Interactive Fly, Drosophila

Evolution of the nuclear receptor superfamily

From a database containing sequences of published nuclear hormone receptors (NRs), an alignment of the C, D and E domains of NR transcription factors was constructed. Using this alignment, tree reconstruction was performed using both distance matrix and parsimony analysis. The robustness of each branch was estimated using bootstrap resampling methods. The trees constructed by these two methods gave congruent topologies. From these analyses six NR subfamilies were derived: (I) a large clustering of thyroid hormone receptors (TRs), retinoic acid receptors (RARs), peroxisome proliferator-activated receptors (PPARs), vitamin D receptors (VDRs) and ecdysone receptors (EcRs) as well as numerous orphan receptors such as RORs or Rev-erbs; (II) retinoid X receptors (RXRs) together with COUP, HNF4, tailless, TR2 and TR4 orphan receptors; (III) steroid receptors; (IV) NGFIB orphan receptors; (V) FTZ-F1 orphan receptors; and finally (vi) only one gene (to date), the GCNF1 orphan receptor. The relationships between the six subfamilies are not known except for subfamilies I and IV, which appear to be related. Interestingly, most of the liganded receptors appear to be derived when compared with orphan receptors. This suggests that the ligand-binding ability of NRs has been gained by orphan receptors during the course of evolution to give rise to the presently known receptors. The distribution into six subfamilies correlates with the known abilities of the various NRs to bind to DNA as homo- or hetero-dimers. For example, receptors heterodimerizing efficiently with RXR belong to the first or the fourth subfamilies. It is suggested that the ability to heterodimerize evolved once, just before the separation of subfamilies I and IV and that the first NR was able to bind to DNA as a homodimer. From the study of NR sequences existing in vertebrates, arthropods and nematodes, two major steps of NR diversification have been defined: one that took place very early, probably during the multicellularization event leading to all the metazoan phyla, and a second occurring later on, corresponding to the advent of vertebrates. In vertebrate species, the various groups of NRs accumulated mutations at very different rates (Laudet, 1997).

Transfer of insect hormone responsiveness to mammals

During metamorphosis of Drosophila, morphological changes occur in a cascade of events triggered by the steroid hormone 20-OH ecdysone via the Ecdysone receptor, a member of the nuclear receptor superfamily. Insect hormone responsiveness has been experimentally transferred to mammalian cells by the stable expression of a modified Ecdysone receptor that regulates an optimized ecdysone responsive promoter. Inductions reaching 4 orders of magnitude have been achieved upon treatment with hormone. Transgenic mice expressing the modified Ecdysone receptor can activate an integrated ecdysone responsive promoter upon administration of hormone. A comparison of tetracycline-based and ecdysone-based inducible systems reveals the ecdysone regulatory system exhibits lower basal activity and higher inducibility. Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene in transgenic mice and for gene therapy (No, 1996).

Structural studies and evolution of Ecdysone receptor

Ecdysteroid hormones are major regulators in reproduction and development of insects, including larval molts and metamorphosis. The functional ecdysone receptor is a heterodimer of ECR (NR1H1) and USP-RXR (NR2B4), which is the ortholog of vertebrate retinoid X receptors (RXR alpha, beta, gamma). Both proteins belong to the superfamily of nuclear hormone receptors, ligand-dependent transcription factors that share two conserved domains: the DNA-binding domain (DBD) and the ligand-binding domain (LBD). In order to gain further insight into the evolution of metamorphosis and gene regulation by ecdysone in arthropods, a phylogenetic analysis was performed of both partners of the heterodimer ECR/USP-RXR. Overall, 38 USP-RXR and 19 ECR protein sequences, from 33 species, have been used for this analysis. Interestingly, sequence alignments and structural comparisons reveal high divergence rates, for both ECR and USP-RXR, specifically among Diptera and Lepidoptera. The most impressive differences affect the ligand-binding domain of USP-RXR. In addition, ECR sequences show variability in other domains, namely the DNA-binding and the carboxy-terminal F domains. These data provide the first evidence that ECR and USP-RXR may have coevolved during holometabolous insect diversification, leading to a functional divergence of the ecdysone receptor. These results have general implications on fundamental aspects of insect development, evolution of nuclear receptors, and the design of specific insecticides (Bonneton, 2003).

