USP is a homolog of the human retinoid X receptor. Since both proteins bind to DNA as heterodimers, the nuclear receptor-based endocrine system as well as its mode of regulation are of ancient origin (Oro, 1990).

A comparative tree of DNA-binding domain amino acid sequences reveals the evolutionary affinities of Drosophila nuclear receptor proteins. Knirps shows no close affinities to other nuclear receptor proteins. Drosophila Ecdysone receptor sequence is most similar to murine RIP14. Tailless has a close affinity to murine Tlx. Drosophila E78 and E75 fall in the same subclass as Rat Reverb alpha and beta, and C. elegans "CNR-14." Drosophila HR3 is in the same subclass as C. elegans "CNR-3." Drosophila HNF-4 is most closely related in sequence to Rat HNF-4. Drosophila Ftz-F1 and Mus ELP show sequence similarity to each other. Drosophila Seven up is closely related to Human COUP-TF. Drosophila Ultraspiracle is in the same subfamily as Human RXRalpha, Human RXRbeta, and Murine RXRgamma. The latter two groups, containing Ultraspiracle and Seven up, show a distant affinity to each other. Four other subfamilies show no close Drosophila affinities. These are: 1) C. elegans rhr-2, 2) Human RARalpha, beta and gamma, 3) Human thyroid hormone receptor alpha and beta, and 4) Human growth hormone receptor, glucocorticoid receptor, and progesterone receptor (Sluder, 1997).

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).

Ultraspiracle in other insects

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).

Two isoforms of the Ultraspiracle homolog (AaUSP) from the mosquito, Aedes aegypti, have been cloned and characterized. The 2.33-kb AaUSPa cDNA has an open reading frame (ORF) of 484 amino acids encoding a polypeptide of 54 kDa, whereas the 2.14-kb AaUSPb ORF of 459 amino acids encodes a 51.3 kDa polypeptide. The AaUSPa and AaUSPb proteins differ only in the N-terminal portion of the variable A/B domain. The AaUSP DNA-binding domain shares 92% and 97% identities with the respective domains of the Drosophila (DmUSP) and Bombyx (BmUSP) Ultraspiracles. However, the AaUSP ligand-binding domain is only 57% and 52% identical to those of DmUSP and BmUSP, respectively. In spite of the relatively low level of sequence conservation, electrophoretic mobility shift assay and hormone-binding assay clearly demonstrate that the products of the AaUSPa and AaUSPb cDNAs are functional heterodimeric partners of the mosquito ecdysteroid receptor. In vitellogenic tissues, each of the two AaUSP isoforms is expressed differently: the AaUSPa is predominant in the fat body and the AaUSPb in the ovary. The kinetics of ovarian AaUSP mRNA coincide with those of the ecdysteroid receptor, being elevated during the previtellogenic period and shortly after the onset of vitellogenesis. In contrast, the level of the AaUSP in the fat body remains relatively constant throughout most of the vitellogenic cycle (Kapitskaya, 1996).

cDNAs were isolated from Manduca sexta that encode two isoforms of an ultraspiracle (USP) homolog (MsUSP-1 and MsUSP-2) with different N-terminal A/B regions. The MsUSP-1 cDNA predicts a protein with 97% and 45% amino acid identity, respectively, to the DNA- and ligand-binding domains of the Drosophila USP, and 89% overall identity with Bombyx mori CF1 (an USP homolog). Northern blot hybridizations with probes specific to MsUSP-1 and MsUSP-2 showed transcripts of an approximately equal size (4.5 kb), but with diverse developmental profiles in Manduca epidermis during the two final larval instars and the onset of the adult molt. The MsUSP-1 mRNA is expressed during the intermolt periods, with higher levels around the time of the larval ecdyses and at the onset of wandering behaviour. In contrast, the MsUSP-2 mRNA is up-regulated at times of high ecdysteroid titer during the larval molts, when the MsUSP-1 mRNA disappears. Together, these conversely regulated isoform mRNAs contribute to the constitutive expression profile of total MsUSP mRNA (Jindra, 1997).

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).

