Chapter Four - Structure and Function of Ecdysone Receptors—Interactions with Ecdysteroids and Synthetic Agonists
Introduction
The initial evidence for hormonal initiation of insect moulting and metamorphosis arose from classical surgical experiments involving larval ligature and removal and transplantation of tissues by Kopec (1922), Wigglesworth (1934) and Fraenkel (1934). It was subsequently shown that an initial signal arises from the insect brain to activate the prothoracic gland in Lepidoptera (Fukuda, 1940, Williams, 1947) or the ring gland in Diptera (Possompès, 1953) to produce the hormone now known as ecdysone, which was eventually isolated by Butenandt and Karlson (1954). Ecdysone is subsequently metabolised to a derivative, 20-hydroxyecdysone (20E), which is less stable during chromatography on alumina; it was isolated from crustaceans by Hampshire and Horn (1966), and from insects by Hocks and Wiechert (1966), Hoffmeister and Grützmacher (1966) and Kaplanis et al. (1966). 20-Hydroxyecdysone is the most commonly occurring and generally most biologically active form of the hormone in arthropods (Horn, 1989). We now know that 20E controls not only moulting and metamorphosis but also other fundamental aspects of arthropod biology, including oogenesis (Raikhel et al., 2005), embryogenesis (Kozlova and Thummel, 2003), cuticle protein synthesis (Doctor et al., 1985), diapause (Denlinger et al., 2005) and polyphenism (Hartfelder and Emlen, 2005).
Ashburner (1972) first postulated a protein receptor for 20E by analogy to the receptors that were beginning to be characterized for mammalian steroid hormones, and on the basis of competition that he observed between 20E and N-ethylmaleimide on a protein target in the pathway that leads to induction of the “ecdysone puffs” on Drosophila melanogaster salivary chromosomes. This receptor protein occupies a central position in the Ashburner model, which was advanced to explain the regulatory network underlying chromosomal puffing in response to 20E (Ashburner, 1974, Thummel, 2002). The physical existence of a receptor for ecdysteroids was put on a firm basis by rigorous characterization of the ability of a protein in extracts of D. melanogaster imaginal discs (Yund et al., 1978) and in tissue-cultured cells (Maroy et al., 1978) to bind tritium-labelled ponasterone A (Pon A); Pon A is an ecdysteroid synthesised by some insects and plants which has greater affinity for the receptor than 20E. Classical biochemical approaches to isolate the receptor protein met with only limited success (Landon et al., 1988, Luo et al., 1991). The breakthrough came with the somewhat adventitious cloning of the gene EcRduring molecular characterization of the early ecdysone-responding puffs (Koelle et al., 1991, Segraves, 1987). Although the product of this gene conferred the anticipated properties of an ecdysone receptor on transfection into a Drosophila cell line (and was thus named the EcR protein), it subsequently emerged that the fully functional ecdysone receptor is a heterodimer of the protein products of the EcR and ultraspiracle (usp) genes (Thomas et al., 1993, Yao et al., 1993). The EcR and USP protein subunits of the ecdysone receptor both belong to the nuclear receptor family whose members consist of up to six canonical domains, denoted A/B (transcriptional activation), C (DNA binding), D (linker) and E/F (ligand-binding) domains (Bain et al., 2007, Jin and Li, 2010, Renaud and Moras, 2000); these are represented diagrammatically in Fig 4.10A.
Over 300 different ecdysteroid derivatives are produced by animals and plants (Dinan et al., 2009; Lafont and Koolman, 2009). Some 5% of plant species have been found to contain phytoecdysteroids, and many more species have the capacity to synthesize these molecules. It is thought that they may contribute to plant defence mechanisms against insect predators, and for some time, the possibility was entertained that ecdysteroids might provide safe insecticides capable of disrupting the endocrine control of pest insect development. For a variety of reasons, including the complexity and expense of ecdysteroid synthesis and the biological lability of these molecules, this hope has not been realized. The discovery of the class of bisacylhydrazine (BAH) insecticides, and the realization that these largely lepidopteran-selective agents act as agonists for ecdysone receptors (Wing, 1988, Wing et al., 1988), reopened interest in ecdysone receptors as targets for safe insecticides (Dhadialla et al., 1998; Chapter 2). The absence of ecdysone receptors from vertebrates and plants not only makes them attractive as targets for environmentally friendly insecticides but also favours their use in ligand-activated switches for transgenes (Graham, 2002, Palli et al., 2005, Yang et al., 1995). More recently, additional chemical families with the ability to bind to ecdysone receptors have been discovered, including the tetrahydroquinolines (Kumar et al., 2004), acylaminoketones (Tice et al., 2003a, Tice et al., 2003b) and methylene lactams (Birru et al., 2010) (see Chapter 3).