Ecdysteroids initiate molting and metamorphosis in insects via a heterodimeric receptor consisting of the Ecdysone receptor (EcR) and Ultraspiracle (Usp). The EcR-USP heterodimer preferentially mediates transcription through highly degenerate pseudo-palindromic response elements, resembling inverted repeats of 5'-AGGTCA-3' separated by 1 bp (IR-1). The requirement for a heterodimeric arrangement of EcR-USP subunits to bind to a symmetric DNA is unusual within the nuclear receptor superfamily. The 2.24 Å structure of the EcR-USP DNA-binding domain (DBD) heterodimer bound to an idealized IR-1 element is described. EcR and USP use similar surfaces, and rely on the deformed minor groove of the DNA to establish protein-protein contacts. Since retinoid X receptor (RXR) is the mammalian homolog of USP, the 2.60 Å crystal structure of the EcR-RXR DBD heterodimer on IR-1 was solved; the dimerization and DNA-binding interfaces are the same as in the EcR-USP complex. Sequence alignments indicate that the EcR-RXR heterodimer is an important model for understanding how the FXR-RXR heterodimer binds to IR-1 sites (Devarakonda, 2003).

The ecdysone receptor is a heterodimer of the two nuclear receptors EcR and Ultraspiracle (USP). The regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter have been defined in Kc cells. The isoform-specific A/B domains of EcR-B1 and B2, but not those of EcR-A or USP, exhibit strong activation activity [activation function 1 (AF1)], both in isolation and in the context of the intact receptor. AF1 activity in isoform B1 derives from dispersed elements; the B2-specific AF1 consists of a 17-residue amphipathic helix. AF2 function was studied using a two-hybrid assay in Kc cells, based on the observation that potent hormone-dependent activation by the EcR/USP ligand-binding domain heterodimer requires the participation of both partners. Mutagenesis reveals that AF2 function depends on EcR helix 12, but not on the cognate USP region. EcR helix 12 mutants (F645A and W650A) exhibit a dominant negative phenotype. Thus, in the setting tested, the ecdysone receptor can activate transcription using the AF1 regions of EcR-B1 or -B2 and the AF2 region of EcR. USP acts as an allosteric effector for EcR, but does not contribute any intrinsic function (Hu, 2003).

Ecdysone responses in other insects

Using the Drosophila melanogaster ecdysone receptor B1 cDNA clone, three genomic clones for EcR were isolated from the tobacco hornworm, Manduca sexta. Subsequent isolation and sequencing of several cDNAs yields a homolog of the B1 isoform with (respectively) 50%, 95% and 70% amino acid identities with Drosophila EcR in the N-terminal A/B, the DNA binding and the ligand binding domains. Unlike Drosophila, an intron occurs between the exons encoding the two zinc fingers of Manduca EcR (MsEcR). A 6.0 kb mRNA encoding MsEcR has been found in both larval wing discs and prothoracic glands and in pupal wings. During the final larval instar, the mRNA is maximal in the wing discs at one day after wandering (W1), whereas in the prothoracic gland EcR mRNA increases rapidly to high levels on day 2 and remains high thereafter. During the onset of adult development, two peaks of EcR mRNA are observed in wings from day 3 to 5 and on day 8 after pupal ecdysis. These two peaks correlate with the time of increasing titers of ecdysone (E) and 20-hydroxyecdysone (20E), respectively. The EcR mRNA peaks always precede the large ecdysteroid peak, suggesting that the transcription of the EcR gene is induced by a low concentration of ecdysteroid in vivo (Fujiwara, 1995).

