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

Abstract

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.Keywords: Drosophila melanogaster; yeast; two-hybrid; ecdysone receptor; dimerization; ultraspiracle.Ecdysteroids are widespread steroid hormones found in invertebrates [1] and plants [2,3] that regulate a variety of developmental, physiological, and reproductive processes [1,3]. Among insects, these hormones regulate the expression of genes through a highly orchestrated and coordinated transcriptional network [4][5][6]. The widespread and diverse effects of ecdysteroids on transcriptional regulation have served as a powerful model for investigating the diverse mechanisms by which steroid hormones, acting via nuclear receptors, exert their effects on a variety of life processes [4,7].The ecdysone receptor (EcR) [8], responsible for mediating these responses, occupies a special position among nuclear hormone receptors because it shows a unique combination of characteristics [9]. Unlike the vertebrate steroid receptors [10][11][12], EcR heterodimerizes with the insect RXR orthologue, ultraspiracle (USP) [13][14][15]. Nevertheless, while other nuclear receptors that dimerize with RXR normally are bound to DNA response elements already in their nonliganded state [11,16], this apparently is not true for the EcR/USP heterodimer (see however, [17]). Immunostaining has shown that the polytene chromosomes of a Chironomid or Sciarid are devoid of EcR/USP signals when prepared from developmental stages associated with low ecdysteroid titers [18,19]. A short in vitro incubation of the tissues with 20-hydroxyecdysone, however, is followed by the appearance of immunostaining signals at known ecdysteroid-responsive gene loci [18,19]. The affinity of EcR/USP dimers for ecdysone response elements (EcREs) clearly increases in the presence of the ecdysteroid muristerone A as demonstrated...

Interaction of proteins involved in ecdysone and juvenile hormone signal transduction

“…One possible reason could be that the EcR as a fusion with DNA binding domains may be able to heterodimerize with USP in the absence of ligand. Similar results were reported previously; GAL4:EcR (375-652) and AD:USP (172-508) containing a complete ligand binding domains but lacking their DNA binding domains were able to heterodimerize in vivo as well as in vitro in the absence of ligand (Lezzi et al, 2002).…”

The X-ray Structure of a Hemipteran Ecdysone Receptor Ligand-binding Domain

“…The ability of EcR-LBD to bind ecdysteroids is intimately related to its heterodimerization with USP-LBD (8,50,51). It has also been pointed out that the primary sequences of dipteran and lepidopteran USPs are distinct from those of other insect orders (40) with the non-lepidopteran/non-dipteran USPs bearing a closer relationship to mammalian retinoid X receptor proteins than to dipteran or lepidopteran USPs.…”

Ligand binding is without effect on complex formation of the ligand binding domain of the ecdysone receptor (EcR)

“…and interaction with DNA (Khorasanizadeh and Rastinejad, 2001;Przibilla et al, 2004;Lezzi et al, 2002). Dimerization/oligomerization of nuclear receptors is a rather complex process, which may involve several receptor domains.…”

“…Wild type and mutated ligand-binding domains of the ecdysteroid receptor and its dimerization partner USP from Drosophila melanogaster, fused to Gal4 domains were examined by two hybrid assays (Lezzi et al, 2002), ligand-binding and gel mobility shift assays (Grebe et al, 2003Przbilla et al, 2004). From these experiments, the minimal receptor fragment necessary for ligand binding of Drosophila EcR, which consists of the C-terminal part of the D-and the complete E-domain, was inferred.…”

“…DNA encoding the C-terminal part of the Dand E-domains of the Drosophila ecdysone receptor EcR (aa 375-652) was cloned into the expression vector pACT2 (Li et al,1994;Lezzi et al, 2002), resulting in a Gal4(AD)-EcR(LBD) fusion. For expression of Gal4(DBD)-USP(LBD), the corresponding domain of Ultraspiracle (aa 172-508) was cloned either into the vector pGBKT7 (Louvet et al, 1997) or into the vector pAS2-1 (Harper et al, 1993;Lezzi et al, 2002).…”