During the final two larval instars, a changing pattern in the distribution of three Ultraspiracle (Usp) proteins (50.5, 52.5, and 57 kDa) was detected in immunoblots of the dorsal abdominal epidermis of the tobacco hornworm, Manduca sexta, by a monoclonal antibody against Drosophila Usp that detects MsUsp. The 57- and 52.5-kDa bands are present during the intermolt periods and the 50.5- and 52.5-kDa bands during the molting phases. The antibody detects a nuclear antigen present in epidermis, muscle, fat body, and the central nervous system from the time of hatching. In the epidermis Usp is present in all cell nuclei but is especially prominent in the tormogen and trichogen cells immediately after ecdysis in both the penultimate and final instars. This latter immunoreactivity disappears within 12 h whereas the remainder of the epidermis retains high levels throughout the feeding period. During the molt immunostaining reappears in the hair cell nuclei. During the wandering stage at the onset of metamorphosis and just before pupal ecdysis, immunoblots show high levels of Usp, but nuclei show little or no staining. This discrepancy is likely due to the loss of one Usp isoform from the nucleus and its dispersal in the cytoplasm in preparation for the appearance of the second isoform (Asahina, 1997)

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).

Cloning and characterization of a Choristoneura fumiferana ultraspiracle (Cfusp) cDNA are described. A PCR fragment and then a cDNA clone (4.4 kb) were isolated from spruce budworm cDNA libraries. Comparison of the deduced amino acid sequence of this cDNA with the sequences in Genbank shows that this sequence has high homology with the ultraspiracle cDNAs cloned from Drosophila melanogaster, Bombyx mori, Manduca sexta, and Aedes aegypti. The Cfusp cDNA contains all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNA binding domain or C region is the most conserved sequence among all the Usps. The A/B, D, and E regions also show high amino acid identity with the amino acid sequences of Usp in each of the other species. The Cfusp 4.5-kb mRNA is present in the embryos, in all larval stages, and in the pupae. The Cfusp mRNA levels in the midgut increase during the sixth-instar larval development and reach peak levels during the ecdysteroid raises for the pupal molt. However, Cfusp mRNA levels remained unchanged in the midgut of fifth-instar larvae, and in the epidermis and fat body of sixth-instar larvae indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression (Perera, 1998).

The steroid hormone 20-hydroxyecdysone is a key regulatory factor, controlling blood-meal triggered egg maturation in mosquitoes. To elucidate the ecdysone hierarchy governing this event, the ecdysone receptor (AaEcR) and the nuclear receptor Ultraspiracle (AaUSP), a retinoid X receptor homolog, from the mosquito Aedes aegypti, were cloned and characterized. These proteins form a functional complex capable of ligand and DNA binding. The DNA-binding properties of the AaEcR.AaUSP heterodimer were analyzed with respect to the effects of nucleotide sequence, orientation, and spacing between half-sites in natural Drosophila and synthetic ecdysone response element (EcREs). By using an electrophoretic gel mobility shift assay, AaEcR.AaUSP has been shown to exhibit a broad binding specificity, forming complexes with inverted (IR) and direct (DR) repeats of the nuclear receptor response element half-site consensus sequence AGGTCA separated by spacers of variable length. A single nucleotide spacer is optimal for both imperfect (IRhsp-1) and perfect (IRper-1) inverted repeats; adding or removing 1 base pair in an IRhsp-1 spacer practically abolishes binding. However, changing the half-site to the consensus sequence AGGTCA (IRper-1) increases binding of AaEcR.AaUSP 10-fold over IRhsp-1 and, at the same time, reduces the stringency of the spacer length requirement, with IRper-0 to IRper-5 showing detectable binding. Spacer length is less important in DRs of AGGTCA (DR-0 to DR-5); although 4 bp is optimal, DR-3 and DR-5 bind AaEcR.AaUSP almost as efficiently as DR-4. Furthermore, AaEcR.AaUSP also binds DRs separated by 11-13 nucleotide spacers. Competition experiments and direct estimation of binding affinity (Kd) indicate that, given identical consensus half-sites and an optimal spacer, the AaEcR.AaUSP heterodimer binds an IR with higher affinity than a DR. Co-transfection assays utilizing CV-1 cells demonstrate that the mosquito EcR.USP heterodimer is capable of transactivating reporter constructs containing either IR-1 or DR-4. The levels of transactivation are correlated with the respective binding affinities of the response elements (IRper-1 > DR-4 > IRhsp-1). Taken together, these analyses predict broad variability in the EcREs of mosquito ecdysone-responsive genes (Wang, 1998).

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).

