Ecdysone: Structures and Functions Guy Smagghe Editor

Ecdysone: Structures and Functions Editor Guy Smagghe Laboratory of Agrozoology Faculty of Bioscience Engineering Ghent University Belgium

ISBN 978-1-4020-9111-7 e-ISBN 978-1-4020-9112-4

Library of Congress Control Number: 2008938015

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Printed on acid-free paper springer.com Preface

The 16th International Ecdysone Workshop took place at Ghent University in Belgium, July 10–14, 2006 and drew some 150 attendees, many of these young students and postdoctoral associates. These young scientists had the opportunity to dis- cuss their work with many senior scientists at meals, breaks and during the several social events, and were encouraged to do so. This book resulting from the meeting is more up-to-date than might be expected since manuscripts were not delivered to the editor until 2007. The workshop itself had 54 oral presentations as well as many posters. This book, and the meeting itself, is comprised of 23 contributed chapters falling into five general categories: Fundamental Aspects of Ecdysteroid Research: The Distribution and Diversity of Ecdysteroids in Animals and Plants; Ecdysteroid Genetic Hierarchies in Insect Growth and Reproduction; Role of Cross Talk and Growth Factors in Ecdysteroid Titers and Signaling; Ecdysteroid Function Through Nuclear and Membrane Receptors; Ecdysteroids in Modern Agriculture, Medicine, Doping and Ecotoxicology. Clearly, all that was presented at the meeting and in this volume cannot be sum- marized here in a single page, but the reader is cordially invited to explore this rela- tively large volume that attempts to synthesize the most current knowledge base for this important category of steroid hormones. I use the plural because the nomencla- ture has indeed undergone a metamorphosis akin to what our experimental animals undergo. In the 1950s when the Butenandt and Karlson laboratory first crystallized and characterized ecdysone, it was thought to be the insect molting hormone since when injected into experimental insects the result was molting. Later, it was found that ecdysone was converted to 20-hydroxyecdysone in tissues peripheral to the prothoracic glands through the mediation of an ecdysone monooxygenase and we then believed that ecdysone was only the precursor for 20-hydroxyecdysone. However, a reading of the older literature as well as new work has demonstrated convincingly that ecdysone does have regulatory roles of its own, and perhaps joins with 20-hydroxyecdysone to initiate the molting process. Further, it must be remembered that the research of Ulrich Clever and Peter Karlson on Chironomus was the very first to demonstrate that a steroid hormone acts at the nuclear level and

v vi Preface indeed Karlson wrote several theoretical papers subsequently urging endocrinolo- gists working on mammals to accept that view. In time they did! Despite the molecular focus of this meeting there remains much research to do at the physiological and biochemical levels, let alone in the areas of chemistry and crystallogra- phy, to further define the roles of these hormones that serve more organisms on this planet than any other steroid hormone. For at least current usage, I suggest that we use the term principal molting hormone for 20-hydroxyecdysone and the terms substrate for the molting hormone and/or hormone for ecdysone (although many Drosophila geneticists still use the term ecdysone for 20-hydroxecdysone; but that is another problem). The Ghent meeting was beautifully organized by Professor Smagghe who seemed to be around 24 h a day insuring that everything proceeded correctly and on schedule. He deserves our thanks for that, and also for this volume that he lab- ored over for many months. I personally believe that this was among the very best of the many ecdysone workshops I have attended and the book is certainly among the most comprehensive outcomes of any of the workshops. I was most honored to have been chosen to give the Karlson lecture on our work on the Halloween genes. I realize that this is not the usual preface in that I haven’t discussed any of the con- tributions, but they are so good that I would do them a disservice by summarizing each in a sentence – they deserve sustained reading and I believe that they will be cited for many years to come. As I approach my 80th birthday after more than half century in this field, I am very pleased to have my new work published here, that I had the chance to attend this very successful meeting in such a very beautiful city, and the opportunity to enjoy the amazing varieties of such fine beers offered to the ‘ecdysonists’ in Ghent.

Chapel Hill, North Carolina, USA Lawrence I. Gilbert September 2008 Contents

Preface ...... v

Color Plates ...... xi

Part I Fundamental Aspects of Ecdysteroid Research: The Distribution and Diversity of Ecdysteroids in Animals and Plants

1 Phytoecdysteroids: Diversity, Biosynthesis and Distribution ...... 3 Laurence Dinan, Juraj Harmatha, Vladimir Volodin, and René Lafont

2 Diversity of Ecdysteroids in Animal Species ...... 47 René Lafont and Jan Koolman

3 Crustacean Ecdysteroids and Their Receptors ...... 73 Penny M. Hopkins

4 Synthetic Ecdysteroidal Compounds ...... 99 Josep Coll Toledano

Part II In the Post-Genomic Era, Ecdysteroid Genetic Hierarchies in Insect Growth and Reproduction

