Ligand-binding properties of a juvenile hormone receptor, Methoprene-tolerant

Abstract

Juvenile hormone (JH) is a sesquiterpenoid of vital importance for insect development, yet the molecular basis of JH signaling remains obscure, mainly because a bona fide JH receptor has not been identified. Mounting evidence points to the basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) domain protein Methoprene-tolerant (Met) as the best JH receptor candidate. However, details of how Met transduces the hormonal signal are missing. Here, we demonstrate that Met specifically binds JH III and its biologically active mimics, methoprene and pyriproxyfen, through its C-terminal PAS domain. Substitution of individual amino acids, predicted to form a ligand-binding pocket, with residues possessing bulkier side chains reduces JH III binding likely because of steric hindrance. Although a mutation that abolishes JH III binding does not affect a Met-Met complex that forms in the absence of methoprene, it prevents both the ligand-dependent dissociation of the Met-Met dimer and the ligand-dependent interaction of Met with its partner bHLH-PAS protein Taiman. These results show that Met can sense the JH signal through direct, specific binding, thus establishing a unique class of intracellular hormone receptors.

Fig. 1.

Tribolium Met and its orthologs bind JH III. (A) Myc-tagged full-length proteins from D. melanogaster (Dm) and T. castaneum (Tc) and the PAS-B region from T. domestica (Td) were translated in vitro (Inset, immunoblot) and incubated with 0.5 pmol of [³H]JH III. Total binding is compared against reticulocyte lysate without DNA (mock). The value obtained for DmMet is significantly higher than mock (Mann–Whitney test, P < 0.05; n = 4). (B) Full-length Tribolium Met was incubated with increasing concentrations of [³H]JH III in the absence (○, total binding) or presence (●, nonspecific binding) of a 100-fold molar excess of unlabeled JH III. Each data point is mean ± SD of two to four assays, and the saturation curve shown is average of four independent experiments. The calculated K_d is 2.94 ± 0.68 nM.

Fig. 2.

The PAS-B domain of Met is necessary and sufficient for JH III binding. (A) Deletion constructs representing the individual domains of Tribolium Met and Taiman proteins tagged with the Myc epitope (black boxes) were translated in vitro (for immunoblot, see Fig. S1). Numbers indicate amino acid positions; 1–516 is the entire Met protein. (B) JH III-binding activities are plotted next to the respective proteins as total radioactivity bound. Values are mean ± SD of several independent repeats (n numbers are in brackets).

Fig. 3.

Met selectively binds JH III and its mimics and mediates the effect of pyriproxyfen in vivo. (A) In vitro-translated Met PAS-B was incubated with [³H]JH III in the absence (○, total binding) or presence (●, nonspecific binding) of a 100-fold molar excess of cold JH III. The saturation curve shown is average of six independent experiments. The calculated K_d is 12.3 ± 0.62 nM. (B) Met PAS-B was incubated with 2 pmol of [³H]JH III in the presence of increasing concentrations of the indicated compounds. The competition curves shown are average of three independent experiments. The calculated dissociation constants (K_i) are 388 ± 52 nM for methoprene and 4.75 ± 0.86 nM for pyriproxyfen; farnesol did not significantly compete for binding. (C) At 3 d after injection with egfp (control) or Met dsRNA, Tribolium pupae were treated with pyriproxyfen and tested for Kr-h1 mRNA expression 12 h later. Inset shows RNAi knockdown of the Met protein in these pupae. Data are mean ± SD from six animals. (D) Kr-h1 mRNA levels were assessed in Tribolium pupae at 8 h after treatment with 0.1 mM solutions of the indicated compounds. Data are mean ± SD from n = 4 pupae; the difference between pyriproxyfen and methoprene is significant at P = 0.03 (Student's t test).

Fig. 4.

Model of the ligand-binding cavity of the Tribolium Met PAS-B domain. (A) Overall structure of Met PAS-B (blue, N terminus; red, C terminus) with the cavity. Position of a hypothetical heterodimeric partner, here represented by Arnt as in the HIF2α–Arnt crystal structure PDB ID 3F1P (22), is shown in gray. (B) A closer view of the pocket relative to the amino acid residues mutated in this study. (C) Docking model of a Met–JH III complex. A hydrogen bond (dotted line) is predicted between the hydroxyl group of Tyr-252 and the epoxide moiety of JH III. Orientation is the same in all models.

Fig. 5.

Ligand-dependent protein interactions of Tribolium Met. Proteins N-terminally tagged with either EGFP (shaded) or the Myc epitope were coexpressed in human HEK293 cells. Methoprene (1 μM) or ethanol was added to cell cultures at 1 h before lysis. Cell lysates were subjected to immunoprecipitation (IP, outlined) with an anti-EGFP serum, and interacting proteins were detected on Western blots (WB) with an anti-Myc antibody. Input panels represent 10% of the initial material. Methoprene disrupted homophilic complexes of full-length Met(1–516) (A) or PAS-B Met(240–516) (B) proteins but not of their V297F mutant versions lacking the JH binding capability. Binding of Met(1–240) bHLH and PAS-A domains to full-length Met was insensitive to methoprene (C). Interaction of full-length Met (D) or its PAS-B domain (E) with Taiman required methoprene and was prevented by mutations that abolish binding of JH.