TBq/mmol) using a filter assay (Turberg & Spindler, 1992). Briefly, cell extracts were incubated routinely with 3 [H]‐Ponasterone for 5 h at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, cell extracts were incubated routinely with 3 [H]‐Ponasterone for 5 h at 4 °C. Free ligand was removed by filtration (Turberg & Spindler, 1992) using NC45 nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) and washed with 3 × 4 ml ice‐cold washing buffer (20 mM HEPES, 20 mM NaCl, 10% glycerol (v/v), 1 mM EDTA, 1 mM β‐mercaptoethanol, pH 7.9). Bound radioactivity was measured with a liquid scintillation counter (Tri‐Carb 1500 1500, Perkin Elmer, Rodgau‐Jügesheim, Germany) and the nonspecific binding, determined by competition with 1 µM 20‐OH‐Ecdysone, subtracted.…”

Section: Methodsmentioning

confidence: 99%

Influence of helix 12 of Ultraspiracle on Drosophila melanogaster ecdysone receptor function

Tremmel

Azoitei

Schaefer

et al. 2011

Insect Molecular Biology

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Although it has no ligand, helix 12 in the ligand binding domain of Ultraspiracle (USP) is locked in an antagonistic position. To investigate whether this position is of functional importance, we enhanced the flexibility of helix 12 by mutating two amino acids (259, located in L1-3 and F491 in helix 12). Mutated USP reduces the stability of USP and all isoforms of the ecdysone receptor (EcR) and impairs nuclear localization and DNA binding of EcR/USP(L259A/F491/A), resulting in lower levels of basal transcriptional activity. Although the affinity of the ligand ponasterone A to EcR/USP(L259/F491) is moderately diminished, hormone-induced stimulation of transcriptional activity is normal. Potentiation of the ecdysone response by juvenile hormone (JH) is selectively increased in mutated heterodimers with EcR-B1, demonstrating that the antagonistic position impairs functional interaction of the EcR complex with JHIII.

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“…Ligand-binding was determined with 3 [H]-Ponasterone A (specific activity 7.9 TBq/mmol) using a filter assay, as described previously (Turberg and Spindler, 1992 …”

Section: Ligand Bindingmentioning

confidence: 99%

DNA‐binding properties of Drosophila ecdysone receptor isoforms and their modification by the heterodimerization partner ultraspiracle

Braun

Azoitei

Spindler-Barth

2009

Arch Insect Biochem Physiol

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Transcriptional activity of ecdysone receptor (EcR) isoforms varies considerably and is modified further by the heterodimerization partner and hormone treatment. To investigate whether differences in DNA binding of receptor complexes are responsible for these variations in transcriptional activity, interaction of Drosophila EcR isoforms, and variants of Ultraspiracle (Usp), the orthologue of RXR, with the ecdysone response elements (EcRE) hsp 27, PAL-1, and DR-1, were determined by electrophoretic mobility shift assays. Receptor proteins were expressed in vertebrate cells (CHO-K1) in order to rule out an influence of endogenous receptor proteins. In the absence of a heterodimerization partner, weak DNA binding of EcR was detected even without hormone with EcR-A and -B1, but not EcR-B2. In the presence of hormone, all three isoforms show increased binding to the hsp 27 EcRE. The heterodimerization partner Usp increased DNA binding considerably. The hormone effect of heterodimers is more pronounced with both EcR-B isoforms compared to EcR-A. Two specific bands were obtained for EcR-A and B1 but only one band is visible with EcR-B2. Deletion of the C-domain of Usp still allows basal DNA binding of the heterodimer, but in contrast to full-length Usp, addition of hormone decreases the intensity of the retarded receptor band of all EcR isoforms and the EcREs hsp27 and DR-1 considerably, whereas interaction with the EcRE PAL-1 is only slightly affected. Synergistic effects on transcriptional activity are associated with the formation of different receptor DNA-complexes observed with 1xhsp27 and 3xhsp27. Comparison of DNA-binding properties of EcR isoforms and EcR/Usp heterodimers revealed that binding of receptor complexes to hsp 27 EcRE is dependent on the AB domain of EcR and the AB-, C-, and D-domains of the heterodimerization partner. Interaction with the hsp 27 EcRE correlates neither with ligand binding nor with transcriptional activity of the various receptor complexes. We, therefore, conclude that the different receptor functions are regulated separately, for example, by interaction with co-modulators or post-transcriptional modifications.

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Properties of nuclear and cytosolic ecdysteroid receptors from an epithelial cell line from Chironomus tentans

Cited by 30 publications

References 33 publications

Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor

Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor

Influence of helix 12 of Ultraspiracle on Drosophila melanogaster ecdysone receptor function

DNA‐binding properties of Drosophila ecdysone receptor isoforms and their modification by the heterodimerization partner ultraspiracle

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