5 Ecdysteroids and Ecdysteroid Signaling Pathways During Insect Oogenesis ...... 127 Luc Swevers and Kostas Iatrou

6 Regulation of Sciarid DNA Puffs by Ecdysone: Mechanisms and Perspectives ...... 165 Nadia Monesi, Juliana Aparecida Candido-Silva, Maria Luísa Paçó-Larson, and Jorge Cury de Almeida

vii viii Contents

7 The Ecdysteroids’ Effects in the Control of Cell Proliferation and Differentiation ...... 185 David Siaussat, Patrick Porcheron, and Stépahne Debernard

8 Applications of RNA Interference in Ecdysone Research ...... 205 Garry N. Hannan, Ronald J. Hill, Skarlatos G. Dedos, Luc Swevers, Kostas Iatrou, Anjiang Tan, R. Parthasarathy, Hua Bai, Zhaolin Zhang, and Subba R. Palli

Part III Role of Cross Talk of Genes and Growth Factors in Ecdysteroid Titers and Signalling

9 The Function and Evolution of the Halloween Genes: The Pathway to the Arthropod Molting Hormone ...... 231 Lawrence I. Gilbert and Kim F. Rewitz

10 Recent Studies on Prothoracic Gland Cell Growth and Ecdysteroidogenesis in the Silkworm, Bombyx mori ...... 271 Shi-Hong Gu and Ju-Ling Lin

11 Diversity in Factors Regulating Ecdysteroidogenesis in Insects ...... 283 Sandrien Van de Velde, Liesbeth Badisco, Elisabeth Marchal, Jozef Vanden Broeck, and Guy Smagghe

12 20-Hydroxyecdysone, Juvenile Hormone and Biogenic Amines: Mechanisms of Interaction in Control of Drosophila Reproduction Under Normal and Stressful Conditions ...... 317 Nataly Gruntenko and Inga Rauschenbach

Part IV Ecdysteroids Function Through Nuclear and Membrane Receptors

13 The Structure and Function of Ecdysone Receptors ...... 335 Isabelle M.L. Billas, Christopher Browning, Michael C. Lawrence, Lloyd D. Graham, Dino Moras, and Ronald J. Hill

14 The Multidimensional Partnership of EcR and USP ...... 361 Vincent C. Henrich, Josh Beatty, Heike Ruff, Jenna Callender, Marco Grebe, and Margarethe Spindler-Barth

15 Functional Analysis of Ecdysteroid Receptor from Drosophila melanogaster “In Vitro” ...... 377 Anca Azoitei, Heike Ruff, Christian Tremmel, Sabine Braun, and Margarethe Spindler-Barth Contents ix

16 Intracellular Localization of the Ecdysteroid Receptor ...... 389 Klaus-Dieter Spindler, Katarzyna Betan´ska, Claudia Nieva, Tomasz Gwóz´dz´, Joanna Dutko-Gwóz´dz´, Andrzej Oz˙yhar, and Margarethe Spindler-Barth

17 Genomic and Nongenomic Actions of 20-Hydroxyecdysone in Programmed Cell Death ...... 411 Masatoshi Iga and Sho Sakurai

18 Rapid, Non-Genomic Responses to Ecdysteroids and Catecholamines Mediated by a Novel Drosophila G-Protein-Coupled Receptor...... 425 Peter D. Evans, D.P. Srivastava, and V. Reale

Part V Ecdysteroids in Modern Agriculture, Medicine, Doping and Ecotoxicology

19 Ecdysone Receptors of Pest Insects – Molecular Cloning, Characterisation, and a Ligand Binding Domain-Based Fluorescence Polarization Screen ...... 447 Lloyd D. Graham, Wynona M. Johnson, Donya Tohidi-Esfahani, Anna Pawlak-Skrzecz, Marianne Bliese, George O. Lovrecz, Louis Lu, Linda Howell, Garry N. Hannan, and Ronald J. Hill

20 SAR and QSAR Studies for In Vivo and In Vitro Activities of Ecdysone Agonists ...... 475 Yoshiaki Nakagawa, Robert E. Hormann, and Guy Smagghe

21 Ecdysone Receptor-Based Gene Switches for Applications in Plants ...... 511 Venkata S. Tavva, Randy D. Dinkins, Glenn B. Collins, and Subba R. Palli

22 Ecdysteroids and Their Importance in Endocrine Disruption Research ...... 539 Thomas Soin, Tim Verslycke, Colin Janssen, and Guy Smagghe

23 Innovative and Future Applications for Ecdysteroids ...... 551 René Lafont and Laurence Dinan

Index ...... 579 Color Plates

Fig. 3.3 Comparison of the growth of a regenerating limb bud from Uca pugilator to amounts of protein in the limb bud and total circulating ecdysteroids, as well as predominant circulating ecdysteroid. At top are molt cycle stages (see legend for Fig. 3.1). Solid line represent growth curve of limb buds as R-values (see legend for Fig. 3.1 for description). PA = ponasterone A and 20E = 20-hydroxyecdysone (Redrawn from Hopkins, 1993) (see also page 82)

xi xxiiii CColorolor PPlateslates maller. Response is maller. page 86) ecdysteroid receptor (UpEcR) to two isoforms of the retinoid-X-receptor (UpRXR). Surface isoforms of the retinoid-X-receptor (UpRXR). Surface ecdysteroid receptor (UpEcR) to two see also Uca pugilator Comparative binding affinities of binding affinities Comparative

