Characterization of the Subunit Nature of Nuclear Estrogen Receptors by Chemical Cross-Linking and Dense Amino Acid Labeling*

Abstract

Chemical cross-linking and dense amino acid labeling of estrogen receptors were used to characterize the subunit nature and rate of turnover of nuclear 5S estrogen-receptor complexes. When MCF-7 human breast cancer cells are incubated with [3H]estradiol or [3H]antiestrogen [.alpha.-[4-pyrrolidinoethoxyl]phenyl-4-hydroxy-.alpha.''-nitrostilbene (C1628M), or (Z)-1-[4-(2-[N-aziridinyl]ethoxy)phenyl]1,2-diphenyl-1-butene (tamoxifen aziridine)] and nuclear estrogen-receptor complexes are extracted with 0.6 M KCl and then chemically cross-linked with the cross-linker 2-iminothiolane, the cross-linked receptor complexes sediment as a 5.4S species on 3 M urea-containing sucrose gradients, while the noncross-linked species are 4S. Sodium dodecyl sulfate-polyacrylamide gel analyses of these cross-linked nuclear receptor complexes labeled with the covalently attaching ligand [3H]tamoxifen aziridine reveal a species of about 130,000 MW, while the noncross-linked or the cross-linked but mercaptan-cleaved receptor is 65,000 MW. Both receptor species are also detectable by interaction with an immunoadsorbent column containing antireceptor monoclonal antibody. For analyses of receptor turnover rates, cells exposed for different time periods to medium containing dense (15N, 13C, and 2H) amino acids were labeled with [3H]antiestrogen [1-[4-(2-dimethylaminoethoxy) phenyl]1-[4-hydroxyphenyl]2-phenyl-but-1-(2)ene (trans-hydroxytamoxifen) or C1628M] or [3H]estradiol, and salt-extracted nuclear estrogen receptors were analyzed on sucrose gradients. The normal density 5S form shifted to a broader, more dense peak at 2 and 4 h and finally, by 8-10 h, to a more dense, sharply sedimenting species. The time course of this shift is the same as that seen for the 4S urea-dissociated nuclear receptor form (t1/2 [half life] .apprx. 4 h), suggesting that the 5.4S nuclear receptor is composed of 2 species which turn over at the same rate. Apparently, the nuclear 5S receptor complex consists of 2 similarly sized units, which turn over with similar half-lives. Evidently, the 5S nuclear receptor complex is a homodimer of 2 4S, 65,000 MW monomers.