Final Report Summary - JHRECEPTOR (Structure and function of the insect Juvenile hormone receptor) Summary description of the project objectivesJuvenile hormone (JH) plays critical roles in insects and crustaceans that constitute most of the animal species and biomass on our planet. JH is a sesquiterpenoid unique to arthropods, and its synthetic analogs have been in use since the 1970's to control insect pests and disease vectors. Yet, the molecular mechanism of JH action remained unknown until our breakthrough finding in 2007 that the developmental effects of JH and its insecticidal mimics are mediated by the Methoprene-tolerant protein (Met), a putative JH receptor.The goal of this project was to prove that Met is a bona fide JH receptor and to understand its function in insect development and reproduction. This objective has been achieved. Three specific aims were proposed to study (1) Structure of the putative hormone-binding domain of Met, (2) Function of partner proteins that interact with Met, and (3) Mechanism of transcriptional activation by Met in response to JH. The first aim is being pursued in collaboration with the Australian Commonwealth Scientific and Industrial Research Organisation (CSIRO) where Dr. Marek Jindra (the Fellow) was seconded for 24 months of the Outgoing phase of the project. This collaboration is still ongoing and it benefits from CSIRO funding and from the expertise of Dr. Ron Hill and his team in crystal structure resolution of insect nuclear receptors. Within this project, three methodological approaches were developed, namely1) protocol for expression and purification of the active JH receptor complex, 2) new and efficient ligand-binding assay for JH and related compounds, and 3) cell-based reporter system to measure JH-receptor mediated transcriptional activation by JH and related compounds. The main results 1) Definition of the hormone-binding pocket of the putative JH receptor Met. Evidence for high-affinity binding of the Met protein to JH and its synthetic mimics. Identification of specific amino acid residues required for Met to bind JH and to engage in a JH-dependent interaction with its partner protein Taiman (Tai). Results were covered in an original paper (Charles et al., PNAS, 2011) and an influential review article (Jindra et al., Annu Rev Entomol, 2013).2) The first successful expression and purification of the active Met-Tai JH receptor complex (unpublished results, ongoing research). 3) Demonstration that the JH receptor complex components, Met and Tai, mediate the effects of JH on reproduction (Smykal et al., Insect Biochem Mol Biol, 2014); and evidence that Met interacts with the circadian clock genes to induce exit from a reproductive diapause (Bajgar et al., PNAS, 2013). 4) Demonstration of two phases of insect postembryonic development. Despite traditional views, we have shown that the juvenile character of early larvae is independent of JH and its receptor; only later during development larvae gain competence to metamorphose and thus require JH signaling to postpone metamorphosis (Smykal et al., Dev Biol, 2014). 5) Primary genetic evidence that the capacity of Met/Gce to bind JH is essential for the protein function in vivo and for JH-dependent transcriptional activation. These results establish Met/Gce as a bona fide JH receptor (Jindra et al., PLoS Genet, under revision). 6) A cell-based reporter system has been deviced to measure JH-dependent transcriptional activation of target genes. Using this assay, the essential components of the JH receptor complex: the Met/Gce and Tai proteins were determined (Jindra et al., PLoS Genet, under revision). Further, evidence was obtained that the JH receptor discriminates between stereisomers of JH and its newly tested synthetic agonists (unpublished results, ongoing research). 7) In a side branch of the project, aimed at a closely related hormonal signaling pathway, the Ecdysoneless protein, which was originally implicated in biosynthesis of the insect steroid hormone ecdysone, was identified as a novel mRNA splicing factor. The results explained that the steroid hormone deficiency in ecdysoneless mutants is caused by intron retention in mRNA encoding one of the essential steroidogenic enzymes (Claudius et al., PLoS Genet, 2014). Conclusions, potential impact and use of the results Our unequivocal identification of the JH receptor resolves a long-standing problem in arthropod biology. This knowledge is essential for understanding of how insect development and reproduction are regulated, and how insecticides mimicking JH exert their effects. As this research has been primarily aimed at gaining new fundamental information, other scientists are the first and most obvious target group that will utilize these results. Indeed, our results have already had a visible impact on the scientific community, with 153 citations to date of the six peer-reviewed papers published within the three years of the project duration. It appears that our findings have inspired a dynamic wave of current research in the field of insect endocrinology. This project has yielded new powerful methods, namely an innovated in-vitro hormone binding assay for JH, and a cell-based system where potential JH agonists and newly discovered antagonists can be efficiently tested. Collaboration aimed at mechanism of action of these compounds has already been established between the Fellow and other institutions in the Czech Republic and abroad (South Korea, United States). Obtaining the crystal structure of the JH receptor has proven extremely difficult, but our effort to resolve it is still ongoing. Together with the outcome of the in-vitro ligand-binding assays and the cell-based system, this information could lead to designing of environmentally friendly insecticides that would not harm beneficial insect species. In the future, such compounds could be adopted by chemical industry and could replace the relatively non-selective JH mimics that are presently in use to protect our crops, stored products, and households from insect pests and disease vectors.