TY - JOUR
AU - Laudet, Vincent
AB - Abstract 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 orthologue of vertebrate retinoid X receptors (RXR α, β, γ). 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, we performed a phylogenetic analysis 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. Our 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. ecdysone receptor, USP-RXR, ECR, insects, coevolution, evolutionary rate Introduction Ecdysteroid hormones regulate many essential processes in reproduction and development of insects. In Drosophila, a single steroid metabolite, 20-hydroxyecdysone (called ecdysone for simplicity), is responsible for controlling the main developmental transitions, including larval molts and metamorphosis (Kozlova and Thummel 2000). It is a remarkable system in which one simple signal triggers specific transcriptional regulation of several genes, at different stages and in different tissues. Extensive genetic and molecular studies have demonstrated that gene cascades regulated by ecdysone play a central role in the developmental timing in Drosophila (Thummel 2001). Evidence from a few other species supports the conservation of this ecdysteroid regulatory pathway in insects (Henrich and Brown 1995). But most of these species belong to the very derived holometabolous orders Diptera and Lepidoptera. Insects present a large range of developmental variability, affecting for example ovarian organization (King and Büning 1985), embryonic germ-band type (Sander 1976; Patel, Condron, and Zinn 1994) or the number of larval molts and the type of metamorphosis (Sehnal, Svàcha, and Zrzavy 1996; Truman and Riddiford 1999). Analysis of this diversity at the molecular level is now possible and constitutes a major objective of evolutionary developmental biology. The functional Drosophila ecdysone receptor is a heterodimer of the products of the ecdysone receptor (EcR) and ultraspiracle (usp) genes, two nuclear receptors (Koelle et al. 1991; Oro, McKeown, and Evans 1992; Yao et al. 1993). Nuclear receptors share a common organization consisting of at least three structural domains: an amino-terminal domain (A/B), a central DNA-binding domain (DBD or C domain), and a ligand-binding domain (LBD or E domain) (Moras and Gronemeyer 1998). In addition, a flexible linker region (D domain) is located between DBD and LBD. Some members of this family also contain a carboxy-terminal tail (F domain). The requirement of heterodimerization between ECR and USP-RXR has been found in other species such as the mosquito Aedes aegypti (Wang et al. 2000), the silkmoth Bombyx mori (Swevers et al. 1996), and even a member of the Chelicerata, the tick Amblyomma americanum (Guo et al. 1998). Understanding the evolution of ecdysone regulation in insects requires comparative analysis of both partners of the heterodimer. Within the superfamily of nuclear receptors, ECR (NR1H1) belongs to the same group as the vertebrate liver X receptors (LXRα and LXRβ: NR1H3 and NR1H2) and farnesoid X receptor (FXR: NR1H4), which are also receptors for steroid hormones (oxysterols and bile acids, respectively) (Laudet and Gronemeyer 2002). Ecdysteroids are not produced by deuterostomes, such as vertebrates. Phylogenies based on 18S rDNA sequences group arthropods and nematodes in the ecdysozoa clade of protostomes sharing the developmental trait of moulting (Aguinaldo et al. 1997). However, ECR horthologues have not been identified in the C. elegans genome but only in some parasitic nematodes, which are sensitive to ecdysteroids (Sluder and Maina 2001). Thus, molting regulation and the primary signal are likely to differ among lineages within ecdysozoa. In fact, a recent analysis of more than 100 nuclear proteins does not support the ecdysozoa hypothesis (Blair et al. 2002), and moulting may have appeared several times during metazoans evolution. USP-RXR (NR2B4) is the orthologue of vertebrate retinoid X receptors (RXRα, β, γ: NR2B1, 2, 3) (Laudet and Gronemeyer 2002). The name USP comes from the phenotype of Drosophila mutants (Perrimon, Engstrom, and Mahowald 1985), whereas RXR (retinoid X receptor) refers to the mammalian ligand (9-cis retinoic acid) (Mangelsdorf et al. 1990). In arthropods, no mutant phenotype is known outside Drosophila, and USP-RXR does not bind 9-cis retinoid acid. Now that this gene has been isolated in a wide variety of metazoans, this nomenclature is sometimes confusing in the literature. In this article, we will use the name USP-RXR for all arthropods and simply RXR for orthologues from other taxa. Unlike ECR, the three-dimensional structure of RXR proteins has been well studied. The crystal structures of the human RXRα LBD (Bourguet et al. 1995; Egea et al. 2000) and DBD (Lee et al. 1993) have been determined, as well as the USP-RXR LBDs of Drosophila melanogaster (Clayton et al. 2001) and of the Lepidoptera Heliothis virescens (Billas et al. 2001). Comparison of these structures reveals that Drosophila and Heliothis USP-RXR LBDs are locked in an inactive conformation. Furthermore, authors of these studies suggest that there may be a natural ligand for this USP-RXR, previously seen as an orphan receptor. In vitro studies have shown that juvenile hormone III can bind Drosophila USP-RXR with a very low affinity (Jones and Sharp 1997; Jones et al. 2001). This hormone is a sesquiterpenoid chemically analog to retinoids and is involved in the control of insect molting and metamorphosis. However, the possibility that juvenile hormone is a natural ligand of USP-RXR awaits further evidence. It has been proposed that arthropods lost the ability to bind 9-cis retinoid acid (Escriva, Delaunay, and Laudet 2000). Then this loss may have been followed by acquisition of a new ligand that remains to be identified. Cloning of ECR or USP-RXR from various arthropods led several authors to observe an intriguing divergence of both proteins in Diptera and Lepidoptera (reviewed in Riddiford, Cherbas, and Truman 2001). In order to gain further insight into the evolution of ecdysone regulation in arthropods, we performed an evolutionary analysis of both partners. Sequence alignments and structural comparisons reveal a combination of variation and conservation in important functional domains for both ECR and USP-RXR. The major structural divergences are specific to Diptera and Lepidoptera. The most impressive differences affect the LBD domain of USP-RXR. ECR sequences also show variability in other domains, namely the DBD and the carboxy-terminal F domain. Furthermore, we show that the LBDs of both proteins are characterized by an acceleration of divergence rates in the Diptera-Lepidoptera lineage. Our data provide the first evidence that ECR and USP-RXR may have coevolved during the course of holometabolous insect diversification, probably leading to a functional