|Jones, Grace - DEPT BIOLOGY, UNIV KY|
|Jones, Davy - DEPT BIOLOGY, UNIV KY|
|Sapa, Agnes - WROCLAW MED. UNIV, POLAND|
|Wozniak, Mietek - WROCLAW MED. UNIV, POLAND|
Submitted to: FEBS Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 11, 2006
Publication Date: October 1, 2006
Citation: Jones, G., Jones, D., Teal, P.E., Sapa, A., Wozniak, M. 2006. The retinoid-x receptor ortholog, ultraspiracle, binds with high nanomolar affinity to an endogenous morphogenetic ligand. FEBS Journal. 1-14. Interpretive Summary: Hormone receptors are important keys for development of ligand agonists and antagonists. Among insects such receptors could be critical tools for development of viable "third generation" insect-selective pesticides. A critical hormone upon which a number of effective "third generation" insect-selective pesticides have been developed is Juvenile Hormone (JH). However, no cloned nuclear receptor that binds to any JH analog or related farnesoid is available. Scientists from the Department of Biology University of Kentucky in collaboration with Center for Medical Agricultural and Veterinary Entomology USDA-ARS have been studying ligand binding of JH related compounds to the USP receptor of Dorsophilla. Their research resulted in documentation that the cloned USP receptor has high binding affinity for the JH precursor methyl farnesoate. The results data identify USP as a potential practical target of selective, high affinity compounds based on a methyl farnesoid structure which may be of significance in developing new alternatives to classical pesticides.
Technical Abstract: The in vivo ligand binding function and ligand-binding activity of the Drosophila melanogaster RXR ortholog, ultraspiracle, toward natural farnesoid products of the ring gland were assessed. Using an equilibrium fluorescence binding assay, farnesoid products in the juvenile hormone biosynthesis pathway, and their epoxy derivatives, measured for their affinity constant for ultraspiracle. Farnesol, farnesal, farnesoic acid and juvenile hormone III exhibited high nanomolar to low micromolar affinity, which in each case decreased upon addition of an epoxide across a double bond of the basic farnesyl structure. Similar analysis of the substitution on C1 of methyl ether, alcohol, aldehyde, and carboxylic acid showed that each conferred weaker affinity than that provided by the methyl ester. Attention was thus focussed for a ring gland farnesoid product that possesses the features of methyl ester and lack of an epoxide. A secreted product of the ring gland was identified possessing these features, that exhibited an affinity for ultraspiracle (40 nanomolar) of similar strength as that of RXR for 9-cis retinoic acid. Mutational analysis of the amino acid residues with side chains extending into the ligand binding pocket cavity (and not interacting with secondary receptor structures nor extending to the receptor surface to interact with coactivators, corepressors or receptor dimer partners) determined that the mutation C472A/H475L most and strongly reduced ultraspiracle binding to this ring gland product and juvenile hormone III, with lesser effect on other ring gland farnesoids and no significant effect on binding by (the unnatural to Drosophila) JH I. This mutated ultraspiracle, expressed from a transgene daily upon heat shock, was deficient compared to a wild type transgene in rescuing survival to adult eclosion of lethal null allele (usp2) males. This is the first report for either vertebrate or invertebrate RXR of reduction in vivo receptor function on account of a ligand binding pocket mutation that significantly reduces receptor binding to an established endogenous compound. Altogether, these results support a model in which ultraspiracle performs a developmental function of binding to an endogenous ligand.