* UMR 5534 du CNRS, Université Claude Bernard Lyon 1, Villeurbanne Cedex, France
UMR 5665 du CNRS, Ecole Normale Supérieure de Lyon, Lyon Cedex, France
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Abstract |
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Key Words: ecdysone receptor USP-RXR ECR insects coevolution evolutionary rate
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Introduction |
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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 (LXR and LXRß: 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 (RXR, ß,
: 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 RXR
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.
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Materials and Methods |
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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.
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Results |
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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.
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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).
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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).
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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).
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Discussion |
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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 H3H12) connected by loops and two short ß-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 H1H3 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 H1H3 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 (ER, HNF-4) the F domain of nuclear receptors can regulate different functions of the LBD. For example, the F domain of human estrogen receptor ER
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.
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Acknowledgements |
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Footnotes |
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E-mail: bonneto{at}univ-lyon1.fr.
William Jeffery, Associate Editor
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