divergence of the ecdysone receptor. They also show that Diptera and Lepidoptera, the most widely used model organisms to analyze ecdysone regulation, are in fact characterized by a very derived ecdysone receptor. Therefore, extreme care must be taken when results from Drosophila or Manduca are generalized, in particular concerning both fundamental aspects of insect development and the design of specific insecticides. Materials and Methods Cloning and Sequencing of cDNAs New USP-RXR and/or ECR sequences were obtained by RT-PCR from the following species: Leptopilina heterotoma (USP-RXR: 850 bp; ECR: 702 bp); Hypera postica (USP-RXR: 854 bp); Periplaneta americana (USP-RXR: 902 bp); Folsomia candida (USP-RXR: 665 bp); and Lithobius forficatus (USP-RXR: 916 bp) (table 1). A 5-μg sample of total RNA was reverse transcribed with random primers and MMLV reverse transcriptase in 20 μl of reaction mixture according to the manufacturer's instruction (GIBCO-BRL, MMLV-RT kit). The resulting cDNA was amplified by PCR in 100 μl volume with 10 mM Tris-Hcl pH = 8.3, 50 mM KCl, 1.5 mM MgCl2 (Perkin-Elmer), 0.25 mM of each dXTP, 2.5 U Taq Gold DNA polymerase (Perkin-Elmer) and 300 ng of each primer. Degenerate primers were designed from an alignment of nucleic sequences for either usp-RXR or EcR. The primers are located within conserved sequences coding the DNA-binding and ligand-binding domains. Four primers were designed for each gene; their orientation and exact position in Drosophila cDNA sequences (usp: X53417; EcR: M74078) are indicated in parentheses: usp51: 5′ GGI AA(a/g) CA(c/t) TA(c/t) GGI GTI TAC AG, (forward, 499 to 421); usp52: 5′ TG(c/t) GA(a/g) GGI TG(c/t) AA(a/g) GGI TT(c/t) TT(c/t) AA, (forward, 423 to 548); usp32: 5′ T(g/t)(c/g) I(g/t)I CGI (c/g)(a/t)(a/g) T(a/g)C TC(c/t) TC, (reverse, 1483 to 1502); usp31: 5′ GTG TCI CCI ATI AG(c/t) TT(a/g) AA, (reverse, 1597 to 1616); EcR51: 5′ ATG TG(c/t) (c/t)TI GTI TG(c/t) GGI GA, (forward, 1855 to 1874); EcR53: 5′ TG(c/t) GAI ATI GA(c/t) AT(c/g) TA(c/t) ATG, (forward, 1984 to 2004); EcR33: 5′ C(g/t)I GCC A(c/t)I C(g/t)(c/g) A(a/g)C ATC AT, (reverse, 2578 to 2597); EcR31: 5′ (c/g)IA (c/t)(a/g)T CCC A(a/g)A (c/t)(c/t)T CIT CIA (a/g)GA A, (reverse, 3001 to 3025). For each gene, all combinations of the four primers were used in seminested PCR amplifications. Reactions were performed in a Perkin-Elmer Thermal Cycler 480, using a modified “Touch Down” protocol (Escriva, Robinson, and Laudet 1999). A brief initial 10 min cycle at 94°C was followed by cycles 1 to 5: 94°C 1 min, 55°C 1 min, 74°C 2 min; cycles 6 to 10: 94°C 1 min, 50°C 1 min, 74°C 2 min; cycles 11 to 15: 94°C 1 min, 45°C 1 min, 74°C 2 min; cycles 16 to 20: 94°C 1 min, 40°C 1 min, 74°C 2 min; cycle 21 to 40: 94°C 1 min, 37°C 1 min, 74°C 2 min; followed by terminal elongation for 10 min at 74°C. Extreme care was taken against contamination: PCR analyses were performed in rooms devoted to ancient DNA studies with overpressure, UV lights, and dedicated hoods. PCR products were cloned into a TA cloning vector (Invitrogen) and transformed into competent cells according to the manufacturer's instructions. Sequencing reactions were performed using a Dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase FS (Applied Biosystems). Protein Sequence Analysis All available sequences were obtained from NUREBASE (Duarte et al. 2002). Species and accession numbers are shown in table 1. Protein-coding sequences were aligned using SEAVIEW (Galtier, Gouy, and Gautier 1996). All positions with gaps were excluded from analyses. Phylogenetic reconstruction was made by use of neighbor-joining (Saitou and Nei 1987), with observed differences as implemented in PHYLO_WIN (Galtier, Gouy, and Gautier 1996). The number of complete aligned sites used for tree reconstruction is 74 for ECR DBD, 221 for ECR LBD, 77 for USP-RXR DBD, and 145 for USP-RXR LBD. Bootstrap analysis with 1,000 replicates was used to assess support for nodes in the tree (Felsenstein 1985). The phylogenetic tree of RXR/USP sequences is rooted by the jellyfish Tripedelia cystophora RXR sequence (Kostrouch et al. 1998). The tree of ECR is rooted by vertebrate LXR and FXR sequences. Evolutionary distances between sequences were mapped on a predefined species consensus tree using Tree-Puzzle (Schmidt et al. 2002), with the JTT substitution model (Jones, Taylor, and Thornton 1992) plus rate heterogeneity between sites, estimated by a gamma law with eight categories. The consensus tree is based on classical taxonomic data, as well as more specific references concerning the following groups: Diptera (Yeates and Wiegmann 1999), Lepidoptera (Weller et al. 1992; Regier et al. 2001), insects (Kristensen 1981; Whiting et al. 1997), and arthropods (Giribet, Edgecombe, and Wheeler 2001; Hwang et al. 2001). In addition, rates were compared between lineages using the relative-rate test on all available sequences (Wilson, Carlson, and White 1977; Robinson et al. 1998), weighting by the predefined tree topology, as implemented in RRTree (Robinson-Rechavi and Huchon 2000), with a Poisson correction for multiple substitutions. Results ECR and USP-RXR Sequences In order to study the role of the ecdysone receptor during evolution of arthropod metamorphosis, we analyzed the evolution of its two components: ECR and USP-RXR. When this work was initiated, most of the sequences available in the public databases had been isolated from Diptera and Lepidoptera species. Therefore, it was necessary to investigate a larger sample of insects and other arthropods. Using an RT-PCR approach with degenerated primers located within the DBD and the LBD, we cloned and sequenced cDNA fragments coding for USP-RXR or ECR from five new species (table 1). These new species give a complete sampling of the different types of metamorphosis in arthropods: holometaboly or complete metamorphosis outside Diptera and Lepidoptera (Hymenoptera and Coleoptera), heterometaboly or incomplete metamorphosis (Dictyoptera), ametaboly or absence of metamorphosis (Collembola), plus one myriapod. Overall, 38 USP-RXR and 19 ECR protein sequences have been used for this analysis, from 33 species. Regarding evolution of these two proteins, as it will be shown further in this article, these 33 species can be distributed into six groups: Diptera (eight species), Lepidoptera (six species), other hexapods (seven species), other arthropods (three species), chordates (eight species), and cnidaria (one species) (table 1). Importantly, the phylogenetic relationships among these six groups are well known and are nonambiguous (see fig. 1). Molecular Phylogeny of ECR and USP-RXR Cloning of ECR or USP-RXR homologues from various arthropods has previously revealed that these proteins are divergent in Diptera and Lepidoptera. This is particularly clear for the LBD of USP-RXR, when sequences