Nuclear localization and DNA binding of ecdysone receptor and ultraspiracle

Abstract

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.

Ecdysteroid hormone action

“…The DNA-binding domain of ultraspiracle causes a deformation of the response element, whereas the C-domain of the ecdysone receptor adds only a slight change to the preformed structure [59]. Nuclear localisation is not necessarily associated with DNA binding [23], which is stimulated by hormone [24]. This is confirmed by EMSA experiments: they revealed weak interaction with DNA, which is reinforced by addition of hormone.…”

“…enhanced interaction with DNA [22] and chromatin [23] or shuttling of the ecdysteroid receptor into the nucleus [24]. Muristerone A stimulates transcriptional activity in vertebrate cells devoid of endogenous EcR and Usp, which are transfected with dmEcR to different degrees depending on the isoform used [25,26].…”

Review of the reproductive biology of amphipods and their endocrine regulation: Identification of mechanistic pathways for reproductive toxicants

“…The ecdysone receptor is a ligand-dependent transcription factor, and it activates transcription of target genes by forming a heterodimer with another nuclear receptor, the ultraspiracle protein [118]. Binding to the DNA helix is mediated by the nuclear receptor that encompasses two zinc-finger polypeptides, where Zn complexes four cysteine amino acids, thereby facilitating the folding of a protein domain involved in the DNA-protein interaction [119]. Displacement of zinc in the DNA binding protein domain by Pb and Cd has been proposed as a mechanism causing toxicity of these metals [120,121], but whether these metals cause reproductive toxicity in amphipods by this mechanism has not been determined.…”

Ecdysone receptor and ultraspiracle proteins are tyrosine phosphorylated during adult development of silkmoths

“…This study suggests that sufficient PTK exists in the nucleus to account for EcR and USP tyrosine phosphorylation. Detection of EcR and USP in the cytoplasmic fractions is not contradictory to the earlier observations regarding the exclusive localization of these functional partners to the nucleus by immuno-histochemical analyses (Talbot et al, 1993;Koch et al, 2003;Cronauer et al, 2007). The protein amounts employed for EcR detection and the sensitivity of the detection procedure used here are orders of magnitude higher than those by immuno-histochemistry.…”

“…The question arose as to where and how EcR and USP were tyrosine phosphorylated because most tyrosine kinases are either membrane bound or in the cytoplasm, while EcR and USP are for the most part nuclear (Talbot et al, 1993;Cronauer et al, 2007; unpublished immuno-histochemical and immuno-fluorescent localization in silkmoth wing epidermis). Hence, PTK activity and extent of PTP were determined on the cytoplasmic and nuclear fractions prepared form polyphemus wing epidermis at two time Fig.…”