Two isoforms of EcR, A and B1 were isolated from the tobacco hornworm, Manduca sexta, and shown to be similar to the corresponding Drosophila EcR isoforms. The developmental profiles of both EcR-A and EcR-B1, however, are different in Manduca epidermis, which produces sequentially the larval, the pupal, and the adult cuticles. EcR-B1 predominates through the larval, pupal, and early developing adult stages with an upregulation early in each molt. By contrast, EcR-A is present only at the onset of new cuticle synthesis during the larval molt, but in the pupal and adult molts is upregulated slightly later than EcR-B1 during the commitment period and is present during the predifferentiative phase. Both isoforms appear in the larval wing discs after pupal commitment and persist through pupal differentiation. The mRNAs for both isoforms are directly induced in larval epidermis in vitro by 20-hydroxyecdysone, but EcR-B1 mRNA accumulates more rapidly, peaking at 3 hr. In the presence of a protein synthesis inhibitor, the accumulation of EcR-B1 mRNA is slower, and its subsequent decline is prevented, but the accumulation of EcR-A mRNA is unaffected. Thus, in this polymorphic epidermis, both isoforms appear in every molt, with EcR-B1 present during the commitment and predifferentiative phases; then, at the onset of cuticle synthesis EcR-A prevails. Additionally, EcR-A is apparently associated with the switching and predifferentiative events necessary for a new synthetic program (Jindra, 1996).

An ecdysone receptor B1 isoform of the silkworm, Bombyx mori has been isolated. The predicted open reading frame encodes 543 amino acids, with 51%, 95% and 71% identities with the Drosophila melanogaster ecdysone receptor B1 isoform in the N terminal A/B region, DNA binding domain (C region) and ligand binding domain (E region), respectively. A single 6.2 kb message for the EcR gene is abundant in wing discs and fat bodies at the onset of metamorphosis. At the same stage, however, either no mRNA or a tiny amount was shown in posterior or middle silk glands, respectively. During the final instar, the mRNA expression in wing discs is maximal on the day of wandering. These data suggest the transcription of the Bombyx EcR gene is regulated in a tissue-specific and a stage-specific manner during metamorphosis (Kamimura, 1996).

Studies of the Bombyx mori ecdysone receptor (BE) have revealed that, unlike the Drosophila melanogaster ecdysone receptor (DE), treatment of BE with the ecdysone agonist tebufenozide stimulates high level transactivation in mammalian cells without adding an exogenous heterodimer partner. Gel mobility shift and transfection assays with the ultraspiracle gene product (Usp) and the retinoid X receptor heterodimer partners indicate that this property of BE stems from significantly augmented heterodimer complex formation and concomitant DNA binding. Bombyx receptor shows an increased capacity for heterodimerization with RXR, when compared with Drosophila Ecdysone receptor. The determinants for high affinity dimerization with either RXR or Usp lie within the BE D and E domains. Although the D domain determinant is sufficient for high affinity heterodimerization with Usp, both determinants are necessary for high affinity interaction with the mammalian retinoid X receptor. Modified BE receptors alone used as replication-defective retroviruses potently stimulate separate "reporter" viruses in all cell types examined, suggesting that BE has the potential for broad utility in the modulation of transgene expression in mammalian cells (Suhr, 1998).

During metamorphosis in the moth, Manduca sexta, the abdominal body-wall muscle DEO1 is remodeled to form the adult muscle DE5. As the larval muscle degenerates, its motoneuron loses its end plates and retracts axon branches from the degenerating muscle. Muscle degeneration is under the control of the insect hormones, the ecdysteroids. Topical application of an ecdysteroid mimic resulted in animals that produced a localized patch of pupal cuticle. Muscle fibers underlying the patch show a gradient of degeneration. The motoneuron shows end-plate loss and axon retraction from degenerating regions of a given fiber but maintains its fine terminal branches and end plates on intact neuromuscular junctions. These results suggest that local steroid treatments that result in local muscle degeneration bring about a loss of synaptic contacts from regions of muscle degeneration (Hegstrom, 1996a).