from a tick (Guo et al. 1998), a crab (Chung et al. 1998), a locust (Hayward et al. 1999), or a beetle (Nicolaï et al. 2000) are compared with Diptera and Lepidoptera sequences. Although less obvious, the same phenomenon affects ECR (Guo et al. 1997; Saleh et al. 1998; Verras et al. 1999). Understanding this evolutionary divergence should give important insights on the evolution of insect metamorphosis and the functional plasticity of nuclear receptors. Therefore, we performed an analysis of all ECR and USP-RXR sequences together, in order to measure their evolutionary rates and to identify precisely the divergent regions. After sequence alignment, identity percentages and phylogenetic trees were determined separately for the DBD and LBD of both proteins. Pairwise comparisons show a clear divergence in the LBD of USP-RXR between Diptera-Lepidoptera and other species (table 2). There is only 49% identity between Diptera-Lepidoptera and other insects, as opposed to 68% between these other insects and other arthropods, and 70% between the other insects and chordates. Thus, the USP-RXR LBD of many insects is less similar to Diptera and Lepidoptera than it is to the chordate RXRs. The same is true of the DBD and LBD domains of ECR (table 2), although the divergence is less pronounced. A Neighbor-Joining analysis with observed differences performed using the full-length LBD of ECR or USP-RXR obtained the trees shown in figure 2. Similar topologies are found by parsimony analysis (data not shown; see also Guo et al. 1997; Guo et al. 1998; Hayward et al. 1999). It should be emphasized that our aim is not to reconstruct the phylogeny of species using RXR or ECR as markers, but rather to characterize the evolution of these receptors using phylogeny as a tool. The trees are therefore presented here to illustrate the aberrant topology with regard to insect phylogeny, and to show the length of the branches. In both trees, it can be seen that Diptera and Lepidoptera sequences constitute a monophyletic group separated from all the other insects. The bootstrap score for the branch that separates Diptera-Lepidoptera from other insects is 100% (boxed). All the other insects are grouped in a branch with a high bootstrap value:100% for ECR (fig. 2A) and 87% for USP-RXR (fig. 2B). These topologies are clearly in contradiction with the phylogeny of the species (fig. 1). For example, the coleoptera Tenebrio molitor belongs to the holometabolous insects, a monophyletic group that includes Diptera and Lepidoptera. However the USP-RXR LBD from this beetle is more similar to that of a chelicerate (Amblyomma americanum), or even of a chordate, than of a Diptera such as Drosophila. The trees of figure 2 also show that Diptera and Lepidoptera sequences share long branches, when compared with other arthropods proteins. This observation is indicative of a rapid rate of divergence. Analysis of Evolutionary rates The phylogenetic analysis suggests that USP-RXR and ECR sequences have undergone accelerated evolution in the Diptera-Lepidoptera lineage. We therefore decided to estimate and to compare the rates of divergence between groups of species. In order to obtain the best estimates of branch lengths, we used a constraint topology based on the known phylogenetic relationships between all the species analyzed in this article (fig. 1). Evolutionary distances between sequences were mapped on this predefined species consensus phylogeny. The trees obtained by this method are shown in figure 3. Moreover, rates were compared between lineages using the relative-rate test on all available sequences. Results are shown in table 3, as differences of substitution rate between groups of species. From these analyses, it appears that both ECR and USP-RXR LBD sequences of Diptera and Lepidoptera have evolved at significantly different rates than other species (fig. 3B and 3D and table 3). The strongest rate difference is with USP-RXR LBDs. Despite the important distances obtained by mapping ECR DBD sequences on the predefined tree for Diptera-Lepidoptera (fig. 3A), rate differences are not significant for DBDs (data not shown). This may be due to the small numbers of sites available for the test (80 amino acids). Our data clearly show that both ECR and USP-RXR experienced a very strong acceleration of evolutionary rate in Diptera and Lepidoptera versus other insects. It is therefore essential to identify which regions of the proteins were affected by this acceleration. Divergence of the Ligand-Binding Domain of USP-RXR It has been shown recently that both crystal structures of a Lepidopteran (Heliothis) and a Dipteran (Drosophila) USP-RXR LBD are locked in an unusual antagonist conformation (Billas et al. 2001; Clayton et al. 2001). Sequence alignment clearly shows the differences between the LBD of USP-RXR proteins from Diptera and Lepidoptera and their homologues in other arthropods. They are grouped into three divergent domains and are not located randomly along the sequence (fig. 4). Interestingly, many differences affect precisely two regions that are implicated in the unusual conformation of Drosophila and Heliothis USP-RXRs: the loop between helices H1 and H3 and the carboxy-terminal end of the LBD (helix H12 and the loop between H11 and H12) (fig. 3). Helix H12 is locked in an inactive position by making contacts with the loop H1-H3, specifically with a conserved domain of 13 residues (boxed in gray in fig. 4). This domain is well conserved within the lineage of Diptera and Lepidoptera but is absent in other arthropods, where the loop H1-H3 is highly variable in length and in sequence. Furthermore, three (Heliothis) or four (Drosophila) residues of the conserved region interact with the phospholipid ligand cocrystallized with the LBD (fig. 4). While helix H12 is highly conserved among most arthropods and chordates, sequences of H12 and of the loop H11-H12 are variable in Diptera and Lepidoptera. Most of the differences are conservative. The loop L5-s1, connecting helix H5 and the β-strand s1 is longer in Diptera and Lepidoptera USP-RXR, with little conservation in the additional residues (fig. 4). Unfortunately, this region could not be modeled because its conformation is not ordered in the crystal (Billas et al. 2001; Clayton et al. 2001). Thus, further experiments are needed to decipher the putative role of this intriguing insertion. Divergent Domains in ECR Despite an increase in evolutionary rates (table 3) the ECR LBDs are rather well conserved in length and sequence (table 2 and data not shown). This conservation enabled Wurtz et al. (2000) to identify the canonical 11 helices and to model 20-hydroxyecdysone binding for the Diptera Chironomus tentans. Thus, contrary to USP-RXR, there is no obvious divergence of the structure of ECR LBD in Diptera and Lepidoptera. This could be due to