Development of an in vitro binding assay for ecdysone receptor of mysid shrimp (Americamysis bahia).

A global effort has been made to establish screening and testing methods that can identify the effects of endocrine-disrupting chemicals (EDCs) on invertebrates. The purpose of our study was to develop an in vitro receptor binding assay for ecdysone receptor (EcR) in mysid shrimp (Americamysis bahia). We cloned mysid shrimp EcR cDNA (2888 nucleotides) and ultraspiracle (USP) cDNA (2116 nucleotides), and determined that they encode predicted proteins of length 570 and 410 amino acids, respectively. The deduced amino acid sequences of these proteins shared 36-71% homology for EcR and 44-65% for USP with those of other arthropods. Phylogenetic analysis revealed that mysid shrimp EcR was classified into an independent cluster together with the EcRs of another mysid species, Neomysis integer and the cluster diverged early from those of the other taxonomic orders of crustaceans. We then expressed the ligand-binding domains (DEF regions) of mysid shrimp EcR (abEcRdef) and USP (abUSPdef) as glutathione S-transferase (GST)-fusion peptides in Escherichia coli. After purifying the fusion peptides by affinity chromatography and removing the GST labels, we subjected the peptides to a ligand-receptor binding assay. [(3)H]-ponasterone A did not bind to abEcRdef or abUSPdef peptides alone but bound strongly to the abEcRdef/abUSPdef mixture with dissociation constant (K(d))=2.14 nM. Competitive binding assays showed that the IC(50) values for ponasterone A, muristerone A, 20-hydroxyecdysone, and α-ecdysone were 1.2, 1.9, 35, and 1200 nM, respectively. In contrast, the IC(50) values for two dibenzoylhydrazine ligands (tebufenozide and chromafenozide) were >1.0 × 10(5)nM. The intra- and inter-assay coefficient of variation values for the IC(50) values of 20-hydroxyecdysone were 14.7% (n=5) and 16.1% (n=8), respectively. Our results indicate that the binding assay with a mixture of abEcRdef and abUSPdef can be used to screen compounds with a broad range of binding affinities for crustacean EcRs.