The degeneration of muscle DEO1 involves the dismantling of its contractile apparatus followed by the degeneration of muscle nuclei. As some nuclei are degenerating, others begin to incorporate 5-bromodeoxyuridine (BrdU), indicating the onset of nuclear proliferation. This proliferation is initially most evident at the site where the motoneuron contacts the muscle remnant. The developmental events involved in muscle remodeling are under the control of the steroid hormones, the ecdysteroids. The loss of the contractile elements of the larval muscle requires the rise and fall of the prepupal peak of ecdysteroids, whereas the subsequent loss of muscle nuclei is influenced by the slight rise in ecdysteroids seen after pupal ecdysis. Incorporation of BrdU by muscle nuclei depends on both the adult peak of the ecdysteroids and contact with the motoneuron. Unilateral axotomy blocks proliferation within the rudiment, but it does not block its subsequent differentiation into a very thin muscle in the adult (Hegstrom, 1996b).

Insect molting and metamorphosis are orchestrated by ecdysteroids with juvenile hormone (JH) preventing the actions of ecdysteroids necessary for metamorphosis. In Drosophila there is only one isoform of Ultraspiracle (USP), but two isoforms have been found in the tobacco hornworm, Manduca sexta and the mosquito Aedex aegypti. During the molt and metamorphosis of the dorsal abdominal epidermis of Manduca, the USP isoforms involved in the ecdysone receptor EcR/USP complex change with the most dramatic switch being the loss of USP-1 and the appearance of USP-2 during the larval and pupal molts. This switch in USP isoforms is mediated by high 20-hydroxyecdysone (20E) and the presence of JH is necessary for the down-regulation of USP-1 mRNA. The decrease of USP-1 mRNA in day 2 fourth instar larval epidermis, in vitro, requires exposure to a high concentration (10^-5 M) of 20E, equivalent to the peak ecdysteroid concentration in vivo, whereas the increase of USP-2 mRNA occurs at lower concentrations (effective concentrations, EC50=6.3x10^-7 M). During the pupal molt of allatectomized larvae which lack JH, USP-2 mRNA increases normally with the increasing ecdysteroid titer, whereas USP-1 mRNA remains high until pupation. When day 2 fifth instar larval epidermis is exposed to 500 ng/ml 20E in the absence of JH to cause pupal commitment of the cells by 24 h, USP-1 RNA remains at its high preculture level for 12 h, then increases two- to three-fold by 24 h. The increase is prevented by the presence of 1 microgram/ml JH I, which also prevents the pupal commitment of the cells. By contrast, USP-2 mRNA increases steadily with the same EC50 as in fourth stage epidermis, irrespective of the presence or absence of JH. Under the same conditions, mRNAs for both EcR-B1 and EcR-A isoforms are up-regulated by 20E, each in its own time-dependent manner, similar to the up-regulaton seen in vivo. These initial mRNA increases are unaffected by the presence of JH I, but those seen after 12 h exposure to 20E are prevented by JH, indicating a difference in response between larvally and pupally committed cells. The presence of JH, which maintains larval commitment of the cells, also prolongs the half-life of the EcR proteins in these cells. These results indicate that both EcR and USP RNAs are regulated by 20E and can be modulated by JH in a complex manner with only that of USP-2 apparently unaffected. These results and the absence of any effect of JH by itself on the levels of either USP mRNA indicate that if USP were the biological receptor for JH, then its behavior is much different from EcR and thyroid hormone receptors, which are upregulated by their ligands (Hiruma, 1999).

Developmental changes in the expression of a FMRFamide-like (Phe-Met-Arg-Phe-NH2) peptide (See Drosophila FMRFamide) or peptides in motoneurons of the tobacco hornworm, Manduca sexta were demonstrated using immunohistochemical techniques. The onset of FMRFamide-like immunoreactivity (FLI) is gradual during larval growth but by the final larval stage, immunoreactivity is present in the majority of motoneurons. FLI then declines during metamorphosis and is absent in all identified adult motoneurons. A novel in vivo culture system was used demonstrate that the steroid hormone, 20-hydroxyecdysone, regulates the loss of FLI in motoneurons during metamorphosis. The small commitment peak of ecdysteroid appears to shut off the program of neuropeptide accumulation that is characteristic of the larval state of the motoneurons. The prepupal peak of steroid release then causes the rapid loss of stored FLI. This steroid-induced change in the neuropeptide content of motoneurons may reflect major changes in neuromuscular functions between the larval and adult stages (Witten 1996).

