Endocrinology, Vol 126, 2190-2195, Copyright © 1990 by Endocrine Society

ARTICLES

Cell-free interaction of the estrogen receptor with mouse uterine nuclear matrix: evidence of saturability, specificity, and resistance to KCl extraction

DA Metzger and KS Korach
Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina 27710.

An integral part of the mechanism of estrogen action is the interaction of estrogen receptor (ER) complexes with specific nuclear acceptor sites to effect alterations in genomic expression. The localization of nuclear acceptor sites has been in question, but an increasing body of indirect evidence implicates the nuclear matrix. To assess the binding characteristics of [3H]estradiol-receptor complexes (3HER) to nuclear matrix, ER from ovariectomized mice was partially purified by ammonium sulfate precipitation and incubated under cell-free conditions with mouse uterine nuclear matrix at 4 C. The binding capacity of the nuclear matrix was determined to be 36.4 +/- 5.7 fmol/100 micrograms DNA, with a Kd of 0.23 +/- 0.03 nM. Binding to nuclear matrix sites was specific, as determined by the ability of increasing concentrations of unlabeled ER complexes to inhibit binding of 3HER. Spleen, used as a nontarget tissue, contained fewer binding sites (n = 4.07 fmol/100 micrograms DNA) than matrix from liver (n = 14.2). The binding affinity was the same in all three tissues. Injection of animals with estradiol before death was associated with loss of assayable nuclear matrix binding sites, implying occupancy of sites by ER in vivo. Unbound receptor (R) also demonstrated the ability to bind to uterine matrix (n = 40.2 +/- 2.7 fmol/100 micrograms DNA; Kd = 0.26 +/- 0.05 nM) as well as to competitively inhibit the binding of 3HER complexes. However, heat-inactivated receptor displayed no binding or competing activity, nor did the progesterone receptor. The two forms of the receptor can be functionally distinguished by extraction with 0.6 M KCl; 43% of ER, but no R, were resistant to KCl extraction. These results indicate that nuclear acceptor sites are associated with the nuclear matrix. Furthermore, these sites demonstrate the criteria expected of specific binding sites, i.e. high affinity, limited capacity, hormone receptor, and relative tissue specificity. The apparent association of uncomplexed receptor to nuclear acceptor sites may explain the uterine tissue nuclear localization of ER in the absence of hormone.

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