Plasmon binding assay used. Sf9 expressed UpEcR (100nM) interacted with verious concentrations of UpRXR isoforms – larger and s concentrations of UpRXR isoforms – larger UpEcR (100nM) interacted with verious Plasmon binding assay used. Sf9 expressed cells ( amount of binding compared to control flow relative Fig. 3.4 Fig. CColorolor PPlateslates xxiiiiii

(A) 20E (B) HR3 VMP30 E75A (1-2(1- h) (vitellin( FTZ-F1-F1 EcR/USP membrane)

Early EcR FTZ-F1 (B1) E75A HR3 (6 h) E75C EcR,USP E75C GATAβ HR3B (12 h) (6 h) Early-Late SH3 HR3C ESP (18 h) (24 h) HNF-4a Chorion genes (24 h)

Late HR3A (48-72 h) ESP Choriogenesis

Vitellogenesis

Fig. 5.1 Regulation of silkmoth (Bombyx mori) oogenesis during pharate adult and adult development by 20E produced by the prothoracic glands. Panel a: Induction of vitellogenesis by rising titers of 20E in the hemolymph. Indicated are different phases in the hormone response: early (repression of FTZ-F1, transient induction of B1-EcR and E75C, permanent induction of E75A), early-late (induction of HR3B and HR3C) and late (induction of HNF-4A, HR3A and ESP). Expression of ESP (egg-specific protein, a yolk protein precursor produced by the follicular epithelium; Sato and Yamashita, 1991) marks the initiation of vitellogenesis. Panel b: Induction of choriogenesis by declining titers of 20E in the hemolymph. At the top of the cascade is shown the nuclear receptor FTZ-F1, which plays a pivotal role in the regulation of developmental events during low titers of ecdysone (Broadus et al., 1999). As in Drosophila, induction of FTZ-F1 may be triggered by changes in the relative levels of the HR3 and E75 receptors (Swevers et al., 2002). Expression of FTZ-F1 is followed by the repression of HR3 and EcR/USP (at 6–12 h), induction of E75C, GATAβ and SH3 (at 12 h), repression of ESP (at 12–18 h) and induction of chorion gene expression (at 18–24 h). Because the vitellin membrane protein VMP30 is co-expressed with FTZ- F1, it was hypothesized that FTZ-F1 is a positive regulator of the expression of VMP30 (Kendirgi et al., 2002). Note that the deduction of the regulatory cascades that trigger vitellogenesis and choriogenesis in the silkmoth is based on expression patterns of mRNAs and remains to be inves- tigated by functional analysis (see also page 130) xxiviv CColorolor PPlateslates

Fig. 5.2 Model to explain the hierarchy of ecdysone response genes regulating apoptosis of stage 8 and 9 follicles in Drosophila melanogaster. Upper Panel: Complete nutrition induces normal development of follicles during oogenesis. In this case, just the Z1 isoform of BR-C is expressed in the follicle cells at stage 8. E75B suppresses E75A expression to prevent apoptosis. Middle Panel: Injection of 20E induces apoptosis in stage 8 and 9 follicles. 20E injection results first in induction of the Z2 and Z3 isoforms of BR-C which in turn decrease E75B and increase E75A expression. While E75B is an apoptosis inhibitor, E75A is an apoptosis inducer. Lower Panel: Starvation induces apoptosis in stage 8 and 9 follicles. During starvation, ecdysone concentrations increase and the Z2 and Z3 isoforms of BR-C become expressed in the follicle cells to suppress E75B and activate E75A expression. The increase in E75A results in induction of apoptosis. JH can counteract the effects of starvation by interference with the increase in ecdysone concentration and by stimulation of the expression of E75B (Reprinted from Terashima and Bownes, (2006). E75A and E75B have opposite effects on the apoptosis/development choice of the Drosophila egg chamber. Cell Death Differ. 13, 454–464. With permission from Macmillan.) (see also page 140)

Fig. 6.4 The BhEcR colocalizes with RNA polymerase II at DNA puff forming sites. Chromo some C from Bradysia hygida larvae at age E7 labeled with antibodies anti-BhEcR (red) and anti-RNA Pol II (green). Yellow signals indicate colocalization of both antibodies. From left to right, images of the same field were captured by confocal microscopy at increasing magnifications. The bars at the bottom left of each picture correspond, from left to right, to 10 μm, 5 μm and 1 μm, respectively (see also page 175) CColorolor PPlateslates xxvv