Unlike Drosophila Ultraspiracle (Usp), for which a single form of mRNA has been identified, two USP isoforms are reported in the mosquito Aedes aegypti (USP-A and USP-B; Kapitskaya, 1996) and in the moth, M. sexta (USP-1 and USP-2; Jindra, 1997). In both insects, USP isoforms exhibit distinct N-terminal A/B domains in accordance with the isoform variation in the EcR isoforms. In A. aegypti, the N-terminal 31 aa in USP-A are different from the first 6 aa in USP-B. Moreover, their 59 untranslated regions are different, suggesting that these isoforms are most likely derived from the same gene via utilization of alternative promoters (Kapitskaya, 1996).

In the Aedes fat body and ovary, USP-A mRNA is highly expressed during the pre- and late vitellogenic stages, corresponding to a period of low ecdysteroid titer, while USP-B mRNA exhibits its highest levels during the vitellogenic period, correlating with a high ecdysteroid titer. Remarkably, 20-hydroxyecdysone (20E) has the opposite effects on USP isoform transcripts in in vitro fat body culture. This steroid hormone upregulates USP-B transcription and its presence is required to sustain a high level of USP-B expression. In contrast, 20E inhibits activation of USP-A transcription. Although the EcR/USP-A dimer recognizes the same ecdysteroid-responsive elements, EcR/USP-B dimer binds them with an affinity twofold higher than that of the EcR/USP-A dimer. Likewise, the EcR/USP-B dimer transactivates a reporter gene in CV-1 cells twofold more strongly than does the EcRz/USP-A dimer. These results suggest that USP-B functions as a major heterodimerization partner for EcR during the vitellogenic response to 20E in the mosquito (Wang, 2000).

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 DNA binding domains of arthropod USPs and their vertebrate homologs, the retinoid X receptor (RXR) family, are highly conserved. However,the ligand binding domain sequences divide into two distinct groups. One group consists of sequences from members of the holometabolous higher insect orders Diptera and Lepidoptera; the other group consists of sequences from vertebrates, a crab and a tick. The sequence of an RXR/USP from the hemimetabolous orthopteran, Locusta migratoria, is reported. The locust RXR/USP ligand binding domain clearly falls in the vertebrate-crab-tick rather than the dipteran-lepidopteran group. The reason for the evolutionarily abrupt divergence of the dipteran and lepidopteran sequences is unknown, but it could be a change in the type of ligand bound or the loss of ligand altogether (Hayward, 1999).

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).

Ligand binding domains and ligand-dependence of Ecdysone receptor/Ultraspiracle heterodimers

Comparison of the ligand binding domain of the locust RXR/USP with the other RXR/USP sequences shows that, as is the case for Amblyomma and Uca, the locust LBD has a greater level of similarity to those of the vertebrate RXRs than it does to those of other insect USPs. The locust LBD exhibits 72%-74% identity to the vertebrate LBDs but only 43%-49% identity to the LBDs of holometabolous insect USPs. The level of identity to the LBDs of non-insect arthropods is slightly lower (70%-72%) than to those of the vertebrates. Interestingly, in this region of the molecule the locust protein is about as diverged from the USPs of the higher insects as are the vertebrate RXRs. For example, there is 49% identity between locust and Drosophila USP in the LBD, as compared to 47% identity between human RXRa and Drosophila. A possibility that needs to be considered is that rather than the dipteran and lepidopteran USPs being homologs of vertebrate RXRs, as they have traditionally been regarded, the situation here cold involve two gene families, one of which codes for USP-like proteins, the other for RXR-like proteins. This is thought to be unlikely, since there are no reports of an organism having nuclear receptor genes coding for both USP-like and RXR-like molecules; rather an organism has one or the other. Also, the two receptor types are found in logical phylogenetic groupings with USPs found only in Diptera and Lepidoptera while RXRs are found elsewhere in the animal kingdom (Hayward, 1999).

