Estrogens play a critical role in mammary gland development, bone homeostasis, reproduction, and the pathogenesis of
breast cancer by activating
estrogen receptors (ERs) alpha and beta.
Ligand-activated ER stimulates the expression of target
proteins by interacting with specific DNA sequences:
estrogen response elements (EREs). We have demonstrated that the ERE sequence and the nucleotide sequences flanking the ERE impact
ERalpha binding affinity and transcriptional activation. Here, we examined whether the sequence of the ERE modulates
ERalpha conformation by measuring changes in sensitivity to
protease digestion.
ERalpha, occupied by
estradiol (E2) or
4-hydroxytamoxifen (4-OHT), was incubated with select EREs and digested by
chymotrypsin followed by a Western analysis with
antibodies to
ERalpha. ERE binding increased the sensitivity of
ERalpha to
chymotrypsin digestion. We found both
ligand-specific and ERE-specific differences in
ERalpha sensitivity to
chymotrypsin digestion. The ERE-mediated increase in
ERalpha sensitivity to
chymotrypsin digestion correlates with E2-stimulated transcriptional activity from the same EREs in transiently transfected cells. Transcriptional activity also correlates with the affinity of
ERalpha-ERE binding in vitro. Our results support the hypothesis that the ERE sequence acts as an allosteric effector, altering ER conformation. We speculate that ERE-induced alterations in
ERalpha conformation modulate interaction with co-regulatory
proteins.