the constraint on all ECRs to presumably bind 20-hydroxyecdysone (Riddiford, Cherbas, and Truman 2001). The DBD of ECR contains six amino acid differences specific for the Diptera-Lepidoptera group (fig. 5A). By contrast, USP-RXR DBD sequences do not show any specific differences (fig. 5B). Among the six differences observed for ECR, only one is not conservative and is located just upstream of the second zinc finger. It is a hydrophobic residue in Diptera (cysteine) and Lepidoptera (isoleucine), but a polar amino acid (glutamine) in other arthropods. Interestingly, four of these substitutions are located in or near the second zinc finger, a region known to form a dimerization interface for some nuclear receptors (Luisi et al. 1991; Schwabe et al. 1993). A surprising originality of Diptera-Lepidoptera ECRs is the presence of a carboxy-terminal F domain (fig. 6). This domain of variable length (226 in Drosophila and 18 in Choristoneura) does not show any sequence conservation between species. Other insect and arthropod ECRs possess only two to four amino acids in the carboxy-terminal of the putative helix H12, which ends the LBD. Most nuclear receptors do not contain any sizable region carboxy-terminal of the LBD, including mammalian LXR and FXR, other proteins from the ECR group. Therefore, it appears that the presence of an F domain in ECR is an evolutionary acquisition of Diptera and Lepidoptera. Discussion This article is the first comprehensive evolutionary analysis of the ecdysone receptor, a major regulatory factor of insect development. Both partners of the ECR/USP-RXR heterodimer, which constitutes the functional ecdysone receptor, experienced a strong acceleration of evolutionary rate in Diptera and Lepidoptera. This acceleration defines a clear separation within holometabolous insects. Diptera and Lepidoptera belong to the clade Panorpida (Kristensen 1981), with Hymenoptera as a sister group. Panorpida also includes Trichoptera (caddisflies), the sister group of Lepidoptera, and Mecoptera (scorpionflies) and Siphonaptera (fleas), which are more closely related to Diptera (fig. 1). The phylogenetic position of Strepsiptera is unclear (Whiting et al. 1997; Rokas, Kathirithamby, and Holland 1999). Thus, the hypothesis of a unique event of acceleration in the ancestor of Diptera and Lepidoptera could be tested by isolation of ECR and USP-RXR sequences from other Panorpida. This event of acceleration could be responsible for the accelerated evolutionary rates at the base of and within these groups. We already know that USP-RXR and ECR sequences from a flea (M. Palmer, personal communication) are more similar to Diptera and Lepidoptera than to other insects (data not shown), which supports our hypothesis. Regarding evolution of metamorphosis, the divergence of the ecdysone receptor does not correlate with the different types of insects' metamorphosis. It may be necessary to isolate more full-length sequences from several species outside Panorpida to decipher a specific trend at this level. We have identified several protein domains for which sequence divergence is specific to Diptera and Lepidoptera. All members of the nuclear hormone receptor family share the canonical LBD structure with 11 helices (H1 and H3–H12) connected by loops and two short β-strands (s1 and s2). The typical activation of nuclear receptor implies the binding of the agonist ligand in the pocket. This binding triggers a repositioning of helix H12 that provides the surface for coactivator interaction and thereby allows the transactivation activity of the nuclear receptor. In the case of an antagonist, helix H12 moves precisely into the hydrophobic furrow where the coactivator interacts in the agonist conformation (Moras and Gronemeyer 1998). In the Drosophila and Heliothis USP-RXR structures, the loop between helices H1 and H3 is located inside the hydrophobic furrow of the LBD, thereby preventing the repositioning of helix H12 and interactions with coactivators, and locking these USP-RXRs in an unusual antagonist conformation (Billas et al. 2001; Clayton et al. 2001). In the light of these results, our observation of Diptera and Lepidoptera specific sequence diversity in both the loop H1–H3 and the helix H12 suggests a form of concerted evolution between these two interacting regions of the USP-RXR LBD. This evolution may have changed the ligand-dependent transactivation activity of the protein. It may also have had an effect on the ligand-binding activity, since the loop H1–H3 contains residues that interact with the phospholipid cocrystallized with Drosophila and Heliothis LBD. On the other hand, given the very strong conservation of helix H10, it is likely that the dimerisation activity of USP-RXR LBD remained unchanged during evolution. It is intriguing that the LBD of ECR underwent a significant increase of substitution rate in Diptera and Lepidoptera, while its structure remained apparently largely unchanged. In all insects, and presumably in all arthropods, ECR LBD binds 20-hydroxyecdysone (Riddiford, Cherbas, and Truman 2001). This fundamental interaction may represent the primary selective constraint acting on this domain. However, nuclear receptor LBDs are also involved in heterodimerization activity. This rapid evolution of ECR can be explained by adaptation to the extremely divergent USP-RXR, and eventually acquisition of new partners. It may be that the stability of the heterodimer required compensatory changes in ECR and USP-RXR, suggestive of coevolution. The differences seen in ECR DBD also suggest functional changes in dimerization. Indeed, four of the six substitutions that are conserved among Diptera and Lepidoptera are located at positions known to be involved in protein dimerization but not in DNA contact or nuclear localization signal (Black et al. 2001; Khorasanizadeh and Rastinejad 2001). Another difference specific to Diptera and Lepidoptera is the presence of a carboxy-terminal F domain. This difference is interesting, since it is known that when present (ERα, HNF-4) the F domain of nuclear receptors can regulate different functions of the LBD. For example, the F domain of human estrogen receptor ERα can modulate transcriptional activity and dimerization signal, probably through interaction with the AF-2 domain (Montano et al. 1995; Nichols, Rientjes, and Stewart 1998; Peters and Khan 1999). An important conclusion of this sequence analysis is that the major structural differences of USP-RXR and ECR are specific to Diptera and Lepidoptera. We hypothesize that these differences changed two functional properties of the heterodimeric ecdysone receptor during insect evolution, namely the ligand-dependent transactivation and the heterodimerization activities of both USP-RXR and ECR. These hypotheses could now be tested by a comparative genetic approach using Drosophila melanogaster and another holometabolous insect chosen outside the Panorpida group. This should help to usefully extend our knowledge concerning the biological role of ecdysone. Indeed, our work indicates that the current model organisms used to analyze the ecdysone pathway are in fact very derived species. Therefore, extreme care must be taken when results obtained from Panorpida are generalized, notably concerning both fundamental aspects of insect development and the design of specific insecticides. 