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Development of a Competitive Binding Assay System with Recombinant Estrogen Receptors from Multiple Species
Citation:
RIDER, C. V., P. C. HARTIG, M. C. CARDON, AND V. S. WILSON. Development of a Competitive Binding Assay System with Recombinant Estrogen Receptors from Multiple Species. TOXICOLOGY LETTERS. Elsevier Science Ltd, New York, NY, 184(2):85-89, (2009).
Impact/Purpose:
Impact Statement We have developed a simple estrogen receptor binding assay system that uses baculovirus expressed receptor constructs from different species. This system has several advantages over other approaches that have been used in multi-species comparisons because it does not require animal tissue as a receptor source, it does not require a purification step, full-length receptors are used, and it should be compatible with any steroid hormone receptor. This system allows for the direct comparison of xenobiotic binding to estrogen receptors from different species in the future.
Description:
ABSTRACT 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 (hERα), quail (qERα), alligator (aERα), salamander (sERα), and fathead minnow (fhERα). Saturation binding analyses indicated 17β-estradiol (E2) dissociation constants (Kd) were 0.22 ± 0.02 nM for hERα, 0.28 ± 0.04 nM for sERα, 0.44 ± 0.04 nM for aERα, 0.58 ± 0.10 nM for qERα, and 0.58 ± 0.05 nM for fhERα. 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 hERα to 1.01 nM with sERα for E2 and from 0.68 nM with sERα to 1.20 nM with qERα for EE. DHT was a weak binder with IC50’s ranging from 3.3 μM with hERα to 39 μM with fhERα, and C did not bind any of the receptors at concentrations up to 100 μM. 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.
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Development of a Competitive Binding Assay System with Recombinant Estrogen Receptors from Multiple Species