In this chapter, we examine the binding of ecdysteroids and other chemical families to recombinant ecdysone receptor proteins using the classical competition assay, which measures the ability of ligands to compete with [3H]-ponasterone A ([3H]-Pon A) for binding. A more recently developed fluorescence polarization (FP) ligand-binding assay will also be described, and its application in chemical library screening will be illustrated by the discovery of the methylene lactam ligand family. We shall describe the atomic structure of the ligand-binding domains (LBDs) of ecdysone receptors and discuss a hypothesis to explain the selectivity of the BAH insecticides for the Lepidoptera. The increased preference for the ecdysteroid makisterone A to act as a moulting hormone in the phytophagous pentatomorphan Nezara viridula will be considered in the light of a specific change in the wall of the N. viridula receptor ligand-binding pocket (LBP). The question of possible ligands for EcR orthologs in nematodes and non-ecdysozoan protosomians will be examined. Finally, we shall consider the extraordinary ability of ecdysone receptors to bind a range of disparate chemistries, a pioneering use of ecdysone receptor atomic structure to guide the optimization of synthetic ligand chemistry, and briefly address broader questions relating to the control spectrum of ecdysone receptor-targeting insecticides.
Section snippets
Molecular cloning and expression of functional ligand-binding domains
A number of approaches have been used to clone the subunits of ecdysone receptors (see, e.g. Fujiwara et al., 1995, Imhof et al., 1993, Koelle et al., 1991, Ogura et al., 2005, Perera et al., 1998, Swevers et al., 1995). For the purposes of this chapter, we shall focus on the procedures employed in our group in CSIRO to clone, express and purify segments of ecdysone receptor proteins. Full-length EcR- and USP-encoding cDNA clones were screened from high-quality pest insect cDNA libraries in
An automated fluorescence polarization chemical library screen
As all steps in the FP ligand-binding assay take place in solution and in a single test tube or microtitre plate well without the need for physical isolation of ligand–protein complexes, the procedure is readily automated for chemical library screening and for making multiple measurements over dilution series of individual compounds for determinations of IC50 (i.e. the concentration of competitor compound required to inhibit binding of the fluorescein-labelled inokosterone by 50%). A Tecan
Overall structure of the ecdysone receptor
The ecdysone receptor complex is a heterodimer of two members of the nuclear receptor family, the EcR and the ultraspiracle (USP) proteins, which assemble into the functional receptor as described in Section 1 (Billas et al., 2009, Renaud and Moras, 2000). All nuclear receptor family members consist of up to six canonical domains, denoted A–F (Bain et al., 2007, Jin and Li, 2010, Renaud and Moras, 2000), as represented diagrammatically in Fig. 4.10A. The A and B domains are highly variable and
EcR Orthologs from Nematodes and from Non-ecdysozoan Protosomians
Recent progress has seen the cloning of EcR and USP homologs from non-arthropod ecdysozoans, such as nematodes, and indeed from non-ecdysozoan species as well, such as leeches. Such developments may be of considerable significance as these receptors, if important to development and/or reproduction, could serve as novel drug targets for parasites that are currently difficult to control. In particular, parasitic nematodes are responsible for many diseases in man, farm animals and crop plants. In
Closing Remarks
20E, and less commonly other closely related ecdysteroids, plays a central role through ecdysone receptors in coordinating the expression of large networks of genes at critical points during the life cycle of insects. 20E, one of the two key non-polypeptide insect hormones, influences gene expression in most tissues and cell types during embryogenesis, larval instars, metamorphosis and gametogenesis. Furthermore, the ecdysone receptor has been demonstrated to be a target for safe,
Acknowledgements
We wish to express our appreciation for support from Australian Wool Innovation Ltd. MCL was supported by the Hazel and Pip Appel Fund, and his contribution was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.
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