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 (hERalpha), quail estrogen receptor alpha (qERalpha), alligator estrogen receptor alpha (aERalpha), salamander estrogen receptor alpha (sERalpha), and fathead minnow estrogen receptor alpha (fhERalpha). Saturation binding analyses indicated 17beta-estradiol (E2) dissociation constants (Kd) were 0.22+/-0.02nM for hERalpha, 0.28+/-0.04nM for sERalpha, 0.44+/-0.04nM for aERalpha, 0.58+/-0.10nM for qERalpha, and 0.58+/-0.05nM for fhERalpha. 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 0.65nM with hERalpha to 1.01nM with sERalpha for E2 and from 0.68nM with sERalpha to 1.20nM with qERalpha for EE. DHT was a weak binder with IC50's ranging from 3.3microM with hERalpha to 39microM with fhERalpha, and C did not bind any of the receptors at concentrations up to 100microM. 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.