1 These two authors contributed equally to this work. E-mail: bonneto@univ-lyon1.fr. William Jeffery, Associate Editor Fig. 1. View largeDownload slide Phylogenetic relationships between the species studied in this article. This consensus tree is based on classical taxonomic data, as well as on more specific references concerning the following groups: Diptera (Yeates and Wiegmann 1999), Lepidoptera (Weller et al. 1992; Regier et al. 2001), insects (Kristensen 1981; Whiting et al. 1997), and arthropods (Hwang et al. 2001; Giribet et al. 2001). Species names underlined indicate that both ECR and USP-RXR sequences are available for these species. Regarding evolution of these proteins, two artificial groups are indicated: “other insects,” for all insects excluding Panorpida, and “other arthropods,” for all arthropods excluding insects Fig. 1. View largeDownload slide Phylogenetic relationships between the species studied in this article. This consensus tree is based on classical taxonomic data, as well as on more specific references concerning the following groups: Diptera (Yeates and Wiegmann 1999), Lepidoptera (Weller et al. 1992; Regier et al. 2001), insects (Kristensen 1981; Whiting et al. 1997), and arthropods (Hwang et al. 2001; Giribet et al. 2001). Species names underlined indicate that both ECR and USP-RXR sequences are available for these species. Regarding evolution of these proteins, two artificial groups are indicated: “other insects,” for all insects excluding Panorpida, and “other arthropods,” for all arthropods excluding insects Fig. 2. View largeDownload slide Phylogenetic trees of LBD domains. (A) ECR, (B) USP-RXR. Trees were constructed with the neighbor-joining method performed with the full-length LBD of ECR (17 sequences) or USP-RXR (36 sequences). Positions with a gap were excluded from the computation, resulting in 221 complete sites for ECR and 145 complete sites for USP-RXR. The RXR protein from the jellyfish Tripedalia cystophora was used as an outgroup to USP-RXRs, and all mammalian LXR and FXR sequences to ECRs. For legibility, outgroups are not shown. Figures at nodes are bootstrap proportions out of 1,000 replicates; only values ≥ 50% are shown. The boxed bootstrap values highlight two important nodes leading to Panorpida and “other insects.” Branch lengths are proportional to sequence divergence; the measure bar represents 0.1 differences per site. Diptera and Lepidoptera species are in bold Fig. 2. View largeDownload slide Phylogenetic trees of LBD domains. (A) ECR, (B) USP-RXR. Trees were constructed with the neighbor-joining method performed with the full-length LBD of ECR (17 sequences) or USP-RXR (36 sequences). Positions with a gap were excluded from the computation, resulting in 221 complete sites for ECR and 145 complete sites for USP-RXR. The RXR protein from the jellyfish Tripedalia cystophora was used as an outgroup to USP-RXRs, and all mammalian LXR and FXR sequences to ECRs. For legibility, outgroups are not shown. Figures at nodes are bootstrap proportions out of 1,000 replicates; only values ≥ 50% are shown. The boxed bootstrap values highlight two important nodes leading to Panorpida and “other insects.” Branch lengths are proportional to sequence divergence; the measure bar represents 0.1 differences per site. Diptera and Lepidoptera species are in bold Fig. 3. View largeDownload slide Predefined trees with evolutionary distances for ECR and USP-RXR. ECRDBD (A), ECR LBD (B), USP-RXR DBD (C), USP-RXR LBD (D). Evolutionary distances between sequences were mapped on a predefined species consensus tree (see fig.1) using Tree-Puzzle (Schmidt et al. 2002), with the JTT substitution model (Jones, Taylor, and Thornton 1992) plus rate heterogeneity between sites, estimated by a gamma law with eight categories. Branch lengths are proportional to evolutionary change; the measure bar represents 0.1 substitutions per site. Diptera and Lepidoptera species are in bold Fig. 3. View largeDownload slide Predefined trees with evolutionary distances for ECR and USP-RXR. ECRDBD (A), ECR LBD (B), USP-RXR DBD (C), USP-RXR LBD (D). Evolutionary distances between sequences were mapped on a predefined species consensus tree (see fig.1) using Tree-Puzzle (Schmidt et al. 2002), with the JTT substitution model (Jones, Taylor, and Thornton 1992) plus rate heterogeneity between sites, estimated by a gamma law with eight categories. Branch lengths are proportional to evolutionary change; the measure bar represents 0.1 substitutions per site. Diptera and Lepidoptera species are in bold Fig. 4. View largeDownload slide Sequence alignment of USP-RXR LBD domains. Sequences are aligned with human RXRα; names of Diptera species are in bold and underlined, names of Lepidoptera species are in bold. The 11 helices and the two β-strands (s1, s2) are boxed. Residues interacting with the ligand in the ligand-binding pocket (LBP) are indicated by asterisks (*) below the alignments. Structural data are from the following sources: human RXRα (Bourguet et al. 1995; Egea et al. 2000), Heliothis USP-RXR (Billas et al. 2001), and Drosophila USP-RXR (Clayton et al. 2001). Note that helices H3 and H12 are shorter in human RXRα, as indicated by a dashed vertical line in the amino-terminal of these helices. The gray box in the loop H1-H3 highlights a region conserved between Diptera and Lepidoptera. RT-PCR clones of five species (Lithobius, Folsomia, Periplaneta, Hypera, Leptopilina) lack some of the carboxy-terminal regions: an X indicates the end of these partial sequences. The few residues (3 to 12) following the helix H12, and therefore outside of the structurally defined LBD, are also shown on this figure Fig. 4. View largeDownload slide Sequence alignment of USP-RXR LBD domains. Sequences are aligned with human RXRα; names of Diptera species are in bold and underlined, names of Lepidoptera species are in bold. The 11 helices and the two β-strands (s1, s2) are boxed. Residues interacting with the ligand in the ligand-binding pocket (LBP) are indicated by asterisks (*) below the alignments. Structural data are from the following sources: human RXRα (Bourguet et al. 1995; Egea et al. 2000), Heliothis USP-RXR (Billas et al. 2001), and Drosophila USP-RXR (Clayton et al. 2001). Note that helices H3 and H12 are shorter in human RXRα, as indicated