Fig. 7.2 Protocol of synchronization by hydroxyurea in the IAL-PID2 cell line. A high degree of synchrony was reached when cells were exposed to two consecutive hydroxyurea treatments at 1 mM for 36 h spaced 16 h apart leading to a massive arrest of cells at the transition G1/S (90%). (a) Only 2 h after the removal of the drug, 70% of cells were recovered in S. This cohort of cells began to enter into G2 at 10 h then kept on progressing through the cell cycle to re-enter progres- sively into G1 after 18 h. (b) Under these conditions, 20E at physiological concentration induced an inhibition of cell growth by an arrest of 90% of the cells in G2/M phase. In the bubble of each figures, corresponding cytometry profiles are presented (see also page 190) xxvivi CColorolor PPlateslates

Fig. 7.10 Schematic representation of control of cell proliferation and differentiation by ecdysteroïds. At the S transition S/G2, the 20E binds to EcR/USP heterodimeric complex and induces a maxi- Period of 20E mum induction of these two sensitivity partners then HR3. This 20E EcR USP to 20E signaling pathway is responsi- ble for G2/M arrest of cells by inhibiting the expression of B cyclin and is also involved HR3 G2/M long term in the morpho- logical differentiation of cells through an increase in the synthesis of β tubulin (see also page 198) B Cyclin

β Tubulin Arrest in G2/M

Differentiation

Fig. 8.3 Dose-dependent decrease in expression of BmEcR at increasing concentrations of BmEcR dsRNA. Panel A: CAT reporter assay. CAT activities are compared with that observed for the reporter construct in the absence of cotransfected dsRNA (100%). Panel B: western blot assay. The membrane was probed for the presence of BmEcR (upper) and BmUSP (lower) proteins. MW markers are indicated at the right (see also page 215) CColorolor PPlateslates xxviivii

Fig. 8.4 Dose-dependent decrease in expression of BmUSP at increasing concentrations of BmUSP dsRNA. Panel A: CAT reporter assay. CAT activities are compared with that observed for the reporter construct in the absence of cotransfected dsRNA (100%). Panel B: western blot assay. The membrane was probed for the presence of BmEcR (upper) and BmUSP (lower) proteins. MW markers are indicated at the right (see also page 215) xxviiiviii CColorolor PPlateslates

Fig. 8.5 Phenotypes observed after knock-down of EcR, TcE75, TcHR4, TcHr39 or Tcbr. dsRNA was injected at 24 h after ecdysis into the final instar larval stage and the phenotypes observed are shown in comparison with control. The larvae injected with TcEcR (b), TcE75 (b) or TcHR4 (c) dsRNA died during quiescent stage. In general, the dsRNA injected larvae were smaller and darker than the control larvae injected with malE dsRNA. About 50% of the larvae injected with TcHR39 successfully pupated but had problems with wing development (d). The larvae injected with Tcbr had problems undergoing larval-pupal ecdysis, the exuvium remained attached to the body (e) and these insects showed antennae, wings, legs and compound eyes similar to those found in adults (f). Scale bar: 1 mm (see also page 220) CColorolor PPlateslates xxixix

Fig. 9.1 Scheme of 20-hydroxyecdysone (20E) biosynthesis in Drosophila. Multiple arrows indicate an uncharacterized pathway, the Black Box. Yellow shade delineates the areas of the sterol that are involved in the transformation to the next compound (see also page 236) xxxx CColorolor PPlateslates

Fig. 9.2 In situ expression of the Halloween genes disembodied, shadow and phantom during late embryonic and larval stages. Shown are stage 17 embryos (a, e and i), late second instar (b, f and j), and both early (c, g and k) and late (d, h and l) third instar brain-ring gland complexes. Note the down regulation of the expression of all three genes between the late second and early third instars and their subsequent up regulation between the early and late third instars. RG, ring gland; Br, brain; VG, ventral ganglion (Data on embryonic dib expression from Chavez et al., 2000. Data on larval dib and sad expression from Warren et al., 2002. Data on phm expression from Warren et al., 2004) (see also page 239) CColorolor PPlateslates xxxixi

Fig. 9.3 Scheme of 20-hydroxyecdysone (20E) biosynthesis in Drosophila with gene names, gene products (enzymes) and CYP designations (see also page 241) xxxiixii CColorolor PPlateslates

W E Fat Body 6 E quivalents ( 6 50 E20MO activity 4

4 m g/ml hemolymph)

25 2 expression (arbitray units) 2 Commitment

shade Peak

0 0 0 1234567890123456 Day of fifth larval instar Day of pupal-adult development

W E 100 Midgut 600 E quivalents ( 6 75 E20MO activity 400 4 m

50 g/ml hemolymph)

Commitment Peak expression (arbitray units) 200 2 25 shade

0 0 1234567890123456 Day of fifth larval instar Day of pupal-adult development

Fig. 9.4 Developmental changes in Manduca shade expression in the fat body and midgut during the fifth instar and through day 6 of pupal-adult development. Expression was analyzed by qPCR and values are means +/−S.E.M. (Data from Rewitz et al. 2006b). Ecdysteroid titer data and enzyme activity are from the Gilbert and Smith laboratories with references given in the above cited publication. W, wandering; E, ecdysis (see also page 244) CColorolor PPlateslates xxxiiixiii

