Exquisite ligand stereoselectivity of a Drosophila juvenile hormone receptor contrasts with its broad agonist repertoire

Abstract

The sesquiterpenoid juvenile hormone (JH) is vital to insect development and reproduction. Intracellular JH receptors have recently been established as basic helix-loop-helix transcription factor (bHLH)/PAS proteins in Drosophila melanogaster known as germ cell-expressed (Gce) and its duplicate paralog, methoprene-tolerant (Met). Upon binding JH, Gce/Met activates its target genes. Insects possess multiple native JH homologs whose molecular activities remain unexplored, and diverse synthetic compounds including insecticides exert JH-like effects. How the JH receptor recognizes its ligands is unknown. To determine which structural features define an active JH receptor agonist, we tested several native JHs and their nonnative geometric and optical isomers for the ability to bind the Drosophila JH receptor Gce, to induce Gce-dependent transcription, and to affect the development of the fly. Our results revealed high ligand stereoselectivity of the receptor. The geometry of the JH skeleton, dictated by two stereogenic double bonds, was the most critical feature followed by the presence of an epoxide moiety at a terminal position. The optical isomerism at carbon C11 proved less important even though Gce preferentially bound a natural JH enantiomer. The results of receptor-ligand-binding and cell-based gene activation assays tightly correlated with the ability of different geometric JH isomers to induce gene expression and morphogenetic effects in the developing insects. Molecular modeling supported the requirement for the proper double-bond geometry of JH, which appears to be its major selective mechanism. The strict stereoselectivity of Gce toward the natural hormone contrasts with the high potency of synthetic Gce agonists of disparate chemistries.

Keywords: Drosophila; basic helix-loop-helix transcription factor (bHLH); development; hormone receptor; insect; juvenile hormone (JH); ligand-binding protein; reproduction; stereoselectivity.

Figure 1.

Structures and names of the tested compounds. A, three known native juvenile hormones in D. melanogaster and the unnatural 10S-JH III enantiomer. B, the native conformation of JH I (top left) and its geometric stereoisomers in both 10R,11S and 10R,11R configurations. C, methyl (2E,6E,10E)-7-ethyl-3,11-dimethyltrideca-2,6,10-trienoate, a nonepoxidated version of JH I, abbreviated as deoxy-JH I. D, carbamate-derived JH mimics. The abbreviated nomenclature (in bold) is preferentially used throughout this study.

Figure 2.

Agonist activities of native insect JHs. A, activation of the JHRE-luc reporter in S2 cells by three JHs known in D. melanogaster and by the lepidopteran JH I. The data (mean from three technical replicates) show a representative example of three or four independent experiments; error bars represent S.D. Average EC₅₀ values were 0.21 ± 0.14 μm for R,S-JH III, 0.37 ± 0.22 μm for JH I, 0.11 ± 0.01 μm for JHB3, and 4.43 ± 0.95 μm for MF. MF was significantly less effective (p < 0.01, t test) than any of the three epoxidated hormones; differences between R,S-JH III and either JH I (p > 0.29; t test) or JHB3 (p > 0.37; t test) were not statistically significant. B, binding of the hormones to the Gce protein in competition assays with R,S-[³H]JH III. Data (mean with error bars representing S.D.) indicate average K_i values of 11.0 ± 2.2 nm (n = 3) for R,S-JH III, 13.8 ± 5.1 nm (n = 5) for JH I, and 83.3 ± 40.8 nm (n = 3) for JHB3; a K_i measured for MF (89.8 ± 26.7 nm) corresponds to a previously determined value (87.9 ± 22.2 nm) (6). Affinities were significantly different (p < 0.01, t test) between the two groups of compounds comprising R,S-JH III and JH I versus JHB3 and MF. C, two-hybrid assay in HEK293T cells reflecting ligand binding to the Gce protein. The data (mean from three technical replicates) show a representative example of three or four independent experiments; error bars represent S.D. Average EC₅₀ values were 58.4 ± 16.9 nm for R,S-JH III, 53.4 ± 13.9 nm for JH I, 0.31 ± 0.15 μm for JHB3, and 0.30 ± 0.06 μm for MF. Differences were significant between the group comprising R,S-JH III and JH I versus JHB3 (p < 0.05, t test) and MF (p < 0.01, t test).

Figure 3.

Agonist activities of JH III enantiomers. A, activation of the JH-inducible JHRE-luc reporter in the Drosophila S2 cell line. The data (mean from three technical replicates) show a representative example of three independent experiments. Although calculated EC₅₀ values (0.93 ± 0.38 μm for R-JH III and 4.46 ± 2.95 μm for S-JH III) indicated about a 4-fold lower activity of the latter enantiomer, the difference was not statistically significant. Comparison of JHRE-luc expression at 1 μm concentration (inset) revealed a 2.2-fold higher activity of R-JH III (*, p < 0.05, t test). The normalized luciferase activities are plotted as -fold increase relative to treatment with solvent alone for which the value is arbitrarily set to 1. B, binding of R-JH III and S-JH III to the Gce protein in vitro as assessed from competition against racemic R,S-[³H]JH III. The data (mean from three measurements for each compound) revealed binding affinities (K_i values) of 4.8 ± 1.3 and 38.3 ± 5.2 nm (p < 0.001, t test) for R-JH III and S-JH III, respectively. C, ligand binding–dependent interaction of Gce and Tai components of the JH receptor complex as assessed in a two-hybrid assay in HEK293T cells. The data (mean from three measurements) indicate EC₅₀ values of 73.1 ± 5.2 and 169.8 ± 5.5 nm (p < 0.001, t test) for R-JH III and S-JH III, respectively. Error bars represent S.D. in all panels.

