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-loophelix (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. structure modeling | insecticide action | metamorphosis | Tribolium | Drosophila J uvenile hormone (JH) prevents adult transition (metamorphosis) of insect larvae until they have attained an appropriate stage (1, 2), and it typically stimulates oogenesis in adult females (3). How JH achieves its function remains unclear, mainly because a JH receptor has long eluded identification (4). The lipophilic nature of the sesquiterpene JH suggests an intracellular receptor, yet none of the known insect nuclear hormone receptors have been linked with the biological function of JH. A screen for Drosophila mutants resistant to methoprene (5), a JH mimic and a widely used insecticide (6), uncovered the Methoprene-tolerant (Met) protein containing a basic helix-loop-helix (bHLH) motif followed by two Per-Arnt-Sim (PAS) domains (7). Recombinant Drosophila Met was shown to bind JH at physiological (nanomolar) concentrations and to mediate a weak JH-and methoprene-dependent transcriptional activation in vitro (8). However, Met-null mutant flies were viable and fertile (5), leaving the notion that Met is a putative JH receptor unsupported with an anticipated developmental phenotype. Latest reports show that, in Drosophila, Met might functionally overlap with its paralog, encoded by the germ cell-expressed (gce) gene. Gce can increase sensitivity of Met-null mutants to methoprene (9), and only simultaneous loss of both Met and Gce is lethal (10). However, the actual mode of interaction between JH/methoprene and Met or Gce still remains unclear.Knockdown of the single Met gene in the flour beetle Tribolium castaneum induced beetle larvae to pupate before reaching their final instar (11), producing a precocious metamorphosis phenotype similar to that caused by loss of JH itself (12).Conversely, removal of Met precluded inhibition of adult ...

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A nimal growth and development rely on coordinated functions of body organs to balance energy consumption and storage under optimal or challenging conditions, such as food scarcity or pathogen attack. When adverse conditions are temporary, animals survive by lengthening development and postponing maturity, reducing their body size (16,65). In contrast, prolonged starvation or chronic inflammation may exhaust energy reserves, causing death (32, 69). The key nutrient-sensing, metabolic, and immune-signaling pathways are functionally conserved across phyla. In the fruit fly Drosophila melanogaster, as in vertebrates, insulin/insulin growth factor (IGF) signaling (IIS) primarily regulates growth and metabolic homeostasis (56). The innate immune response of the fly relies on the Toll receptor and the immune deficiency (Imd) pathways (35). In response to bacterial or fungal infection, the Toll and Imd pathways engage downstream transcription factors of the NF-B-like family (Dif, Dorsal, and Relish) that in turn trigger synthesis of a battery of antimicrobial peptides (AMPs).It has become evident that the metabolic and immune system responses are tightly interconnected. Selective activation of Toll signaling in the Drosophila fat body attenuates IIS to reduce nutrient stores and overall growth (14). Mycobacterial infection causes energy wasting due to the systemic activation of the Forkhead transcription factor FOXO (15). Activation of the Jun N-terminal kinase (JNK) pathway is a well-established example of antagonistic regulation of IIS in both flies and vertebrates (29). Chronic inflammation, accompanied by high JNK activity, is at the heart of the metabolic syndrome and type 2 diabetes (54). Interestingly, genetic removal of IIS pathway components, starvation, and DNA damage all induce AMP expression in the absence of infection. This infection-independent AMP upregulation requires both FOXO (5) and Relish (30,70), further supporting the notion that maintenance of metabolic and innate immune balance are intimately linked.The Drosophila fat body and gut integrate metabolic and inflammatory signals to coordinate energy use. The fly alimentary tract digests and absorbs nutrients, while the fat body metabolizes and stores them. In addition, both organs can mount an immune response. The fat body requires both the Toll and Imd pathways for AMP production, whereas the gut immune response relies solely on Imd signaling (35,62). In contrast to the low basal immune activity within the unchallenged fat body, the presence of commensal bacteria in the gut lumen keeps the gut epithelium permanently alert with activated nuclear Relish. In this case, additional factors, such as the homeobox gene caudal (49), control AMP expression. Tightly regulated production of positive and negative Imd modulators prevents overgrowth of pathogens while preserving beneficial commensal bacteria. Uncontrolled inflammatory response of the intestinal epithelium has a dramatic impact on gut physiology and homeostasis in both flies and humans (43,47). A ...

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.