Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.     

Functional studies on the ligand-binding domain of Ultraspiracle from Drosophila melanogaster

The functional insect ecdysteroid receptor is comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP). The ligand-binding domain (LBD) of USP was fused to the GAL4 DNA-binding domain (GAL4-DBD) and characterized by analyzing the effect of site-directed mutations in the LBD. Normal and mutant proteins were tested for ligand and DNA binding, dimerization, and their ability to induce gene expression. The presence of helix 12 proved to be essential for DNA binding and was necessary to confer efficient ecdysteroid binding to the heterodimer with the EcR (LBD), but did not influence dimerization. The antagonistic position of helix 12 is indispensible for interaction between the fusion protein and DNA, whereas hormone binding to the EcR (LBD) was only partially reduced if fixation of helix 12 was disturbed. The mutation of amino acids, which presumably bind to a fatty acid evoked a profound negative influence on transactivation ability, although enhanced transactivation potency and ligand binding to the ecdysteroid receptor was impaired to varying degrees by mutation of these residues. Mutations of one fatty acid-binding residue within the ligand-binding pocket, 1323, however, evoked enhanced transactivation. The results confirmed that the LBD of Ultraspiracle modifies ecdysteroid receptor function through intermolecular interactions and demonstrated that the ligand-binding pocket of USP modifies the DNA-binding and transactivation abilities of the fusion protein.     

Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo we established transgenic animals that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of beta-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. We show that the patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. We also show that GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, we observe no GAL4-LBD activation in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. We conclude that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. Finally, we show that GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development.     

Interaction of proteins involved in ecdysone and juvenile hormone signal transduction

Ecdysteroids and juvenile hormones (JH) regulate a variety of developmental, physiological, behavioral, and metabolic processes. Ecdysteroids function through a heterodimeric complex of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP). An 85 kDa protein identified in Drosophila melanogaster methoprene-tolerant (Met) mutant binds to JH III with high affinity, and the mutant flies are resistant to juvenile hormone analog (JHA), methoprene. Reporter assays using the yeast two-hybrid system were performed in order to study the molecular interactions between EcR, USP and Met. As expected, EcR fused to the B42 activation domain and USP fused to the LexA DNA binding domain interacted with each other and supported induction of the reporter gene in the presence of stable ecdysteroid analog, RG-102240 or steroids, muristerone A and ponasterone A. The USP:USP homodimers supported expression of the reporter gene in the absence of ligand, and there was no significant increase in the reporter activity after addition of a JHA, methoprene. Similarly, Met:Met homodimers as well as Met:EcR and Met:USP heterodimers induced reporter activity in the absence of ligand and addition of ecdysteroid or JH analogs did not increase the reporter activity regulated by either homodimers or heterodimers of Met protein. Two-hybrid assays in insect cells and in vitro pull-down assays confirmed the interaction of Met with EcR and USP. These data suggest that the proteins that are involved in signal transduction of ecdysteroids (EcR and USP) and juvenile hormones (Met) interact to mediate cross-talk between these two important hormones. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.     

昆虫蜕皮激素受体及其类似物的杀虫机制研究进展

昆虫的蜕皮、变态和繁殖受到蜕皮激素的严格调控。蜕皮激素作用靶标由蜕皮激素受体（ecdysteroid receptor, EcR）和超气门蛋白（ultraspiracle protein, USP）组成,蜕皮激素与EcR/USP作用启动蜕皮级联反应过程。昆虫EcR具有种类或类群的特异性,研究其结构、功能和调控机理在开发环境友好型新药剂和基因调控开关等方面具有重要指导作用。该文介绍了昆虫EcR的结构和功能特点,蜕皮激素及其类似物与EcR/USP的分子作用方式,以及基于EcR/USP的新杀虫剂创制和基因调控开关设计等方面的重要进展。     

Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster

Mutants created by site-directed mutagenesis were used to elucidate the function of amino acids involved in ligand binding to ecdysteroid receptor (EcR) and heterodimer formation with ultraspiracle (USP). The results demonstrate the importance of the C-terminal part of the D-domain and helix 12 of EcR for hormone binding. Some amino acids are involved either in ligand binding to EcR (E476, M504, D572, I617, N626) or ligand-dependent heterodimerization as determined by gel mobility shift assays (A612, L615, T619), while others are involved in both functions (K497, E648). Some amino acids are suboptimal for ligand binding (L615, T619), but mediate ligand-dependent dimerization. We conclude that the enhanced regulatory potential by ligand-dependent modulation of dimerization in the wild type is achieved at the expense of optimal ligand binding. Mutation of amino acids (K497, E648) involved in the salt bridge between helix 4 and 12 impair ligand binding to EcR more severely than hormone binding to the heterodimer, indicating that to some extent heterodimerization compensates for the deleterious effect of certain mutations. Different effects of the same point mutations on ligand binding to EcR and EcR/USP (R511, A612, L615, I617, T619, N626) indicate that the ligand-binding pocket is modified by heterodimerization.     