The responsiveness of several nuclear transcription factor genes to 20-hydroxyecdysone (20E) was characterized in an embryonic cell line, GV1, from Manduca sexta. The mRNA for the Manduca ecdysone receptor (MsEcR) is present in the GV1 cells and transiently increases 2.3-fold by 5 h after the addition of 20-hydroxyecdysone (20E). In contrast, Manduca ultraspiracle (MsUSP) mRNA level in the GV1 cells decreases slowly to half of its initial level by 12 h when exposed to 20E. The mRNAs for two putative transcription factors, MsE75 and MHR3, are induced in the GV1 cells by 20E; the mRNA for E75 appears within 1 hour whereas that for MHR3 appears within 2 hours (Lan, 1997).

MHR3, a homolog of the retinoid orphan receptor (ROR), is a transcription factor in the nuclear hormone receptor family that is induced by 20-hydroxyecdysone (20E) in the epidermis of the tobacco hornworm, Manduca sexta. Its 2.7-kb 5' flanking region was found to contain four putative ecdysone receptor response elements (EcREs) and a monomeric (GGGTCA) nuclear receptor binding site. Activation of this promoter by 20E per ml in Manduca GV1 cells is similar to that of endogenous MHR3, with detectable response by 3 h. When the ecdysone receptor B1 (EcR-B1) and Ultraspiracle 1 (USP-1) are expressed at high levels under the control of a constitutive promoter, expression levels after a 3-h exposure to 20E increases two- to six-fold. In contrast, high expression of EcR-B1 and USP-2 cause little increase in reporter levels in response to 20E. Moreover, expression of USP-2 prevents activation by EcR-B1-USP-1. Deletion experiments show that the upstream region, including the three most proximal putative EcREs, is responsible for most of the 20E activation, with the EcRE3 at -671 and the adjacent GGGTCA being most critical. The EcRE1 at -342 is necessary but not sufficient for the activational response but is the only one of the three putative EcREs to bind the EcR-B1-USP-1 complex in gel mobility shift assays and is responsible for the silencing action of EcR-B1-USP-1 in the absence of hormone. EcRE2 and EcRE3 each specifically bind other protein(s) in the cell extract, but not EcR and USP, and so are not EcREs in this cellular context. When cell extracts were used, the EcR-B1-USP-2 heterodimer shows no binding to EcRE1, and the presence of excess USP-2 prevents the binding of EcR-B1-USP-1 to this element. In contrast, in vitro-transcribed-translated USP-1 and USP-2 both form heterodimeric complexes with EcR-B1 and bind to both EcRE1 and heat shock protein 27 EcRE. Thus, factors present in the cell extract appear to modulate the differential actions of the two USP isoforms (Lan, 1999).

The homolog of the ecdysteroid-induced transcription factor E75A of Drosophila was cloned from the tobacco hornworm, Manduca sexta, and its developmental expression and hormonal regulation were analyzed. The intron-exon structure of Manduca E75A is identical to that of Drosophila except for the loss of the small intron separating exons 4 and 5. The transcription unit of Munduca E75A appears to be only 16 kb in length, approximately one-third the length of Drosophila E75A. Both E75A and E75B mRNAs of Manduca are found in the abdominal epidermis during both the larval and the pupal molts, with E75A appearing before E75B, coincident with the rise of ecdysteroid. Exposure of either fourth or fifth instar epidermis to 20E in vitro causes the rapid, transient induction of E75A RNA, with peaks at 6 and 3 h, respectively, followed by maintenance at low levels until 24 h. Epidermis from fourth instar larvae with high endogenous juvenile hormone (JH) shows a 10-fold higher sensitivity to 20E. The presence of the protein synthesis inhibitor anisomycin has no effect on the induction but prevents the decline, indicating that E75A RNA is directly induced by 20E, but its down-regulation depends on protein synthesis. Exposure of day 2 fifth instar epidermis to 20E in the presence of JH I, which prevents the 20E-induced pupal commitment, causes an increased accumulation of E75A RNA throughout the culture period although the temporal pattern is unaffected. These findings show for the first time that JH plays a role in 20E-induced early gene expression and suggest that the higher levels of E75A may be required for maintenance of larval commitment of this epidermis (Zhou, 1998).