Fig. 9.6 Conservation of microsynteny for phm in Drosophila melanogaster (Diptera), Apis mellifera (Hymenoptera) and Daphnia pulex (Crustacea). In these species, CYP18A1 and phm are paralogs closely linked on the same chromosome (see also Claudianos et al., 2006). Arrows indicate transcriptional orientation of genes on the genome DNA sequence (horizontal lines). The size of the chromosomal regions is shown in parenthesis but distances are not to scale (see also page 255) xxxivxiv CColorolor PPlateslates ), a nvd ( neverland adjacent to mation of 20E (Yoshiyama et al., 2006). mation of 20E (Yoshiyama in Drosophilidae) is page 258) spok see also and 2007) ( Anopheles and species of the Sophophoran subgenus and Drosophila (see Fig. 9.7), Daphnia Drosophila in ydrocholesterol (7dC) which is the first step in the for ydrocholesterol (7dC) which is the first spo ) genes in CYP307A cholesterol to 7- deh cholesterol to 7- -like ( -like spo . The intron-containing gene ( Daphnia pulex and Conservation of microsynteny for of microsynteny Conservation

Anopheles gambiae of in the conversion to be involved gene believed with permissionfrom Sztal et al. Symbols otherwise as in Fig. 9.6 (Data on Drosophilids modified Fig. 9.8 Fig. CColorolor PPlateslates xxxvxv

Fig. 9.11 Conservation profiles of ecdysteroidogenic CYP products of the Halloween genes and CYP6 proteins with the predicted substrate recognition sites (SRSs) 1–6. Note that the area encompassing SRS2 and SRS3 is shown as 2/3. n = number of aligned sequences. — indicates the position of the conserved heme-binding domain (Reproduced from Rewitz et al. 2007) (see also page 264) xxxvixvi CColorolor PPlateslates

Fig. 10.5 Immunoperoxidase-stained whole mounts of silkworm salivary glands (a, b), and cor- pora allata (c, d) showing the incorporation of BrdU. (a) Salivary gland from day 0 last instar larvae incubated in 50 μl Grace’s medium containing BrdU. (b) Salivary gland from day 0 last instar larvae incubated in 50 μl Grace’s medium containing BrdU and 10% hemolymph. (c) Corpus allatum from day 0 last instar larvae incubated in 50 μl of control medium containing BrdU. (d) Corpus allatum from day 0 last instar larvae incubated in 50 μl prothoracic gland- preconditioned medium. Each incubation was maintained for 2 days, and the number of BrdU-labeled cells was counted after a 2-day incubation. BrdU-labeled cells are identified by the intense black precipita- tion of diaminobenzidine. Scale bar, 75 μm (Adapted from Gu, 2006) (see also page 279) Fig. 13.2 Two types of USP LBD structures. Depicted are the LBD structures of HvUSP (in blue) and TcUSP (in brown). (a) The activation helices H12 (in red and orange for HvUSP and TcUSP, respectively) adopt an antagonist conformation. The global fold of the two types of USP structures is conserved, but significant structural differences occur, consistent with the ligand-binding status of the domain (phospholipid-bound HvUSP and ligand-free TcUSP). The phospholipid bound to HvUSP is shown in a ball representation with atoms coloured as follows: carbon atoms magenta, oxygen atoms red and phosphorus atom orange. The ligand-binding pocket is shown as a light blue semi-transparent surface. Helices H6 and H11 of HvUSP are replaced by loops in TcUSP. An important shift in the positioning of helix H3, and to a lesser degree of helix H1, can be observed. (b) Enlarged view of the region corresponding to helices H6 and H11 of HvUSP that in the case of TcUSP are reorganized into two loops (L6 and L11, respectively) that are folded onto the surface the LBD, stabilizing the apo conformation. The colour scheme of the activation helices and the bound ligand atoms follows that of Panel (a) (see also page 341) xxxviiixviii CColorolor PPlateslates

Fig. 13.3 The ligand-binding domains of EcR/USP. (a) The overall structures of the EcR/USP LBD heterodimer in complex with an ecdysteroid are depicted in two orientations for Heliothis virescens (Hv) and Tribolium castaneum (Tc), insect species belonging to the orders Lepidoptera and Coleoptera, respectively. The EcR/USP heterodimerization interface is conserved between the different species. However, differences are seen between that of the lepidopteran/dipteran EcR/ USP and those of other insects. Globally, the EcR/USP LBD heterodimer is more compact and more symmetric in lepidopteran and dipteran species (exemplified here by H. virescens) as compared to that of other insects (exemplified here by T. castaneum) as explained in the text. HvEcR and HvUSP are shown as dark green and blue ribbons respectively, and TcEcR and TcUSP as light green and brown ribbons respectively). The activation helices H12 of HvEcR and TcEcR are depicted as a red and orange helical ribbon, respectively. (b) Enlarged view of the L9-10 loop region of HvEcR and TcEcR LBDs in the context of their heterodimer with the corresponding USP LBD partner. The differences both in sequence (a two amino-acid insertion) and in conformation are indicated for the loop L9-10 of lepidopteran/dipteran EcRs (exemplified here by HvEcR) as compared to that of EcRs of other insects (exemplified here by TcEcR). Residues of loop L9-10 and a few residues of loop L7-8 that interact with this element are shown in stick representation, with carbon atoms in dark green and light green for HvEcR and TcEcR respectively, oxygen atoms in red and nitrogen atoms in blue. Yellow dotted lines indicate hydrogen bonds between residues of the protein and the colour scheme of the ribbons follows that of Panel (a) (see also page 343) CColorolor PPlateslates xxxixxix