Purification and characterization of recombinant ligand-binding domains from the ecdysone receptors of four pest insects

Cloned EcR and USP cDNAs encoding the ecdysone receptors of four insect pests (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were manipulated to allow the co-expression of their ligand binding domains (LBDs) in insect cells using a baculovirus vector. Recombinant DE/F segment pairs (and additionally, for H. armigera, an E/F segment pair) from the EcR and USP proteins associated spontaneously with high affinity to form heterodimers that avidly bound an ecdysteroid ligand. This shows that neither ligand nor D-regions are essential for the formation of tightly associated and functional LBD heterodimers. Expression levels ranged up to 16.6mg of functional apo-LBD (i.e., unliganded LBD) heterodimer per liter of recombinant insect cell culture. Each recombinant heterodimer was affinity-purified via an oligo-histidine tag at the N-terminus of the EcR subunit, and could be purified further by ion exchange and/or gel filtration chromatography. The apo-LBD heterodimers appeared to be more easily inactivated than their ligand-containing counterparts: after purification, populations of the former were <40% active, whereas for the latter >70% could be obtained as the ligand-LBD heterodimer complex. Interestingly, we found that the amount of ligand bound by recombinant LBD heterodimer preparations could be enhanced by the non-denaturing detergent CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate). Purity, integrity, size and charge data are reported for the recombinant proteins under native and denaturing conditions. Certain intra- and intermolecular disulfide bonds were observed to form in the absence of reducing agents, and thiol-specific alkylation was shown to suppress this phenomenon but to introduce microheterogeneity.     

DNA binding properties of the ecdysteroid receptor in the salivary gland of the female ixodid tick, Amblyomma hebraeum

Salivary gland degeneration in the female tick, Amblyomma hebraeum Koch (Acari: Ixodidae) is controlled by an ecdysteroid hormone. In an earlier study (Mao, H., McBlain, W.A., Kaufman, W.R., 1995. Some properties of the ecdysteroid receptor in the salivary gland of the ixodid tick, Amblyomma hebraeum. Gen. Comp. Endocrinol. 99, 340–348), we demonstrated that a protein component of a salivary gland extract binds to ponasterone A (Pon A) with high affinity (Kd1 nM), suggesting a tick ecdysteroid receptor (EcR). In this study, the Pon A binding protein bound to calf thymus DNA; this binding could be dissociated by Drosophila hsp27 EcRE. The binding protein shifted the [³²P]hsp27 EcRE band on a gel mobility shift assay; formation of the complex with hsp27 EcRE required KCl (optimal concentration was approximately 75 mM). A number of physiologically effective ecdysteroids enhanced the binding with the following order of potency: Pon A>Mur A>Mak A>20E>ecdysone, whereas vertebrate steroids (estradiol, cholesterol, corticosterone, progesterone, testosterone) had no such effect. Using monoclonal antibodies against Drosophila EcR and USP, we found that AG10.2 recognized three bands (90.5, 87.3 and 84 kDa for EcR) and AB11 recognized at least two major bands (50.3 and 47.1 kDa for USP) in the salivary gland extract by western blot analysis. In addition, AB11 supershifted the tick EcR-hsp27 EcRE band on a gel mobility shift assay, indicating that the tick EcR heterodimerized with a USP-like protein for DNA binding. Furthermore, selective mutations to the 15-basepair palindrome of hsp27 EcRE at positions −5, +2, or adding a base to the spacer, resulted in considerably reduced affinity to the tick EcR/USP. We thus propose a sequence similarity of EcREs between A. hebraeum and its insect counterpart.     

