Characterization of the Estrogen Receptor and Its Dynamics in MCF-7 Human Breast Cancer Cells Using a Covalently Attaching Antiestrogen*

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Abstract
A covalently attaching antiestrogen, tamoxifen aziridine [TA; (Z)-(1-[4-(2-[N-aziridinyl] ethoxy)phenyl])1,2-diphenyl-1-butene], was used to analyze the structure and dynamics of the estrogen receptor in MCF-7 human breast cancer cells. The labeling of receptor with [3H]TA is specific, being blocked only by estrogens and antiestrogens; the labeling is very efficient in that TA labels covalently the same number of receptors that are labeled reversibly by estradiol. In cells exposed to [3H]TA for 1 h, most of the covalently associated radioactivity is found in the 0.6 M KCl extract of the nuclear fraction; this receptor has an apparent MW of 63,000 .+-. 2000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI [isoelectric point] of 5.7 by gel isoelectric focusing in the presence of 8 M urea. The MW and pI of cytosol receptor labeled with [3H]TA are identical. In cells labeled with [3H]TA (20 nM) for 1 h and then exposed to a chase of 10-6 M estradiol, [3H]TA-labeled nuclear receptor disappears with a half-life of 4 h. Analysis of nuclear receptor by sodium dodecyl sulfate-gels during the chase period reveals that this loss reflects a decrease in the 63,000 MW species; no significant quantities of lower MW TA-labeled fragments are observed in the nuclear, cytosol, or membrane fractions. Affinity labeled receptor interacts with several monoclonal antibodies to MCF-7 estrogen receptor, and it can be purified extensively by immunoadsorbent chromatography. TA has a low affinity (8% that of tamoxifen) for microsomal antiestrogen-binding sites that are distinct from the estrogen receptor, but TA reacts reversibly, rather than covalently, with these sites. The findings of similar MW and pI for soluble cytosol and nuclear extracted receptors, under strongly denaturing and disaggregating conditions, reveal that nuclear localization of receptor after ligand binding is not associated with major structural alterations in the receptor component labeled by TA. In addition, the receptor, even when occupied by a covalently attached ligand, is rapidly turned over in these cells.