Fig. 13.4 Opened-out view of the major and ancillary pockets of BtEcR LBD. In these Panels the pocket surface (grey) has been open out into two “shells” by slicing in a plane parallel to that of the page. Residues lying beyond the plane of slicing are shown in each Panel, with carbon atoms in orange if they highly conserved across all insect species, otherwise with carbon atoms in green. PonA, bound in the major pocket of the LBD, is shown in stick representation with carbon atoms shown in black. Oxygen atoms in both PonA and BtEcR are shown in red and nitrogen atoms in blue. Red dotted lines indicate hydrogen bonds between the protein and the ligand. For clarity, side chain or main chain groups of individual residues are omitted from the Figure where these are not involved in the formation of the pocket surface (see also page 346) Fig. 13.5 Residues in the region of the ancillary pocket of the EcR LBD. (a) The existence of an ancillary pocket, as observed in the BtEcR LBD, is intimately linked to the conformation of the side chains of residues (in particular HvEcR-E309/BtEcR-E199 and HvEcR-Y437/BtEcR-Y325) that fill or do not fill the volume between helices H1 and H8. HvEcR and BtEcR LBDs are depicted as dark green and yellow ribbons, respectively. Residues are shown in a stick representation, with carbon atoms in grey and yellow for HvEcR and BtEcR, respectively, oxygen atoms in red and nitrogen atoms in blue. The ligand- binding pocket of HvEcR and TcEcR are represented by a blue and yellow translucent molecular surface, respectively. In HvEcR the entrance to the cavity corresponding to the ancillary pocket of BtEcR is occluded by the side chains of HvEcR-E309 and HvEcR-Y437. (b) In the TcEcR LBD, the volume corresponding to the ancillary pocket of BtEcR is occupied by water molecules located between helices H1 and H8 and is not occluded by side chain atoms. The ligand-binding pocket was calculated without the three water molecules and is represented by a light green translucent molecular surface. The TcEcR LBD is depicted as a light green ribbon. The residues E330 and Y456 and the bound ponA ligand are shown in a stick representation with carbon atoms in green, oxygen atoms in red and nitrogen atoms in blue. The three water molecules are represented by red balls (see also page 347) Fig. 13.6 Stereoviews of water-mediated interaction network between the ecdysteroid and EcR residues. (a) In the case of 20E bound to the HvEcR LBD, the hydrogen-bond interaction network involves the 20-, 22-, and 25-hydroxyl groups of the 20E alkyl side-chain and three structural water molecules. (b) In the case of ponA bound to the TcEcR LBD, the interaction network involves the 20- and 22-hydroxyl groups of ponA alkyl side chain and up to five structural water molecules. The HvEcR and TcEcR LBDs are depicted as dark green and light green ribbon, respectively, with individual atoms within the LBD and ligand shown in stick representation and with carbon atoms coloured in light blue and green, respectively, oxygen atoms in red, nitrogen atoms in blue and sulphur atoms in yellow. The water molecules are represented by red balls. Hydrogen bonds are indicated by yellow and pink dotted lines in (a) and (b) respectively (see also page 349) xxxxiixxii CColorolor PPlateslates