The reasons for the divergence of the LBDs of the USPs of higher insects from those of RXRs are unknown. The LBD of nuclear receptors is a complex region that may or may not bind a specific ligand; the LBD is necessary for receptor dimerization, and binds co-activators and co-repressors. In terms of ligand binding, either a loss of ligand or a change in ligand structure might be associated with the altered LBD found in the USPs of Lepidoptera and Diptera. At this time the information on their ligand binding properties is fragmentary and inconclusive. In the arthropods no endogenous retinoids have been identified, although treatment with retinoic acid disrupts regeneration in Uca and up-regulates Uca RXR mRNA. The sesquiterpenoid methyl farnesoate has structural similarities to retinoids and acts as a hormone in the regulation of both molting and reproduction in crustaceans. In insects, juvenile hormone (JH) is necessary to allow larval molting instead of metamorphosis in response to ecdysteroids; JH also regulates reproduction. In most insects, including the lower dipteran mosquitoes, JH III, the epoxide of methyl farnesoate, is the primary JH involved. Lepidopteran larvae use only JH I and II, whereas Drosophila and other higher Diptera use both JH III and JH III bisepoxide. Although JH III does not bind and activate vertebrate RXR, the JH mimics methoprene and hydroprene do activate, apparently due to conversion to their acids that bind to RXR. Recently Drosophila USP has been shown to bind JH III with low affinity. If this binding is physiologically significant, the structural differences in these various JHs may have driven the changes seen in the LBD of the USPs of the higher insects, since the lepidopteran LBDs show about 82% identity whereas there is only 57% identity between the LBDs of Aedes and Drosophila and 44%-50% between those of Lepidoptera and either Aedes or Drosophila (Hayward, 1999).

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).

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).

Crustacean retinoid-X receptor isoforms: distinctive DNA binding and receptor-receptor interaction with a cognate ecdysteroid receptor

cDNA clones have been identified that encode homologs of the ecdysteroid receptor (EcR) and retinoid-X receptor (RXR)/USP classes of nuclear receptors from the fiddler crab Uca pugilator (UpEcR and UpRXR). Several UpRXR cDNA splicing variants were found in coding regions that could potentially influence function. A five-amino acid (aa) insertion/deletion is located in the 'T' box in the hinge region. Another 33-aa insertion/deletion is found inside the ligand-binding domain (LBD), between helix 1 and helix 3. Ribonuclease protection assays (RPA) have shown that four UpRXR transcripts [UpRXR(+5+33), UpRXR(-5+33), UpRXR(+5-33) and UpRXR(-5-33)] are present in regenerating limb buds. UpRXR(-5+33) is the most abundant transcript present in regenerating limb buds in both early blastema and late premolt growth stages. Expression vectors for these UpRXR variants and UpEcR were constructed, and the proteins expressed in E. coli and in vitro expression systems. The expressed crab nuclear receptors were then characterized by electrophoretic mobility shift assay (EMSA) and glutathione S-transferase (GST) pull down experiments. EMSA results showed that UpEcR/UpRXR(-5+33) heterocomplexes bind with appreciable affinity to a series of hormone response elements (HREs) including eip28/29, IRper-1, DR-4, and IRhsp-1. Competition EMSA also showed that the affinity decreases as sequence composition deviates from a perfect consensus element. Binding to IRper-1 HREs occurs only if the heterodimer partner UpRXR contains the 33-aa LBD insertion. UpRXR lacking both the 5-aa and 33-aa insertion binds to a DR-1G HRE in the absence of UpEcR. The results of GST-pull down experiments show that UpEcR interacts only with UpRXR variants containing the 33-aa insertion, and not with those lacking the 33-aa insertion. These in vitro receptor protein-DNA and receptor protein-protein interactions occur in the absence of hormone (20-hydroxyecdysone and 9-cis retinoid acid, 9-cis RA). Transactivation studies using a hybrid UpEcR ligand-binding domain construct and UpRXR (+/-33) ligand-binding domain constructs also show that the 33-aa insertion is indispensable in mediating ecdysteroid stimulated transactivation (Wu, 2004).

