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Uterine Estrogen Receptor Interaction with Estrogen-Responsive DNA Sequences in Vitro: Effects of Ligand Binding on Receptor-DNA Complexes

Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences Research Triangle Park, North Carolina 27709

Address requests for reprints to: Dr. Kenneth S. Korach, National Institute of Environmental Health Sciences, Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, P.O. Box 12233, Research Triangle Park, North Carolina 27709.

Abstract

Interaction between the mouse estrogen receptor (ER) and its responsive element (ERE) were examined in vitro using tissue extracts and an oligonucleotide containing a Vitellogenin A2 ERE (VRE) in a gel retardation assay. Three specific complexes were formed when nuclear extracts were prepared from estrogen agonist-treated uteri or when ligandfree ER from cytosol was used. ER protein was associated with the three complexes, as demonstrated by their ability to bind anti-ER antibody H222. Specific complexes were formed only when double stranded nonheat-denatured VRE was used. Mutation of the ERE in VRE abolished specific binding. The complexes formed by nuclear extracts were qualitatively identical when obtained from mice treated with a variety of estrogenic compounds with different potencies. Nuclear extracts obtained from mice treated with an estrogen antagonist LY117018 formed complexes that migrated slower than the complexes formed by the other estrogenic compounds examined. The dissociation rates (k_–1) and equilibrium dissociation constants (K_d) of the ERVRE complexes formed by estradiol- or estriolbound ER or ligand-free ER were measured and were found to be very similar, although the ligandfree specific ER complexes associate and dissociate less rapidly than those of estradiol- or estriolbound ER. In addition, one of the specific complexes formed by the estriol nuclear extract dissociated more slowly than the equivalent estradiol complex. Heat activating ligand-free ER did not increase its binding to VRE. It appears that the ligand-binding domain of the ER does not exert its regulatory effects at the level of sequence-specific DNA binding, since its occupancy does not alter binding to the ERE. The subtle differences we observed in association and dissociation of multiple complexes when unoccupied ER and ER boundto ligands of varying potencies are compared may reflect differences in ER association with other protein factors that govern transcriptional activity.

Received for publication October 4, 1989. Revision received November 13, 1989. Accepted for publication November 13, 1989.

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