Although most insects reproduce in the adult stage, facultative larval or pupal reproduction (paedogenesis) has evolved at least six times independently in insects, twice in gall midges of the family Cecidomyiidae (Diptera). Paedogenesis in gall midges involves the precocious growth and differentiation of the ovary in an otherwise larval form. In the paedogenetic life cycle, the ovaries differentiate and grow precociously in the early larval stage. The eggs activate parthenogenetically, and the embryos are brooded inside the mother larva's hemocoel. Ultimately the larvae hatch, consume the histolyzing tissues of the mother, and emerge from the mother's empty cuticle. In paedogenetic gall midges, if fungal food resources remain plentiful, the larvae will repeat the paedogenetic life cycle. When conditions worsen, the larvae will develop through metamorphosis, and fly away to find another good fungal patch. Because reproduction occurs precociously, the paedogenetic life cycle is very rapid; in some species, as short as four days. The timing of expression of the Ecdysone Receptor (EcR) and Ultraspiracle (USP), the two proteins that constitute the functional receptor for the steroid hormone 20-hydroxyecdysone, regulates the timing and progression of ovarian differentiation in Drosophila (Diptera: Drosophilidae). The hypothesis that precocious activation of EcR and USP in the ovaries of paedogenetic gall midges allows for precocious ovarian differentiation has been tested. Using monoclonal antibodies directed against insect EcR and USP proteins, it has been shown that when these gall midges are reared under conditions that promote typical, metamorphic development, up-regulation of EcR and USP occurs in the final larval stage. By contrast, in the paedogenetic life cycle, EcR and USP are up-regulated early in the first larval stage. A similar pattern is seen for two independently-evolved paedogenetic gall midges, Heteropeza pygmaea and Mycophila speyeri (Hodin, 2000).

The following hypothesis is proposed for the restricted evolutionary distribution of paedogenesis. The evolution of paedogenesis must be associated with several necessary pre-adaptations. One pre-adaptation must be parthenogenesis. In addition, both the somatic and the germ cell differentiation programs need to be precociously activated, and these two programs may be under separate developmental control (as evidenced by the evolutionary dissociability of these two processes, as well as the absence of EcR and USP expression in the differentiating germ cells of D. melanogaster and both paedogenetic gall midge species). There are undoubtedly other requirements as well. Therefore, assembling all the necessary pre-adaptations for larval reproduction may simply be a situation that arises infrequently in insect evolution. The early determination of germ cells in lower Diptera may predispose these taxa for paedogenesis. Thus, perhaps it is not surprising to find that within the Cecidomyiidae, the mechanisms of paedogenesis have evolved in parallel. Since the germ cells were already differentiating early in a hypothetical ancestral non-paedogenetic gall midge, the most important change necessary to evolve paedogenesis may have been the precocious activation of the ovarian somatic cell differentiation program. Since the timing of somatic cell differentiation appears to be regulated by the ecdysone system, different gall midges may be predisposed to evolve paedogenesis by a common mechanism. This may be appropriately seen as a developmental constraint on the evolution of paedogenesis. There may only be a limited set of possible ways to evolve this life cycle, and it may be essentially unavailable to many taxa due to the vagaries of evolutionary history (namely, which taxa have the appropriate pre-adaptations, such as parthenogenesis and early germ cell differentiation). Clearly a detailed examination of the known, phylogenetically disparate cases of paedogenesis is warranted, since it would address these hypotheses, and in so doing provide insight into the mechanisms underlying life history evolution (Hodin, 2000).