Xenopus RXR and the Thyroid Receptor

The precocious induction of amphibian metamorphosis is an ideal system for analyzing the developmental action of TH, while the hormonal activation of tadpole tail regression offers the further advantage of studying programmed cell death. One of the striking features of thyroid hormone (TH)-induced tail regression in Xenopus (as with the morphogenetic responses of all tadpole tissues) is the rapid autoinduction of TRbeta gene, but it is not known how TH affects the expression of the genes encoding TR's heterodimeric partner, retinoid X receptor (RXR). The synthetic glucocorticoid dexamethasone (Dex) potentiates and prolactin (PRL) suppresses the 3,3',5-triiodothyronine (T3)-induced regression of pre-metamorphic Xenopus tadpole tails in organ culture. T3 strongly upregulates (11-35-fold) the concentration of Xenopus TRbeta (xTRbeta) mRNA in these cultures while downregulating by 50% that of Xenopus RXRgamma (xRXRgamma) mRNA in the same samples of tail RNA. DEX and PRL either enhance or diminish (respectively) the T3-regulated expression of these two transcripts, which parallels their other effects in whole tadpoles or cultured tails. The contrasting effects of the three hormones on the steady-state levels of xTRbeta and XRXRgamma mRNAs are time- and dose-dependent. T3 and DEX also strongly upregulate the transcription of xTRbeta gene transfected into Xenopus XTC-2 cells but PRL fails to prevent this autoinduction. The actions of these three hormones involved in amphibian metamorphosis, as judged by the expression of xTRbeta and xRXRgamma genes, reveal a new facet of hormonal interplay underlying their developmental actions (Iwamuro, 1995).

Thyroid hormone (T3) plays a causative role in amphibian metamorphosis. This regulation is thought to be mediated by heterodimers of T3 receptors (TRs) and retinoid X receptors (RXRs). Xenopus TRs can form strong heterodimers with Xenopus RXRs on the T3 response element (TRE) present in Xenopus TR beta genes. Using a T3-responsive in vivo transcription system established by introducing TRs and RXRs into Xenopus oocytes, it has been demonstrated that TR-RXR heterodimers repress the TR beta gene promoter in the absence of T3 and activated the promoter in the presence of the hormone. Furthermore, by analyzing the expression of TR and RXR genes, it has been shown that TR and RXR genes are coordinately regulated in different tissues during metamorphosis. Thus high levels of their mRNAs are present in the limb during early stages of limb development when morphogenesis occurs and in the tail toward the end of metamorphosis, when the tail is being resorbed. Such correlations coupled with the TRE-binding and in vivo transcriptional activation experiments provide strong evidence that TRs and RXRs function together to mediate the effects of T3 during metamorphosis. These results further suggest a possible molecular basis for the temporal regulation of tissue-specific metamorphosis (Wong, 1995).

Chromatin disruption and transcriptional activation are both thyroid hormone-dependent processes, regulated by the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). In the absence of hormone, the TR-RXR dimer binds to nucleosomal DNA, locally disrupts histone-DNA contacts and generates a DNase I-hypersensitive site. Chromatin-bound unliganded TR-RXR silences transcription of the Xenopus TRbetaA gene within a canonical nucleosomal array. On addition of hormone, the receptor directs the extensive further disruption of chromatin structure over several hundred base pairs of DNA and activates transcription. A domain of the TR protein, the C-terminal nine amino acids, is necessary for directing this extensive hormone-dependent chromatin disruption. Particular TR-RXR heterodimers containing mutations in this domain are able to bind both hormone and their thyroid hormone receptor recognition element (TRE) within chromatin, yet are unable to direct the extensive hormone-dependent disruption of chromatin or to activate transcription. The hormone-dependent disruption of chromatin and transcriptional activation are independently regulated events, distinguished through the mutagenesis of basal promoter elements and by altering the position and number of TREs within the TRbetaA promoter. Chromatin disruption alone on a minichromosome is shown to be insufficient for transcriptional activation of the TRbetaA gene (Wong, 1997a).

The Xenopus thyroid hormone receptor betaA (TRbetaA) gene contains an important thyroid hormone response element (TRE) that is assembled into a positioned nucleosome. A determination was made of the translational position of the nucleosome containing the TRE and the rotational positioning of the double helix with respect to the histone surface. Histone H1 is incorporated into the nucleosome leading to an asymmetric protection to micrococcal nuclease cleavage of linker DNA, relative to the nucleosome core. Histone H1 association is without significant consequence for the binding of the heterodimer of thyroid hormone receptor and 9-cis retinoic acid receptor (TR/RXR) to nucleosomal DNA in vitro, or for the regulation of TRbetaA gene transcription following microinjection into the oocyte nucleus. Small alterations of 3 and 6 bp in the translational positioning of the TRE in chromatin are also without effect on the transcriptional activity of the TRbetaA gene, whereas a small change in the rotational position of the TRE (3 bp) relative to the histone surface significantly reduces the binding of TR/RXR to the nucleosome and decreases transcriptional activation directed by TR/RXR. These results indicate that the specific architecture of the nucleosome containing the TRE may have regulatory significance for expression of the TRbetaA gene (Wong, 1997b).

