Non-genomic ecdysone effects and the invertebrate nuclear steroid hormone receptor EcR--new role for an "old" receptor?

The ecdysteroids (Ec), invertebrate steroid hormones, elicit genomic but also non-genomic effects. By analogy to vertebrates, non-genomic responses towards Ec may be mediated not only by distinct membrane-integrated but also by membrane-associated receptors like the classical nuclear ecdysteroid receptor (EcR) of arthropods. This is supported by a comparison of physiological properties between invertebrate and vertebrate steroid hormone systems and recent findings on the subcellular localization of EcR. The measured or predicted high degree of conformational flexibility of both Ec and the ligand binding domain (LBD) of EcR give rise to a conformational compatibility model: the compatibility between conformations of the cognate receptor's ligand binding domain and structures or conformations of the ligand would determine their interaction and eventually the initiation of genomic versus non-genomic pathways. This model could also explain why specific non-genomic effects are generally not observed with non-steroidal agonists of the bisacylhydrazine group.

Ligand control of interaction in vivo between ecdysteroid receptor and ultraspiracle ligand-binding domain.

Ecdysteroids (Ecs) enhance the formation of Ec receptor-ultraspiracle protein (EcR-USP) heterodimers which regulate gene transcription. To study EcR-USP heterodimerization, fusion proteins were constructed from the LBDs (ligand-binding domains) of Drosophila EcR or USP and the activation or DNA-binding region of GAL4 respectively. Reporter gene ( lacZ ) activation was fully dependent on the co-expression of the two fusion proteins and thus constitutes a reliable measure for the interaction in vivo between the two LBDs in the yeast cell. To identify structures involved in heterodimerization, a total of 27 point mutations were generated in the EcR and USP LBDs at selected sites. Heterodimerization and its inducibility by ligand were mainly affected by mutations in the dimerization interface and in the ligand-binding pocket of EcR respectively. However, also mutations not located in these structures or even in the LBD of USP influenced ligand-dependent heterodimerization. Together with previously reported ligand-binding studies, the existence of such local, intra- and inter-molecular mutation effects suggest that ligand-induced dimerization results from a synergistic interaction between ligand-binding and heterodimerization functions in EcR LBD, and that it depends on global features of the LBDs of EcR and USP and on their mutual surface compatibility.

DNA in the centromeric heterochromatin of polytene chromosomes is topologically open.

Heterochromatin differs from euchromatin by a set of specific features. We suggested earlier that specific features of heterochromatin result from differences in DNA topology of these two chromatin types and provided explanations for the majority of them (Gruzdev 2000). We proposed that, unlike topologically closed euchromatic DNA, the DNA of heterochromatin is topologically open, i.e. it likely contains single- or doublestrand breaks. In this work, we studied the topological state of DNA in a block of centromeric heterochromatin and in a euchromatic banded region of Chironomus melanotus polytene chromosomes by microfluorimetric methods using the fluorescent intercalating dye ethidium bromide (EB). It was demonstrated that the fraction of topologically closed DNA in heterochromatin blocks is five-fold smaller than in the banded region. The data obtained support the hypothesis proposed.

Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle.

The insect ecdysteroid receptor consists of a heterodimer between EcR and the RXR-orthologue, USP. We addressed the question of whether this heterodimer, like all other RXR heterodimers, may be formed in the absence of ligand and whether ligand promotes dimerization. We found that C-terminal protein fragments that comprised the ligand binding, but not the DNA binding domain of EcR and USP and which were equipped with the activation or DNA binding region of GAL4, respectively, exhibit a weak ability to interact spontaneously with each other. Moreover, the heterodimer formation is greatly enhanced upon administration of active ecdysteroids in a dose-dependent manner. This was shown in vivo by a yeast two-hybrid system and in vitro by a modified electromobility shift assay. Furthermore, the EcR fragment expressed in yeast was functional and bound radioactively labelled ecdysteroid specifically. Ligand binding was greatly enhanced by the presence of a USP ligand binding domain. Therefore, ecdysteroids are capable of inducing heterodimer formation between EcR and USP, even when the binding of these receptor proteins to cognate DNA response elements does not occur. This capability may be a regulated aspect of ecdysteroid action during insect development.