The molecular basis of ecdysteroid function during development has been analyzed in detail in holometabolous insects, especially in Drosophila melanogaster, but rarely in hemimetabolous. Using the hemimetabolous species Blattella germanica (German cockroach) as model, it has been shown that the ecdysone receptor isoform-A (BgEcR-A) mRNA is present throughout the penultimate and last nymphal instars in all tissues analyzed (prothoracic gland, epidermis and fat body). To study the functions of BgEcR-A, its expression was reduced using systemic RNAi in vivo, and knockdown specimens were obtained. Examination of these specimens indicated that BgEcR-A during the last nymphal instar is required for nymphal survival, and that reduced expression is associated with molting defects, lower circulating ecdysteroid levels and defects in cell proliferation in the follicular epithelium. Some BgEcR-A knockdown nymphs survive to the adult stage. The features of these specimens indicate that BgEcR-A is required for adult-specific developmental processes, such as wing development, prothoracic gland degeneration and normal choriogenesis (Cruz, 2006).

Ligand binding of Ecdysone receptors

In insects, a steroid hormone 20-hydroxyecdysone has an important role in regulating critical events such as development and reproduction. The action of 20-hydroxyecdysone is mediated by its binding to the ecdysteroid receptor (EcR), which requires a heterodimeric partner, ultraspiracle protein (USP), a homolog of the retinoid X receptor (RXR). The EcR-USP heterodimer represents a functional receptor complex capable of initiating transcription of early genes. A ligand-dependent transactivation system was established in yeast utilizing an insect EcR-USP heterodimer. This has been achieved using mosquito Aedes aegypti AaEcR-USP. Expression of AaEcR alone, but not USP, results in constitutive transcription of the ecdysone reporter gene coupled with the Drosophila heat shock protein-27 ecdysone response elements. Removal of the N-terminal A/B domain of AaEcR abolishes its constitutive transcription. Constitutive transcription was also eliminated in the presence of its heterodimeric partner, AaUSPa, AaUSPb or mammalian RXR. This suggests that the A/B domain is essential for the EcR ligand-independent transactivation and its interaction with the yeast transcription complex. A ligand-mediated transactivation of Aa(Delta A/B)EcR-USP or Aa(Delta A/B)EcR-RXR heterodimers in response to an ecdysteroid agonist RH-5992 was observed only in the presence of GRIP1, a mouse co-activator. In the presence of a co-repressor, SMRT, Aa(Delta A/B)EcR-USP heterodimer exhibits a ligand-dependent repression activity. In addition, ligand-dependent transactivation systems for spruce budworm and fruit fly ecdysone receptors are also reported. This is the first report establishing the requirements of co-factors for a highly efficient ligand-dependent function of the insect EcR-USP in yeast. These findings open a way to study insect EcR-USP structure and function and to identify ligands that are specific for a certain group of insects, such as mosquitoes (Tran, 2001).

EcR binds to ecdysteroids and regulates transcription of genes that contain ecdysone response elements. The EcR has been used to develop inducible gene switches for efficient regulation of foreign genes in applications such as gene therapy, protein production, and functional genomics. An EcR [Choristoneura fumiferana EcR (CfEcR)] homology model was constructed, and 17 amino acid residues were identified as critical for 20-hydroxyecdysone binding. Mutation of these amino acids followed by analysis of these mutants in transactivation (in insect and mammalian cells and in vivo in mice) and ligand-binding assays identified one particular mutant (A110P) that failed to respond to steroids, but its response to the diacylhydrazine nonsteroidal ligands RG-102240 (GS(TM)E) and RG-102317 is unaffected. This steroid-insensitive EcR mutant has potential gene switch applications in insects and plants that have endogenous ecdysteroids. In addition, this mutant would be also useful for developing orthogonal EcR-ligand pairs for simultaneous regulation of multiple genes in the same cell (Kumar, 2002).

