Development of a competitive binding assay system with recombinant estrogen receptors from multiple species

In the current study, we developed a new system using full-length recombinant baculovirus-expressed estrogen receptors which allows for direct comparison of binding across species. Estrogen receptors representing five vertebrate classes were compared: human estrogen receptor alpha (hER alpha), quail estrogen receptor alpha (qER alpha), alligator estrogen receptor alpha (aER alpha), salamander estrogen receptor alpha (sER alpha), and fathead minnow estrogen receptor alpha (fhER alpha). Saturation binding analyses indicated 17 beta-estradiol (E2) dissociation constants (Kd) were 0.22 +/- 0.02 nM for hER alpha, 0,28 +/- 0.04 nM for sER alpha, 0.44 +/- 0.04 nM for aER alpha, 0.58 +/- 0.10 nM for qER alpha, and 0.58 +/- 0.05 nM for fhER alpha. Binding specificity to each of the receptors was evaluated using E2, dihydrotestosterone (DHT), corticosterone (C), and ethinylestradiol (EE). E2 and EE were strong binders in all species with IC50's ranging from 0.65 nM with hER alpha to 1.01 nM with sER alpha for E2 and from 0.68 nM with sER alpha to 1.20 nM with qERa for EE. DHT was a weak binder with IC50's ranging from 3.3 mu M with hER alpha to 39 mu M with fhER alpha, and C did not bind any of the receptors at concentrations up to 100 mu M. This system provides a convenient in vitro approach for directly comparing chemical binding to estrogen receptors across multiple species Without the need to sacrifice animals. Published by Elsevier Ireland Ltd.