by a dashed vertical line in the amino-terminal of these helices. The gray box in the loop H1-H3 highlights a region conserved between Diptera and Lepidoptera. RT-PCR clones of five species (Lithobius, Folsomia, Periplaneta, Hypera, Leptopilina) lack some of the carboxy-terminal regions: an X indicates the end of these partial sequences. The few residues (3 to 12) following the helix H12, and therefore outside of the structurally defined LBD, are also shown on this figure Fig. 4 View largeDownload slide (Continued) Fig. 4 View largeDownload slide (Continued) Fig. 5. View largeDownload slide Sequences alignment of DBD domains. (A) ECR and (B) USP-RXR. ECR sequences are aligned with Celuca ECR. USP-RXRs are aligned with human RXRα. Structural data are from Lee et al. (1993) and Khorasanizadeh and Rastinejad (2001). The two zinc fingers are underlined on each sequence of reference, and they are also indicated below the alignments; critical cysteine residues of the zinc fingers are identified with asterisks. Names of Diptera species are in bold and underlined; names of Lepidoptera species are in bold. The gray boxes indicate ECR divergent positions between Diptera-Lepidoptera and other arthropods Fig. 5. View largeDownload slide Sequences alignment of DBD domains. (A) ECR and (B) USP-RXR. ECR sequences are aligned with Celuca ECR. USP-RXRs are aligned with human RXRα. Structural data are from Lee et al. (1993) and Khorasanizadeh and Rastinejad (2001). The two zinc fingers are underlined on each sequence of reference, and they are also indicated below the alignments; critical cysteine residues of the zinc fingers are identified with asterisks. Names of Diptera species are in bold and underlined; names of Lepidoptera species are in bold. The gray boxes indicate ECR divergent positions between Diptera-Lepidoptera and other arthropods Fig. 6. View largeDownload slide Sequence alignment of ECR F domains. Sequences are aligned with Drosophila ECR. Names of Diptera species are in bold and underlined; names of Lepidoptera species are in bold.Putative helix H12 (Wurtz et al. 2000) is boxed. Amino acids following the helix H12 are numbered above Drosophila sequence. The total number of residues in carboxy-terminal domains of ECR proteins is indicated at the end of each sequence Fig. 6. View largeDownload slide Sequence alignment of ECR F domains. Sequences are aligned with Drosophila ECR. Names of Diptera species are in bold and underlined; names of Lepidoptera species are in bold.Putative helix H12 (Wurtz et al. 2000) is boxed. Amino acids following the helix H12 are numbered above Drosophila sequence. The total number of residues in carboxy-terminal domains of ECR proteins is indicated at the end of each sequence Table 1 Accession Number and Phylogenetic Origin of Proteins Used in This Study. Group  Species  USP-RXR  ECR (and Related)  Diptera  Drosophila melanogaster  P20153  P34021    Ceratitis capitata    CAA11907    Lucilia cuprina    O18531    Calliphora vicina    AAG46050    Sarcophaga crassipalpis  AAF44674a  AAF44673a    Aedes aegypti  AAG24886  P49880    Aedes albopictus  AAF19033  AAF19032    Chironomus tentans  AAC03056  P49882  Lepidoptera  Bombyx mori  S44490  P49881    Manduca sexta  P54779  P49883    Heliothis virescens  14278415a  O18473    Junonia coenia    CAB63485a    Bicyclus anynana    CAB63236a    Choristoneura fumiferana  AAC31795  AAC61596  Hymenoptera  Apis mellifera  AAF73057    Leptopilina heterotoma  AY157931a,b  AY157932a,b  Coleoptera  Tenebrio molitor  CAB75361  CAA72296    Hypera postica  AY157933a,b  Orthoptera  Locusta migratoria  AAF00981  AAD19828  Dictyoptera  Periplaneta americana  AY157928a,b  Collembola  Folsomia candida  AY157930a,b  Crustacea  Celuca pugilator  AAC32789  AAC33432  Chelicerata  Amblyomma americanum  RXR1: AAC15588  AAB94566      RXR2: AAC15589  Myriapoda  Lithobius forficatus  AY157929a,b  Vertebrata  Homo sapiens  RXRα: CAA36982  LXRα: Q13133      RXRβ: AAA60293  LXRβ: P55055      RXRγ: AAA80681  FXR: AAB08107    Rattus norvegicus  RXRα: AAA42093  LXRα: Q62685        LXRβ: Q62755        FXR: A56918    Rattus rattus  RXRβ: AAA42025    Mus musculus  RXRα: AAA40080  LXRα: Q9Z0Y9      RXRβ: CAA46963  LXRβ: Q60644      RXRγ: CAA46964  FXR: NP_033134    Gallus gallus  RXRγ: CAA41743    Xenopus laevis  RXRα: P51128      RXRβ: S73269      RXRγ: P51129    Danio rerio  RXRα: AAC59719      RXRβ1: AAC59722      RXRβ2: AAC59721      RXRγ: AAC59720  Urochordata  Polyandrocarpa misakiensis  BAA82618a  Cnidaria  Tripedalia cystophora  AAC80008  Group  Species  USP-RXR  ECR (and Related)  Diptera  Drosophila melanogaster  P20153  P34021    Ceratitis capitata    CAA11907    Lucilia cuprina    O18531    Calliphora vicina    AAG46050    Sarcophaga crassipalpis  AAF44674a  AAF44673a    Aedes aegypti  AAG24886  P49880    Aedes albopictus  AAF19033  AAF19032    Chironomus tentans  AAC03056  P49882  Lepidoptera  Bombyx mori  S44490  P49881    Manduca sexta  P54779  P49883    Heliothis virescens  14278415a  O18473    Junonia coenia    CAB63485a    Bicyclus anynana    CAB63236a    Choristoneura fumiferana  AAC31795  AAC61596  Hymenoptera  Apis mellifera  AAF73057    Leptopilina heterotoma  AY157931a,b  AY157932a,b  Coleoptera  Tenebrio molitor  CAB75361  CAA72296    Hypera postica  AY157933a,b  Orthoptera  Locusta migratoria  AAF00981  AAD19828  Dictyoptera  Periplaneta americana  AY157928a,b  Collembola  Folsomia candida  AY157930a,b  Crustacea  Celuca pugilator  AAC32789  AAC33432  Chelicerata  Amblyomma americanum  RXR1: AAC15588  AAB94566      RXR2: AAC15589  Myriapoda  Lithobius forficatus  AY157929a,b  Vertebrata  Homo sapiens  RXRα: CAA36982  LXRα: Q13133      RXRβ: AAA60293  LXRβ: P55055      RXRγ: AAA80681  FXR: AAB08107    Rattus norvegicus  RXRα: AAA42093  LXRα: Q62685        LXRβ: Q62755        FXR: A56918    Rattus rattus  RXRβ: AAA42025    Mus musculus  RXRα: AAA40080  LXRα: Q9Z0Y9      RXRβ: CAA46963  LXRβ: Q60644      RXRγ: CAA46964  FXR: NP_033134    Gallus gallus  RXRγ: CAA41743    Xenopus laevis  RXRα: P51128      RXRβ: S73269      RXRγ: P51129    Danio rerio  RXRα: AAC59719      RXRβ1: AAC59722      RXRβ2: AAC59721      RXRγ: AAC59720  Urochordata  Polyandrocarpa misakiensis  BAA82618a  Cnidaria  Tripedalia cystophora  AAC80008  aPartial sequences. bThis paper. View Large Table 2 Average Identity Percentages of Pairwise Comparisons for DBD and LBD Domains of USP-RXR and ECR.     