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Title: Ligand binding pocket function of drosophila USP is necessary for metamorphosis

item JONES, GRACE - University Of Kentucky
item Teal, Peter
item HENRICH, VINCENT - University Of North Carolina
item KRZYWONOS, ANNA - Wroclaw Medical University
item SAPA, AGNES - Wroclaw Medical University
item WOZNIAK, MIETEK - Wroclaw Medical University
item SMOLKA, JOHN - University Of Kentucky
item JONES, DAVY - University Of Kentucky

Submitted to: General and Comparative Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/19/2012
Publication Date: 1/15/2013
Citation: Jones, G., Teal, P.E., Henrich, V., Krzywonos, A., Sapa, A., Wozniak, M., Smolka, J., Jones, D. 2013. Ligand binding pocket function of drosophila USP is necessary for metamorphosis. General and Comparative Endocrinology. 182:73-82.

Interpretive Summary: The predominant theory in insect physiology is that juvenile hormone is the sole sesquiterpene hormone released into the circulatory system of insects and that juvenile hormone is the sole sesquiterpene that regulates biological functions and events including reproductive competence, diapause, and even division of labor in social insects. Scientists at the University of Kentucky, Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville Fl., Wroclaw Medical University, Poland and Univ. of North Carolina at Greensboro, NC conducted studies on other horones that might regulate these functions. They identified methyl farnesoate as a circulating hormone in fruit flies and mosquitoes and demonstrated that indeed levels of endogenous methyl farnesoate are both sufficient and necessary to load the receptor and thus regulate metamorphosis from the larva to the adult form. In establishing a methyl farnesoate/RXR endocrine axis that is distinct from JH/MET protein, the study offers a long sought solution to issues that have bedeviled the field of insect endocrinology. There are a number of physiological processes that have been attributed, for lack of an alternative, to JH (and more recently its receptor MET), but for which the JH/MET axis has not provided a satisfactory mechanistic explanation. The results define new venues for study in insect physiology and provide a focal point for development of new bio-safe methods of pest insect control. Additionally, the research has broad ranging implications for comparative physiology and endocrinology as well as on the evolution of hormone signaling systems.

Technical Abstract: The widely accepted paradigm that epoxidized methyl farnesoates (“juvenile hormones,” JHs) are the principle sesquiterpenoid hormones regulating insect metamorphosis was assessed in Drosophila melanogaster. GC-MS analysis showed that methyl farnesoate, rather than methyl epoxyfarnesoate (= JH III), was the predominant circulating methyl farnesoid prior to metamorphosis. The circulating concentration of methyl farnesoate (reaching nearly 500 nM), was easily high enough on a kinetic basis to load the Drosophila nuclear hormone receptor RXR (also known as “ultraspiracle,” USP), whereas the circulating concentrations of JH III and methyl bisepoxyfarnesoate were not. The hypothesis that the ligand pocket of RXR/USP necessarily binds an endogenous ligand for differentiation of the immature to the adult was tested with USP/RXR mutated at residue that normally extends a side chain into the ligand binding pocket. An equilibrium binding assay confirmed that the mutation (Q288A) strongly altered methyl farnesoate interaction with RXR/USP, while a heterologous cell-line transfection assay confirmed that the mutation did not allosterically alter the transcriptional response of the ultraspiracle/ecdysone receptor heterodimer to ecdysteroid signaling. Transgenic wildtype RXR/USP driven by the cognate natural promoter rescued null animals to develop to the adult inside a normally formed puparium, while in contrast animals transgenically expressing instead the ligand pocket mutant exhibited developmental derrangement at the larval to pupal transition, including failure to sclerotize and properly form a puparium. Other point mutations to the pocket strongly reducing affinity for methyl farnesoate similarly disrupted the larval to pupal metamorphosis. These results suggest that normal larval to pupal maturation in this mecopteran model insect requires the involvement of a distinct endocrine axis of RXR/USP binding to its own endogenous terpenoid ligand.