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Dissertation
Molecular Basis of Estrogenic Endocrine
Disruptor-Estrogen Receptor
Interactions: A Comparison Among Species
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-hERadef),
mouse alpha (GST-mERadef), chicken (GST-cERdef), green anole
(GST-aERdef) and rainbow trout ERs (GST-rtERadef). Although,
the fusion proteins exhibited similar binding preferences for
many EEDs, several differences were observed. The GST-rtERadef,
which has the greatest amino acid sequence variability in its
ligand binding domain compared to hERadef, 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-rtERadef was 2 orders of magnitude lower compared to
the other receptors examined. Much of this effect was due to
temperature, since when compared to hERa the 280-fold difference
at 37oC was reduced to only 9-fold at 20oC. A comparison of
their ligand binding pockets identified two conservative amino
acid substitutions in rtERa (M317, I496) and hERa (L349, M528).
The effect of these substitutions on ligand binding and transactivation
was examined by constructing reciprocal mutants. The rtERadef
M317L:I496M mutant exhibited a hERa phenotype with increased
E2 binding affinity and transactivation ability at higher temperatures.
The hERa L349M:M528I mutant also exhibited a modest trend towards
adopting the rtERa 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.
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