Fig. 13.7 Inter-species comparison of side-chain packing in walls of the EcR ligand-binding pocket. (a) B. tabaci EcR with ponA bound, (b) H. virescens EcR with ponA bound, (c) H. virescens EcR with BYI06830 bound and (d) T. castaneum EcR with ponA bound. The orientations of the EcR-LBDs in Panels (a–d) are identical, with the dashed line indicating a common line of in-plane register. Panel (a) shows (in rod representation) the side chains of B. tabaci residues I230, M272, T304, L308, M389, T393 and V404, whilst Panels (b) and (c) show their counterparts of these residues in H. virescens EcR (namely M342, V384, V416, L420, Q503, M507 and L518, respectively), and Panel (d) their counterparts in T. castaneum (namely I361, M403, T435, L439, Q520, M524 and L535, respectively). Atoms in these residues are coloured as follows: carbon atoms green, oxygen atoms red, nitrogen atoms blue and sulphur atoms yellow, with the position of the Cα atom of each residue being highlighted by a sphere for clarity. Also shown in each panel is the ligand-binding pocket (coloured transparent gold and calculated with a 1.2 Å probe radius), encap- sulating the respective bound ligand. Ligand atoms are coloured according to the same scheme as that of the EcR side chains, except that carbon atoms are shown in black. Panels (e), (f), (g) and (h) show the atomic packing of the ligand-binding cavity wall in the vicinity of the residues highlighted to the left of the ligand in Panels (a–d) respectively, but viewed from the interior of the binding pocket and looking in the direction of the arrows. Atoms are shown in CPK representations and include both main and side chain atoms from all residues contributing to the immediate wall of the binding pocket in the direction of the view. The site of the protrusion into the protein atomic volume of the ligand-binding pocket walls of H. virescens EcR and T. castaneum EcR in the vicinity of the alkyl tail of bound ponA (see text) is indicated by an asterisk in Panels (b), (d), (f) and (h). The site of deeper protrusion into the protein atomic volume of ponA-bound HvEcR LBD in the vicinity of the C4 atom of the ligand is indicated by a solid star in Panel (b); this deeper protrusion with respect to the pocket walls of ponA-bound BtEcR LBD and TcEcR LBD is a consequence of the substitution of BtEcR I230 and TcEcR I361 by methionine in HvEcR (M342, see text) (see also page 350) CColorolor PPlateslates x xxxiiixxiii

Fig. 13.8 Stereoviews of the differential conservation of the pocket residues HvEcR-V384 and HvEcR-V416 in Lepidoptera. (a) HvEcR-V384, which is replaced by methionine in non-lepidopteran EcRs, allows the specific side chain conformation of HvEcR-L420 and a closer van der Waals’ interaction with the ecdysteroid 22-hydroxyl group. (b) HvEcR-V416, which is replaced by threonine in TcEcR and BtEcR, is also closer to the ecdysteroid 22-hydroxyl group due to the concomitant shift of helix H7 towards the interior of the receptor pocket. The stereoviews depict HvEcR in blue, TcEcR in green and BtEcR in yellow. Amino acid residues of HvEcR, TcEcR and BtEcR are shown in stick representation colour with carbon atoms in light blue, green and yellow for the three species respectively and oxygen atoms in red. The ligands 20E (for HvEcR) and ponA (for TcEcR and BtEcR) are shown in a stick representation with carbon atoms in blue (20E), green (ponA in TcEcR) or wheat (ponA in BtEcR) and oxygen atoms in red. The activation helix H12 is also shown as a red ribbon (see also page 351) xxxxivxxiv CColorolor PPlateslates

Fig. 13.9 Flexible region in the EcR LBD exploited by DBH compounds. (a) Ribbon diagram showing the superimposition of the structures of HvEcR-LBD in complex with 20E (dark green ribbons) and with BYI06830 (orange ribbons). The view is restricted to the region differing the most between the two EcR-LBDs that includes helices H2, H6, H7 and the β-sheet. 20E and BYI06830 are shown in stick representation with carbon atoms in cyan and light grey, respectively, oxygen atoms in red and nitrogen atoms in blue. (b) Superimposition of 20E and BYI06830 bound to HvEcR. Oxygen atoms are shown in red, nitrogen atoms in blue, 20E carbon atoms in cyan and BYI06830 carbon atoms in olive. The three structural water molecules observed in the structure of 20E-bound HvEcR, shown by red balls, superimpose well with the region of the B-ring of the DBH compound (see also page 353) CColorolor PPlateslates x xxxvxxv

Fig. 13.10 The steric relationship between the alkyl side chain of ponA in the binding pocket of the B. tabaci ecdysone receptor LBD and the side chain sulphydryl group of BtEcR C394. The distance between the van der Waals’ surfaces of the terminal side chain carbon atom of the steroid and the sulphur atom of the cysteine residue C394 in the pocket wall is 0.5 Å. This cysteine residue is strictly conserved among all known EcR sequences (see also page 356) xxxxvixxvi CColorolor PPlateslates

Steroid

a c b GPCR

TMTM 3 TM 5 4 TM 6 cyclic AMP TM 1 Nuclear receptor TMTM 2 7 at plasma Calcium α γ membrane β MAPK GDP Nuclear receptor

mRNA

Protein Synthesis

Fig. 18.1 Steroid hormone signaling. (a) Conventionally steroid hormones are thought to be lipophylic hormones and to be able to enter the cell easily. They then bind with a range of ‘nuclear’ receptors which transport them into the nucleus where the complex binds to the DNA and initiates changes in gene expression. Non-genomic actions of steroids are very rapid and mediate changes in second messenger levels without any changes in gene expression or protein synthesis. (b) It has been postulated that these rapid non-genomic effects could be mediated via steroid interactions with the nuclear binding proteins that migrate to become closely associated with the plasma membrane. Once activated the complex is then proposed to bring about rapid changes in second messenger levels. (c) It has also been proposed that steroids may interact with specific seven transmembrane-spanning GPCRs to activate second messenger pathways via the activated G-proteins (see also page 426) CColorolor PPlateslates x xxxviixxvii

