Ecdysone Receptors of Pest Insects–Molecular Cloning, Characterisation, and a Ligand Binding Domain-Based Fluorescence Polarization Screen

EcR- and USP-encoding cDNAs of four pest insects (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were cloned from high-quality lambda cDNA libraries and sequenced. Cognate EcR-USP cDNA pairs were shown to express functional ecdysone receptors in transfected cells. The amino acid sequences of the EcR ligand binding domains (LBDs) were employed in conjunction with those known for other arthropods to construct a phylogenetic tree. Affinity tagged EcR-USP LBD heterodimers were co-expressed efficiently in insect cells using a baculovirus vector. The recombinant EcR and USP DE/F segments from each species associated spontaneously to form heterodimers that bound ecdysteroids with high affinity. An E/F segment pair (constructed only for H. armigera) also associated spontaneously to form a functional heterodimer, but one with ligand binding affinities several times lower than its DE/F counterpart. A fluorescein-inokosterone conjugate was synthesized and used to develop a novel ligand binding assay based on fluorescence polarization. This assay can be used in place of the classical [3H]-ponasterone A binding assay, and is ideally suited to high-throughput screening. The ligand binding data obtained in vitro using recombinant LBD heterodim-ers reflect the ability of agonists to induce ecdysone receptor controlled transgene expression in recombinant mammalian cells; in vitro binding data can also reflect the potency of ligands to act as insecticides.