The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signaling pathway

Ecdysteroid pulses trigger the major developmental transitions during the Drosophila life cycle. These hormonal responses are thought to be mediated by the ecdysteroid receptor (EcR) and its heterodimeric partner Ultraspiracle (USP). We provide evidence for a second ecdysteroid signaling pathway mediated by DHR38, the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors. DHR38 also heterodimerizes with USP, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR. This response is unusual in that it does not involve direct binding of ecdysteroids to either DHR38 or USP. X-ray crystallographic analysis of DHR38 reveals the absence of both a classic ligand binding pocket and coactivator binding site, features that seem to be common to all NGFI-B subfamily members. Taken together, these data reveal the existence of a separate structural class of nuclear receptors that is conserved from fly to humans.     

Expression of ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta, Diptera) in E. coli and purification in a functional state.

Full length clones of ecdysteroid receptor (EcR) and Ultraspiracle (USP) from Chironomus tentans were expressed as GST fusion proteins in E. coli and purified by affinity chromatography. The absence of detergents during the purification procedure is essential for retaining receptor function, especially ligand binding. Presence of USP is mandatory for ligand binding to EcR, but no other cofactors or posttranslational modifications seem to be important, since Scatchard plots revealed the same characteristics (two high affinity binding sites for Ponasterone A with K(D1)=0.24+/-0.1nM and K(D2)=3.9+/-1.3.nM) as found in 0.4 M NaCl extracts of Chironomus cells. Gel mobility shift assays showed binding of the heterodimer to PAL and DR5 even after removal of the GST-tag, whereas EcR binding to PAL1 is GST-dependent. USP binds preferentially to DR5. Addition of unprogrammed reticulocyte lysate improves ligand binding only slightly. Removal of GST has no effect on (3)H-ponasterone A binding, but alters DNA binding characteristics. Calculation of specific binding (5.3+3.0 nmol/mg GST EcR) revealed that 47+/-26% of purified receptor protein was able to bind ligand. The addition of purified EcR to cell extracts of hormone resistant subclones of the epithelial cell line from C. tentans, which have lost their ability to bind ligand, restores specific binding of (3)H-ponasterone A.     

The influence of heterodimer partner ultraspiracle/retinoid X receptor on the function of ecdysone receptor

A pair of nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP), heterodimerize and transduce ecdysteroid signals. The EcR and its nonsteroidal ligands are being developed for regulation of transgene expression in humans, animals and plants. In mammalian cells, EcR:USP heterodimers can function in the absence of ligand, but EcR/retinoid X receptor (EcR:RXR) heterodimers require the presence of ligand for activation. The heterodimer partner of EcR can influence ligand sensitivity of EcR so that the EcR/Locusta migratoria RXR (EcR:LmRXR) heterodimers are activated at lower concentrations of ligand when compared with the concentrations of ligand required for the activation of EcR/Homo sapiens RXR (EcR:HsRXR) heterodimers. Analysis of chimeric RXRs containing regions of LmRXR and HsRXR and point mutants of HsRXR showed that the amino acid residues present in helix 9 and in the two loops on either end of helix 9 are responsible for improved activity of LmRXR. The EcR:Lm-HsRXR chimera heterodimer induced reporter genes with nanomolar concentration of ligand compared with the micromolar concentration of ligand required for activating the EcR:HsRXR heterodimer. The EcR:Lm-HsRXR chimera heterodimer, but not the EcR:HsRXR heterodimer, supported ligand-dependent induction of reporter gene in a C57BL/6 mouse model.     

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

The ligand-binding domain (LBD) encompassing the C-terminal parts of the D- and the complete E-domains of the ecdysteroid receptor (EcR) fused to Gal4(AD) is present in two high molecular weight complexes (600 and 150 kDa) in yeast extracts according to size exclusion chromatography (Superdex 200 HR 10/30). Hormone binding is mainly associated with 150-kDa complexes. Complex formation is not influenced by hormone, but the ligand stabilizes the complexes at elevated salt concentrations. Mutational analysis of Gal4(AD)-EcR(LBD) revealed that formation of 600-kDa, but not 150-kDa, complexes depends on dimerization mediated by the EcR(LBD). Deletion of helix 12 is without effect. Mutation of K497 in helix 4, known to be essential for comodulator binding, abolishes 600-KDa complexes, but does not interfere with the formation of 150-kDa complexes. In contrast, the DE-domains of USP fused to Gal4(DBD) elute as monomer after elimination of the dimerization capacity of the ligand-binding domains by mutation of P463 in helix 10. The data presented here reveal that the complex formation of ligand-binding domains EcR and USP ligand is different.