Dynamic of ligand binding to Drosophila melanogaster ecdysteroid receptor

Grebe, Marco; Fauth, Torsten; Spindler-Barth, Margarethe

doi:10.1016/j.ibmb.2004.06.007

Cited by 34 publications

(20 citation statements)

References 50 publications

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“…As shown previously, ecdysteroids bind to EcR already in the absence of a heterodimerization partner (Grebe et al, 2003(Grebe et al, , 2004 leading to increased nuclear localization of EcR in the presence of ligand. Only the increased nuclear fraction of EcR observed after hormone treatment is NO sensitive, indicating association with DNA.…”

Section: Nuclear Localization Of Ecr and Uspsupporting

confidence: 62%

Nuclear localization and DNA binding of ecdysone receptor and ultraspiracle

Cronauer¹,

Braun²,

Tremmel³

et al. 2007

Arch Insect Biochem Physiol

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The Ecdysone receptor (EcR) is distributed between cytoplasm and nucleus in CHO cells. Nuclear localization is increased by the ligand Muristerone A. The most important heterodimerization partner Ultraspiracle (Usp) is localized predominantly in the nucleus. We used the diethylentriamine nitric oxide adduct DETA/NO, which releases NO and destroys the zinc-finger structure of nuclear receptors, to investigate whether nuclear EcR and Usp interact with DNA. If expressed separately, Usp and EcR in the absence of hormone do not interact with DNA. The hormone-induced increase in nuclear EcR is due to enhanced DNA binding. In the presence of Usp, EcR is shifted nearly quantitatively into the nucleus. Only a fraction (approximately 30%) of the heterodimer is sensitive to DETA/NO. Interaction of the heterodimer with DNA is mediated mainly by the C-domain of EcR. Deletion of the DNA-binding domain of Usp only slightly reduces nuclear localization of EcR/Usp, although the nuclear localization signal of Usp is not present anymore. The results indicate that EcR and Usp can enter the nucleus independently, but cotransport of both receptors mediated by dimerization via the ligand binding domains is possible even in the absence of hormone.

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Section: Nuclear Localization Of Ecr and Uspsupporting

confidence: 62%

Nuclear localization and DNA binding of ecdysone receptor and ultraspiracle

Cronauer¹,

Braun²,

Tremmel³

et al. 2007

Arch Insect Biochem Physiol

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“…In the absence of a heterodimerization partner, hormone binding is rather weak (Grebe et al, 2004); however, ligand binding is enhanced considerably in the presence of HREs (Azoitei and Spindler-Barth, 2009) and is in accordance with the pronounced increase in DNA binding in the presence of ligand as shown by EMSA. Because the same amount of receptor protein was applied with and without hormone, the enhanced DNA binding is not due to increased receptor concentration observed in hormone-treated cells.…”

Section: Influence Of Receptor Concentration On Hormonal Response Of mentioning

confidence: 61%

“…To ensure that not only was the concentration of EcR and Usp identical, but that the amount of heterodimers applied on each lane of the EMSA gels is the same for all receptor combinations, ligandbinding tests were performed and the number of binding sites determined (Scatchard, 1949). Because ligand binding in the absence of Usp is negligible, the number of binding sites corresponds to the amount of heterodimers (Grebe et al, 2004). This method ensured that not only the same amount of EcR but also the same amounts of heterodimers were applied on each EMSA gel lane.…”

Section: Determination Of Receptor Concentrationsmentioning

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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“…Although EcR can bind ecdysone alone, optimal binding to the ecdysone response elements (EcRE) and activation of transcriptional targets requires the addition of USP (Grad et al 2001;Grebe, Fauth, and Spindler-Barth 2004). USP exhibits a strong structural and functional similarity to the orthologous vertebrate retinoid X receptor (RXR) (Yao et al 1992;Oro, McKeown, and Evans 1990).…”

Section: Ecdysone Ecr and Usp Structure And Functionmentioning

confidence: 99%