Figure 4.

Agonist activities of geometric isomers of JH I. A, binding of the native S-(E,E)-JH I, its stereoisomers, and nonepoxidated deoxy-JH I to the Gce protein in vitro as assessed from competition against R,S-[³H]JH III. The data (mean from three measurements for each compound) revealed the binding affinities (K_i values) listed in Table 1. B, ligand binding–dependent Gce–Tai dimerization in the two-hybrid assay in HEK293T cells. The data (mean from three technical replicates) show a representative example of three independent experiments that together indicated EC₅₀ values of 53.4 ± 13.9 nm for S-(E,E), 1.24 ± 0.09 μm for S-(E,Z), and 1.60 ± 0.04 μm for S-(Z,E) JH I isomers; S-(Z,Z)-JH I was inactive. C, induction of JHRE-luc in the Drosophila S2 cell-based reporter assay by the indicated compounds at 1 μm concentration. The data are mean values from four independent experiments (each in three technical replicates). Different letters above the data indicate that activity of the individual compounds differed significantly (p < 0.05) as determined by one-way analysis of variance of log₁₀-transformed data with Tukey's multiple post hoc comparison. Error bars represent S.D. in all panels.

Figure 5.

Activity of the JH I geometric isomers in vivo. A, capacity of the native S-(E,E)-JH I, its stereoisomers, and deoxy-JH I to induce ectopic expression of Kr-h1 mRNA in Drosophila pupae. Animals were treated at the white puparium stage and collected 24 h later. Shown are normalized mean qRT-PCR data from four biological replicates, each comprising three individual pupae for each compound. The mRNA levels are plotted on a logarithmic scale as -fold increase relative to treatment with solvent (acetone) alone for which the value was arbitrarily set to 1. Error bars represent S.D. Different letters above the data indicate that activity of the individual compounds differed significantly (p < 0.05) as determined by one-way analysis of variance of log₁₀-transformed data with Tukey's multiple post hoc comparison. B, effect of the JH I stereoisomers and deoxy-JH I on the ability of flies to complete development. Animals were again treated with the compounds as white puparia (n = 15–20 specimens per treatment).

Figure 6.

Modeling of JH interaction with the Gce protein. A, docking of the native conformations R-JH-III (green) and S-(E,E)-JH I (cyan) and of the JH I isomers S-(E,Z) (yellow), S-(Z,E) (magenta), R-(E,Z) (gray), and R-(Z,E) (pink) in the model of the PAS-B–binding pocket of Drosophila Gce. B, two-dimensional interaction diagrams of the native JH I (left) and JH III (right) with the PAS-B domain of Drosophila Gce. Numbers are amino acid positions within the modeled region; corresponding positions within the Drosophila Gce protein (NCBI Reference Sequence NP_511160.1) are the displayed numbers plus 261 (i.e. Tyr-9 is Tyr-270). C, loss of the hydroxyl group in the mutated Gce^Y270F protein reduced the amount of bound [³H]JH III to 40% of the WT (Gce^WT) protein (n = 8; p < 4.83 × 10⁻¹⁷, t test). Data are mean values; error bars represent S.D. Inset, immunoblot of the in vitro expressed, Myc-tagged Gce proteins used in the binding assay.

Figure 7.

Comparison of agonist activities between carbamate JH mimics and a native JH. A, binding of the insecticide fenoxycarb and of the carbamate juvenoid W330 to the Gce protein determined in a competition assay with R,S-[³H]JH III. The data (mean with error bars representing S.D.) indicate a K_i of 2.8 ± 1.1 nm (n = 3) for fenoxycarb, which differed significantly (t test) from the K_i values of 13.8 ± 5.1 (n = 5) and 17.4 ± 5.7 nm (n = 4) determined for JH I (p < 0.05) and W330 (p < 0.01), respectively. The difference between W330 and JH I was not statistically significant. B, fenoxycarb and W330 were both significantly (p < 0.001, t test) more effective in stimulating Gce–Tai dimerization with EC₅₀ values of 5.4 ± 0.8 (n = 4) and 17.7 ± 4.9 nm (n = 4), respectively. Error bars represent S.D. C, with EC₅₀ values reaching 2.29 ± 0.65 (n = 4) and 5.42 ± 0.41 nm (n = 3), respectively, fenoxycarb and W330 were much stronger than JH I in activating the JHRE-luc reporter in Drosophila S2 cells. Error bars represent S.D. D, fenoxycarb, W330, and JH I all induced Kr-h1 mRNA expression in Drosophila pupae to a similar degree (differences not statistically significant). Animals were treated at the white puparium stage and collected 24 h later. Shown are normalized mean qRT-PCR data from four biological replicates, each comprising three individual pupae; error bars represent S.D. The mRNA levels are plotted as -fold increase relative to treatment with solvent (acetone) alone for which the value was arbitrarily set to 1. E, capacity of the carbamates to prevent normal fly development as assessed in the white puparium bioassay (n = 15–20 specimens per treatment). fenox, fenoxycarb.