Histone deacetylase and chromatin assembly contribute to the control of transcription of the Xenopus TRbetaA gene promoter by the heterodimer of Xenopus thyroid hormone receptor and 9-cis retinoic acid receptor (TR-RXR). Addition of the histone deacetylase inhibitor Trichostatin A (TSA) relieves repression of transcription due to chromatin assembly following microinjection of templates into Xenopus oocyte nuclei, and eliminates regulation of transcription by TR-RXR. Expression of Xenopus RPD3p, the catalytic subunit of histone deacetylase, represses the TRbetaA promoter, but only after efficient assembly of the template into nucleosomes. In contrast, the unliganded TR-RXR represses templates only partially assembled into nucleosomes; addition of TSA also relieves this transcriptional repression. This result indicates the distinct requirements for chromatin assembly in mediating transcriptional repression by the deacetylase alone, as compared with those needed in the presence of unliganded TR-RXR. Whereas hormone-bound TR-RXR targets chromatin disruption (as assayed through changes in minichromosome topology) and loss of a regular nucleosomal ladder on micrococcal nuclease digestion, addition of TSA relieves transcriptional repression but does not disrupt chromatin. Thus, TR-RXR can facilitate transcriptional repression in the absence of hormone by means of other mechanisms, in addition to the recruitment of deacetylase, and it can disrupt chromatin structure by means of other mechanisms, in addition to the inhibition or release of deacetylase (Wong, 1998).

Xenopus thyroid hormone (xTR) and retinoid X (xRXR) were overexpressed in cells and the response to various ligands was studied. 3,3'5-triiodothyronine (T3) strongly upregulates xTR beta mRNA in XTC-2 cells, but not xTR alpha or xRXR alpha mRNAs, while xRXR gamma transcripts cannot be detected. 9-cis-retinoic acid (9-cis-RA) does not substantially influence the expression of any of these four receptor genes. Measurements of transcription activity were taken from three different thyroid response elements (TREs): a palindromic TREpal, an inverted repeat +6 [F2] and a direct repeat +4[DR+4], as present in the promoter of xTR beta gene. Only T3 upregulates transcription, while 9-cis-RA, either alone or together with T3, is ineffective. 9-cis-RA however enhances transcription from an RXR responsive element (RXR-RE). A second approach involved overexpression of xTR beta and xRXR alpha in premetamorphic Xenopus tadpole tail muscle followed by measuring the response of the tails to T3 in organ culture. T3 enhances transcription from the xTR beta DR +4 TRE in tails in which xTR beta is overexpressed, but the overexpression of xRXR alpha fails to modify this response. It is concluded that in both XTC cells and tadpole tails exogenous 9-cis-RA is ineffective, and that overexpressed xRXR fails to modify the enhanced transcriptional response of endogenous and overexpressed xTR beta to T3 (Ulisse, 1997).

Heterodimers of thyroid hormone receptors (TRs) and 9-cis retinoic acid receptors (RXRs) are the likely in vivo complexes that mediate the biological effects of thyroid hormone, 3,5,3'-triiodothyronine (T3). However, direct in vivo evidence for such a hypothesis has been lacking. There is a close correlation between the coordinated expression of TR and RXR genes and tissue-dependent temporal regulation of organ transformations during Xenopus laevis metamorphosis. By introducing TRs and RXRs either individually or together into developing Xenopus embryos, it has been demonstrated that RXRs are critical for the developmental function of TRs. Precocious expression of TRs and RXRs together, but not individually, leads to drastic, distinct embryonic abnormalities, depending upon the presence or absence of T3; these developmental effects require the same receptor domains as those required for transcriptional regulation by TR-RXR heterodimers. More importantly, the overexpressed TR-RXR heterodimers faithfully regulate endogenous T3 response genes that are normally regulated by T3 only during metamorphosis. That is, they repress the genes in the absence of T3 and activate them in the presence of the hormone. On the other hand, the receptors have no effect on a retinoic acid (RA) response gene. Thus, RA- and T3 receptor-mediated teratogenic effects in Xenopus embryos occur through distinct molecular pathways, even though the resulting phenotypes have similarities (Puzianowska-Kuznicka, 1997).

Mammalian RXR

Continued see Evolutionary Homologs: part 2/4 | part 3/4 | part 4/4

ultraspiracle: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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