N-tert-Butyl-N,N'-dibenzoylhydrazine and its analogs are nonsteroidal ecdysone agonists that exhibit insect molting hormonal and larvicidal activities. The interaction mode of those ecdysone agonists with the heterodimer of the Ecdysone receptor and Ultraspiracle has not been fully elucidated. Ecdysone receptor B1 and the Ultraspiracle of the lepidopteran, Chilo suppressalis, were expressed using an in vitro transcription/translation system, and using gel-shift assays, it was confirmed that the proteins function as ecdysone receptors. Their ligand-binding affinity was analyzed. A potent ecdysteroid, ponasterone A, specifically binds to the ecdysone receptor with low affinity (KD = 55 nm), and the specific binding was dramatically increased (KD = 1.2 nm) in the presence of the Ultraspiracle. For seven nonsteroidal ecdysone agonists and five ecdysteroids, the binding activity to the in vitro-translated Ecdysone receptor-Ultraspiracle complex is linearly correlated with the binding activity to the inherent receptor protein in the cell-free preparation of C. suppressalis integument. The binding to the ecdysone receptor-Ultraspiracle complex for a series of compounds is highly correlated with their molting hormonal activity, indicating that the binding affinity of nonsteroidal ecdysone agonists to the ecdysone receptor-Ultraspiracle complex primarily determines the strength of their molting hormonal activity (Minakuchi, 2003).

The ecdysteroid hormones coordinate the major stages of insect development, notably molting and metamorphosis, by binding to the ecdysone receptor (EcR); a ligand-inducible nuclear transcription factor. To bind either ligand or DNA, EcR must form a heterodimer with Ultraspiracle (Usp), the homolog of retinoid-X receptor. The crystal structures of the ligand-binding domains is reported of the moth Heliothis virescens EcR-USP heterodimer in complex with the ecdysteroid ponasterone A and with a non-steroidal, lepidopteran-specific agonist BYI06830 used in agrochemical pest control. The two structures of EcR-USP emphasize the universality of heterodimerization as a general mechanism common to both vertebrates and invertebrates. Comparison of the EcR structures in complex with steroidal and non-steroidal ligands reveals radically different and only partially overlapping ligand-binding pockets that could not be predicted by molecular modelling and docking studies. These findings offer new perspectives for the design of insect-specific, environmentally safe insecticides. The concept of a ligand-dependent binding pocket in EcR provides an insight into the molding of nuclear receptors to their ligand, and has potential applications for human nuclear receptors (Billas, 2003).

Insect development is guided by the combined actions of ecdysteroids and juvenile hormones (JHs). The transcriptional effects of ecdysteroids are mediated by a protein complex consisting of the ecdysone receptor (EcR) and its heterodimeric partner, Ultraspiracle (USP), but a corresponding JH receptor has not been defined conclusively. Given that the EcR ligand binding domain (LBD) is similar to that of the JH-responsive rat farnesoid-X-activated receptor (FXR), attempts were made to define experimental conditions under which EcR-dependent transcription could be promoted by JH. Chinese hamster ovary (CHO) cells were transfected with a plasmid carrying an ecdysteroid-inducible reporter gene, a second plasmid expressing one of the three amino-terminal variants of Drosophila EcR or an EcR chimera, and a third plasmid expressing either the mouse retinoid X receptor (RXR), or its insect ortholog, Usp. Each of the EcR variants responded to the synthetic ecdysteroid, muristerone A (murA), but a maximal response to 20-hydroxyecdysone (20E) was achieved only for specific EcR combinations with its heterodimeric partner. Notably, the Drosophila EcR isoforms were responsive to 20E only when paired with USP, and only EcRB2 activity was further potentiated by JHIII in the presence of 20E. EcR chimeras that fuse the activator domains from VP16 or the glucocorticoid receptor to the Drosophila EcR DNA-binding and ligand-binding domains are responsive to ecdysteroids. Again, the effects of JHIII and 20E are associated with specific partners of the chimeric EcRs. In all experiments, the LBD of EcR proved to be the prerequisite component for potentiation by JHIII, and in this conformation may resemble the FXR LBD. These results indicate that EcR responsiveness is influenced by the heterodimeric partner and that both the N-terminal domain of EcR and the particular ecdysteroid affect JHIII potentiation (Henrich, 2003).

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