DBD  LBD  Groups of Species    USP-RXR  ECR  USP-RXR  ECR  Diptera-Lepidoptera >  Other insects  94.6 ± 1.5  88.75 ± 1.1  49.1 ± 3.1  64.4 ± 3.1    Other arthropods  93.6 ± 2  88.8 ± 1.8  43.65 ± 2.1  58.2 ± 3.2    Chordates  82.7 ± 1.8  #  46.7 ± 2  #  Other Insects >  Other arthropods  93.3 ± 2.1  96.9 ± 0.7  68 ± 4.6  67.7 ± 2.7    Chordates  84.4 ± 1.7  #  69.8 ± 3.3  #  Other Arthropods >  Chordates  82.4 ± 1.5  #  70.1 ± 1.9  #      DBD  LBD  Groups of Species    USP-RXR  ECR  USP-RXR  ECR  Diptera-Lepidoptera >  Other insects  94.6 ± 1.5  88.75 ± 1.1  49.1 ± 3.1  64.4 ± 3.1    Other arthropods  93.6 ± 2  88.8 ± 1.8  43.65 ± 2.1  58.2 ± 3.2    Chordates  82.7 ± 1.8  #  46.7 ± 2  #  Other Insects >  Other arthropods  93.3 ± 2.1  96.9 ± 0.7  68 ± 4.6  67.7 ± 2.7    Chordates  84.4 ± 1.7  #  69.8 ± 3.3  #  Other Arthropods >  Chordates  82.4 ± 1.5  #  70.1 ± 1.9  #  Note.—# indicates no chordate ECR sequences to compare. View Large Table 3 Comparison of Evolutionary Rates for USP-RXR and ECR LBDs Between Three Groups of Arthropods. Groups of Species    USP-RXR LBD  ECR LBD  Diptera-Lepidoptera >  Other insects  0.307 ± 0.068***  0.122 ± 0.054*  Diptera-Lepidoptera >  Other arthropods  0.365 ± 0.082***  0.129 ± 0.057*  Other insects >  Other arthropods  0.0574 ± 0.041 NS  0.0064 ± 0.049 NS  Groups of Species    USP-RXR LBD  ECR LBD  Diptera-Lepidoptera >  Other insects  0.307 ± 0.068***  0.122 ± 0.054*  Diptera-Lepidoptera >  Other arthropods  0.365 ± 0.082***  0.129 ± 0.057*  Other insects >  Other arthropods  0.0574 ± 0.041 NS  0.0064 ± 0.049 NS  Note.—Values indicated are substitution rate difference ± standard deviation. The probability associated to the test is indicated as follows: not significant (NS) > 5%; * ≤ 5%; ** ≤ 1%; *** ≤ 0.5%. View Large We thank Laure Debure for samples of Periplaneta americana, Guillaume Balavoine for Folsomia candida, Frédéric Fleury for Leptopilina heterotoma, Elisabeth Rull for help in RT-PCR experiments, and Melanie Palmer and two reviewers for their very helpful comments. CNRS, ENS, ARC Région Rhône-Alpes, and MENRT funded this work. Literature Cited Aguinaldo, A. M., J. M. Turbeville, L. S. Linford, M. C. Rivera, J. R. Garey, R. A. Raff, and J. A. Lake. 1997. Evidence for a clade of nematodes, arthropods and other moulting animals. Nature   387: 489-493. Google Scholar Billas, I. M., L. Moulinier, N. Rochel, and D. Moras. 2001. Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid x receptors in insects. J. Biol. Chem.   276: 7465-7474. Google Scholar Black, B. E., J. M. Holaska, F. Rastinejad, and B. M. Paschal. 2001. DNA binding domains in diverse nuclear receptors function as nuclear export signals. Curr. Biol.  11: 1749-1758. Google Scholar Blair, J. E., K. Ikeo, T. Gojobori, and S. B. Hedges. 2002. The evolutionary position of nematodes. BMC Evol. Biol.   2: 1-7. Google Scholar Bourguet, W., M. Ruff, D. Bonnier, F. Granger, M. Boeglin, P. Chambon, D. Moras, and H. Gronemeyer. 1995. Purification, functional characterization, and crystallization of the ligand-binding domain of the retinoid X receptor. Protein Expr. Purif.   6: 604-608. Google Scholar Chung, A. C.-K., D. S. Durica, S. W. Clifton, B. A. Roe, and P. M. Hopkins. 1998. Cloning of crustacean ecdysteroid receptor and retinoid-X receptor gene homologs and elevation of retinoid-X receptor mRNA retinoic acid. Mol. Cell. Endocrinol.  139: 209-227. Google Scholar Clayton, G. M., S. Y. Peak-Chew, R. M. Evans, and J. W. R. Schwabe. 2001. The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation. Proc. Natl. Acad. Sci. USA   98: 1549-1554. Google Scholar Duarte, J., G. Perriere, V. Laudet, and M. Robinson-Rechavi. 2002. NUREBASE: database of nuclear hormone receptors. Nucleic Acids Res.   30: 364-368. Google Scholar Egea, P. F., A. Mitschler, N. Rochel, M. Ruff, P. Chambon, and D. Moras. 2000. Crystal structure of the human RXRα ligand-binding domain bound to its natural ligand : 9-cis retinoic acid. EMBO J.   19: 2592-2601. Google Scholar Escriva, H., F. Delaunay, and V. Laudet. 2000. Ligand binding and nuclear receptor evolution. BioEssays   22: 717-727. Google Scholar Escriva, H., M. Robinson, and V. Laudet. 1999. Evolutionary biology of the nuclear receptor superfamily. Pp. 1–28 in D. Picard ed. Steroid/nuclear receptor superfamily: practical approach. Academic Press, Oxford. Google Scholar Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution  39: 738-791. Google Scholar Galtier, N., M. Gouy, and C. Gautier. 1996. SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput. Appl. Biosci.   12: 543-548. Google Scholar Giribet, G., G. D. Edgecombe, and W. C. Wheeler. 2001. Arthropod phylogeny based on eight molecular loci and morphology. Nature   413: 157-161. Google Scholar Guo, X., M. A. Harmon, V. Laudet, D. J. Mangelsdorf, and M. J. Palmer. 1997. Isolation of a functional ecdysteroid receptor homologue from the Ixodid tick, Amblyomma americanum (L.). Insect Biochem. Mol. Biol.   27: 945-962. Google Scholar Guo, X., Q. Xu, M. A. Harmon, X. Jin, V. Laudet, D. J. Mangelsdorf, and M. J. Palmer. 1998. Isolation of two functional retinoid X receptor subtypes from the Ixodid tick, Amblyomma americanum (L.). Mol. Cell. Endocrinol.   139: 45-60. Google Scholar Hayward, D. C., M. J. Bastiani, J. W. Trueman, J. W. Truman, L. M. Riddiford, and E. E. Ball. 1999. The sequence of Locusta RXR, homologous to Drosophila ultraspiracle, and its evolutionary implications. Dev. Genes Evol.   209: 564-571. Google Scholar Henrich, V. C., and N. E. Brown. 1995. Insect nuclear receptors: a developmental and comparative perspective. Insect Biochem. Mol. Biol.   25: 881-897. Google Scholar Hwang, U. W., M. Friedrich, D. Tautz, C. J. Park, and W. Kim. 2001. Mitochondrial protein phylogeny joins myriapods with chelicerates. Nature   413: 154-157. Google Scholar Jones, D. T., W. R. Taylor, and J. M. Thornton. 1992. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci.  8: 275-282. Google Scholar Jones, G., and P. A. Sharp. 1997. Ultraspiracle: an invertebrate nuclear receptor for juvenile hormones. Proc. Natl. Acad. Sci. USA  94: 13499-51303. Google Scholar Jones, G., M. Wozniak, Y. Chu, S. Dhar, and D. Jones. 2001. Juvenile hormone III-dependent conformational changes of the nuclear receptor ultraspiracle. Insect Biochem. Mol. Biol.   32: 33-49. Google Scholar Khorasanizadeh, S., and F. Rastinejad. 2001. Nuclear-receptor interactions on DNA-response elements. Trends Biochem. Sci.   26: 384-390. Google Scholar King, R. C., and J. Büning. 1985. The origin and functioning of insect oocytes and nurse cells. Pp. 37–82 in G. A. Kerkurt and L. I. Gilbert, eds. Comprehensive insect physiology biochemistry and pharmacology. Pergamon Press, Oxford. Google Scholar Koelle, M. R., W. S. Talbot, W. A. Segraves, M. T. Bender, P. Cherbas, and D. S. Hogness. 1991. The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell   67: 59-77. Google Scholar Kostrouch, Z., M. Kostrouchova, W. Love, E. Jannini, J. Piatigorsky, and J. E. Rall. 1998. Retinoic acid X receptor in the diploblast, Tripedalia cystophora. Proc. Natl. Acad. Sci. USA   95: 13442-13447. Google Scholar Kozlova, T., and C. S. Thummel. 2000. Steroid regulation of postembryonic development and reproduction in Drosophila. Trends Endocrinol. Metab.   