| In recent years, there has been heightened concern that environmental exposure to estrogen mimicking chemicals, known as estrogenic endocrine disruptors (EEDs) may cause adverse health effects in humans and wildlife. Many of the effects of EEDs are mediated through the estrogen receptor (ER). Although the physiological actions of the ER are conserved among species, variation within the amino acid sequence of ligand binding domains suggests that species may exhibit different responses and sensitivities to EEDs.; Species-specific responses to EEDs were first examined in competitive binding assays using glutathione-S-transferase (GST)-ER fusion proteins from several different species. Fusion proteins consisted of the ER D, E, and F domains of human alpha (GST-hERαdef), mouse alpha (GST-mERαdef), chicken (GST-cERdef), green anole (GST-aERdef) and rainbow trout ERs (GST-rtERαdef). Although, the fusion proteins exhibited similar binding preferences for many EEDs, several differences were observed. The GST-rtERαdef, which has the greatest amino acid sequence variability in its ligand binding domain compared to hERαdef, exhibited the most striking differences compared to the other GST-ERs. The ability of several of these EEDs to induce gene expression mediated by the various ERs was then examined in MCF-7 cells transiently transfected with Gal4-ERdef chimeric receptors. Overall, the data in the gene expression assay correlated with the competitive binding results. However, there were examples where EEDs bound to GST-ERs but were unable to significantly induce ER-mediated gene expression. Intriguingly, the E2-induced response mediated by Gal4-rtERαdef was 2 orders of magnitude lower compared to the other receptors examined. Much of this effect was due to temperature, since when compared to hERα the 280-fold difference at 37°C was reduced to only 9-fold at 20°C. A comparison of their ligand binding pockets identified two conservative amino acid substitutions in rtERα (M317, I496) and hERα (L349, M528). The effect of these substitutions on ligand binding and transactivation was examined by constructing reciprocal mutants. The rtERαdef M317L:I496M mutant exhibited a hERα phenotype with increased E2 binding affinity and transactivation ability at higher temperatures. The hERα L349M:M528I mutant also exhibited a modest trend towards adopting the rtERα phenotype. The lack of a complete exchange of phenotypes indicates that factors outside of the ligand binding pocket are also involved.; Taken together these results demonstrate that ERs from different vertebrate species exhibit different affinities and transactivation responses to EEDs. Since few differences were observed, these data do not preclude the use of a single surrogate ER to examine estrogenic responses for all vertebrate species. The present report also highlights the impact of temperature when comparing functional characteristics of proteins from poikilothermic species, such as rainbow trout, and humans. |