Rapid Non-Genomic EDA Effects CG18314 G1 G2

MAPKinase Cyclic AMP E AKT (PI3Kinase ) Early Genomic Effects Development ER USP Late

Fig. 18.3 The orphan Drosophila GPCR (CG18314) responds to both ecdysteroids and to the catecholamine dopamine and has been renamed DmDopEcR (Srivastava et al., 2005). DmDopEcR shows ‘agonist-specific coupling’ whereby the catecholamine dopamine can couple the receptor to an increase in cyclic AMP levels together with an activation of the PI3Kinase pathway, as judged by the increased phosphorylation of Akt. Ecdysteroids can produce rapid non-genomic effects through this receptor by coupling it to the activation of the MAPKinase pathway and by inhibiting the actions of dopamine. The receptor has a much higher affinity for ecdysteroids compared to dopamine (see also page 435)

Fig. 20.4 CoMFA electrostatic and steric field contour plots for DAH toxicity toward C. suppressalis, S. exigua, and L. decemlineata. The diacylhydrazine with X = 2-Cl and Y = 2,3-di-Cl is depicted. Green/yellow polyhedra represent sterically-favored/disfavored regions; blue/red polyhedra represent regions where positive charge is favored/disfavored (Reproduced with the permission of Wiley-VCH Verlag GmbH & Co. KGaA) (see also page 489) Fig. 20.5 CoMFA hydrogen-bond field contour plots for toxicity toward C. suppressalis, S. exigua , and L. decemlineata. The diacylhydrazine with X = 2-Cl and Y = 2,3-di-Cl (Fig. 20.3) is depicted. Green/yellow polyhedra represent hydrogen-bond acceptor-favored/disfavored regions; blue/red polyhedra represent hydrogen-bond donor-favored/disfavored regions (Reproduced with the permission of Wiley-VCH Verlag GmbH & Co. KgaA) (see also page 490)

Fig. 20.7 Stereoviews of CoMFA fields with tebufenozide. (a) The contours are shown to surround regions where a positive (blue) and a negative (red) electrostatic potential increases the activity. (b) The contours are shown to surround regions where a higher steric bulk increases (green) or decreases (yellow) the activity (Reproduced with the permission of Elsevier) (see also page 499) CColorolor PPlateslates x xxxixxxix

Fig. 20.8 The docking of tebufenozide to the cavity of BmEcR -LBD. Hydrophobicity gradient is shown from blue (lowest/–0.1) to brown (highest/0.1). Brown indicates receptor surface that is most hydrophobic, and blue indicates the surface area that is least hydrophobic. Hydrophobic amino acid residues (M409, M503, L507, L515) are surrounding the t-butyl group (Reproduced with the permission of Elsevier) (see also page 501)

Fig. 20.9 The docking of tebufenozide to the LBD of BmEcR with CoMFA steric fields (Reproduced with the permission of Elsevier) (see also page 501) xxll CColorolor PPlateslates

Fig. 20.10 Stereoview of the tebufenozide in the binding cavity of modeled BmEcR with the neighboring hydrophilic amino acids (Reproduced with the permission of Elsevier) (see also page 502) CColorolor PPlateslates xxlili

Fig. 21.3 Methoxyfenozide-inducible AtZFP11 phenotype in Arabidopsis and tobacco. Seed collected from the transgenic Arabidopsis and tobacco plants expressing AtZFP11 gene under the control of CfEcR + LmRXR two-hybrid gene switch were plated on agar media supplemented with different concentrations of methoxyfenozide. (a) Wild-type Arabidopsis seedlings growing on agar media. (b) T3 transgenic Arabidopsis seed germinated on agar media containing DMSO and 50 mg/l kanamycin. (c) T3 transgenic Arabidopsis seed germinated on agar media containing

400 nM methoxyfenozide and 50 mg/l kanamycin. (d–g) Microscopic pictures of the T3 transgenic Arabidopsis seedlings collected from different methoxyfenozide treatments. (d) 0 (DMSO), (e) 16,

(f) 80 and (g) 400 nM. (h–m) Photographs of the T2 transgenic tobacco seedlings and plants treated with different concentrations of methoxyfenozide. (h) Wild-type tobacco seedlings growing on agar media. (i) T2 tobacco seed germinated on agar media containing DMSO and 300 mg/l kanamycin. (j) T2 tobacco seed germinated on agar media containing 400 nM methoxyfenozide and 300 mg/l kanamycin. (k–m) Transgenic tobacco plants growing in the greenhouse. (k) DMSO, (l) and (m) 400 nM methoxyfenozide (see also page 533) xxliilii CColorolor PPlateslates

crude plant extracts (elixirs or powders)

purified ecdysteroids ecdysteroids + other substances (or ecdysteroid mixtures) (e.g. isoflavones) and/or proteins

transdermal applicator and cosmetic uses

Fig. 23.2 More or less pure phytoecdysteroids as dietary supplements for farm and domesticated animals and for humans as food supplements for healthy or sick people for use as additives in cosmetics (see also page 571)