11: 276-280. Google Scholar Kristensen, N. P. 1981. Phylogeny of insect orders. Annu. Rev. Entomol.   26: 135-157. Google Scholar Laudet, V., and H. Gronemeyer. 2002. The nuclear receptor factsbook, Academic Press, London. Google Scholar Lee, M. S., S. A. Kliewer, J. Provencal, P. E. Wright, and R. M. Evans. 1993. Structure of the retinoid X receptor alpha DNA binding domain: a helix required for homodimeric DNA binding. Science   260: 1117-1121. Google Scholar Luisi, B. F., W. X. Xu, Z. Otwinowski, L. P. Freedman, K. R. Yamamoto, and P. B. Sigler. 1991. Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature   352: 497-505. Google Scholar Mangelsdorf, D. J., E. S. Ong, J. A. Dyck, and R. M. Evans. 1990. Nuclear receptor that identifies a novel retinoic acid response pathway. Nature   345: 224-229. Google Scholar Montano, M. M., V. Muller, A. Trobaugh, and B. S. Katzenellenbogen. 1995. The carboxy-terminal F domain of the human estrogen receptor: role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists. Mol. Endocrinol.   9: 814-825. Google Scholar Moras, D., and H. Gronemeyer. 1998. The nuclear receptor ligand-binding domain: structure and function. Curr. Opin. Cell Biol.   10: 384-391. Google Scholar Nichols, M., J. M. Rientjes, and A. F. Stewart. 1998. Different positioning of the ligand-binding domain helix 12 and the F domain of the estrogen receptor accounts for functional differences between agonists and antagonists. EMBO J.   17: 765-773. Google Scholar Nicolaï, M., H. Bouhin, B. Quennedey, and J. Delachambre. 2000. Molecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20-hydroxyecdysone. Insect Mol. Biol.   9: 241-249. Google Scholar Oro, A. E., M. McKeown, and R. M. Evans. 1992. The Drosophila retinoid X receptor homologue ultraspiracle functions in both female reproduction and eye morphogenesis. Development  115: 449-462. Google Scholar Patel, N. H., B. G. Condron, and K. Zinn. 1994. Pair-rule expression patterns of even-skipped are found in both short- and long-germ beetles. Nature   367: 429-434. Google Scholar Perrimon, N., L. Engstrom, and A. P. Mahowald. 1985. Developmental genetics of the 2C-D region of the Drosophila X chromosome. Genetics   111: 23-41. Google Scholar Peters, G. A., and S. A. Khan. 1999. Estrogen receptor domains E and F: role in dimerization and interaction with coactivator RIP-140. Mol. Endocrinol.   13: 286-296. Google Scholar Regier, J. C., C. Mitter, T. P. Friedlander, and R. S. Peigler. 2001.. Mol. Phylogenet. Evol.   20: 311-325. Google Scholar Riddiford, L. M., P. Cherbas, and J. W. Truman. 2001. Ecdysone receptors and their biological actions. Vitam. Horm.   60: 1-73. Google Scholar Robinson, M., M. Gouy, C. Gautier, and D. Mouchiroud. 1998. Sensitivity of the relative-rate test to taxonomic sampling. Mol. Biol. Evol.  15: 1091-1098. Google Scholar Robinson-Rechavi, M., and D. Huchon. 2000. RRTree: relative-rate tests between groups of sequences on a phylogenetic tree. Bioinformatics   16: 296-297. Google Scholar Rokas, A., J. Kathirithamby, and P. W. Holland. 1999. Intron insertion as a phylogenetic character: the engrailed homeobox of Strepsiptera does not indicate affinity with Diptera. Insect Mol. Biol.   8: 527-530. Google Scholar Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol.   4: 406-425. Google Scholar Saleh, D. S., J. Zhang, G. R. Wyatt, and V. K. Walker. 1998. Cloning and characterization of an ecdysone receptor cDNA from Locusta migratoria. Mol. Cell. Endocrinol.  143: 91-99. Google Scholar Sander, K. 1976. Specification of the basic body pattern in insect embryogenesis. Adv. Insect. Physiol.   12: 125-238. Google Scholar Schmidt, H. A., K. Strimmer, M. Vingron, and A. von Haeseler. 2002. Tree-Puzzle: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics   18: 502-504. Google Scholar Schwabe, J. W. R., L. Chapman, J. T. Finch, and D. Rhodes. 1993. The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell   75: 567-578. Google Scholar Sehnal, F., P. Svàcha, and J. Zrzavy. 1996. Evolution of insect metamorphosis. Pp. 3–58 in L. I. Gilbert, J. R. Tata, and B. G. Atkinson, eds. Metamorphosis: postembryonic reprogramming of gene expression in amphibian and insect cells. Academic Press, San Diego. Google Scholar Sluder, A. E., and C. V. Maina. 2001. Nuclear receptors in nematodes: themes and variations. Trends Genet.   17: 206-213. Google Scholar Swevers, L., L. Cherbas, P. Cherbas, and K. Iatrou. 1996. Bombyx EcR (BmEcR) and Bombyx USP (BmCF1) combine to form a functional ecdysone receptor. Insect Biochem. Mol. Biol.   26: 217-221. Google Scholar Thummel, C. S. 2001. Molecular mechanisms of developmental timing in C. elegans and Drosophila. Dev. Cell   1: 453-465. Google Scholar Truman, J. W., and L. M. Riddiford. 1999. The origins of insect metamorphosis. Nature   401: 447-452. Google Scholar Verras, M., M. Mavroidis, G. Kokolahis, P. Gourzi, A. Zacharopoulou, and A. C. Mintzas. 1999. An ecdysone receptor homologue from the Mediterranean fruit fly Ceratitis capitata. Eur. J. Biochem.   265: 798-808. Google Scholar Wang, S. F., S. Ayer, W. A. Segraves, D. R. Williams, and A. S. Raikhel. 2000. Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors. Mol. Cell Biol.  20: 3870-3879. Google Scholar Weller, S. J., T. P. Friedlander, J. A. Martin, and D. P. Pashley. 1992. Phylogenetic studies of ribosomal RNA variation in higher moths and butterflies (Lepidoptera: Ditrysia). Mol. Phylogenet. Evol.   1: 312-337. Google Scholar Whiting, M. F., J. C. Carpenter, Q. D. Wheeler, and W. C. Wheeler. 1997. The streptisera problem: phylogeny of the holometabolous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Syst. Biol.   46: 1-68. Google Scholar Wilson, A. C., S. S. Carlson, and T. J. White. 1977. Biochemical evolution. Annu. Rev. Biochem.  46: 573-639. Google Scholar Wurtz, J. M., B. Guillot, J. Fagart, D. Moras, K. Tietjen, and M. Schindler. 2000. A new model for 20-hydroxyecdysone and dibenzoylhydrazine binding: a homology modeling and docking approach. Protein Sci.  9: 1073-1084. Google Scholar Yao, T. P., B. M. Forman, Z. Jiang, L. Cherbas, J. D. Chen, M. McKeown, P. Cherbas, and R. M. Evans. 1993. Functional ecdysone receptor is the product of EcR and ultraspiracle genes. Nature   366: 476-479. Google Scholar Yeates, D. K., and B. M. Wiegmann. 1999. Congruence and controversy: toward a higher-level phylogeny of Diptera. Annu. Rev. Entomol.   44: 397-428. Google Scholar Society for Molecular Biology and Evolution
TI - Rapid Divergence of the Ecdysone Receptor in Diptera and Lepidoptera Suggests Coevolution Between ECR and USP-RXR
JF - Molecular Biology and Evolution
DO - 10.1093/molbev/msg054
DA - 2003-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/rapid-divergence-of-the-ecdysone-receptor-in-diptera-and-lepidoptera-CznMDsZ0zd
SP - 541
EP - 553
VL - 20
IS - 4
DP - DeepDyve
ER -