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

Characterization of the Subunit Nature of Nuclear Estrogen Receptors by Chemical Cross-Linking... We have used chemical cross-linking and dense amino acid labeling of estrogen receptors to characterize the subunit nature and rate of turnover of nuclear 5S estrogenreceptor complexes. When MCF-7 human breast cancer cells are incubated with [3H]estradiol or [3H]antiestrogen [a-[4-pyrrolidinoethoxy] phenyl-4-hydroxy-a-nitrostilbene (CI628M) or (Z)- l-[4-(2-[iV-aziridinyl]ethoxy)phenyl]l,2-diphenyl-l-butene (tamoxifen aziridine)] and nuclear estrogen-receptor complexes are extracted with 0.6 M KC1 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 mol wt, while the noncross-linked or the cross-linked but mercaptan- cleaved receptor is 65,000 mol wt. Both receptor species are also detectable by interaction with an immunoadsorbent column containing antireceptor monoclonal antibody. For analyses of receptor turnover rate, cells exposed for different time periods to medium containing dense (16N, 13C, and 2H) amino acids were labeled with [3H]antiestrogen [l-[4-(2- dimethylaminoethoxy) phenyl ] 1-[ 4-hydroxypheny 1 ]2-pheny 1 - but-l-(2)ene (trans-hydroxytamoxifen) or CI628M] 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 (t½∼4h), suggesting that the 5.4S nuclear receptor is composed of two species which turn over at the same rate. We conclude from these cross-linking and density shift experiments that the nuclear 5S receptor complex consists of two similarly sized units, which turn over with similar half-lives. These data provide strong evidence that the 5S nuclear receptor complex is a homodimer of two 4S, 65,000 mol wt monomers. (Endocrinology117: 515–522,1985) This content is only available as a PDF. Author notes * This work was supported by NIH Grants CA-18119 and CA-31870 (to B.S.K.) and CA-02897, American Cancer Society Grant BC86, and Abbott Laboratories (to G.L.G.). A preliminary report of portions of this work was presented at the Seventh International Congress of Endocrinology, Quebec, July 1984 (Ref. 46). Copyright © 1985 by The Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Endocrinology Oxford University Press

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

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References (40)

Publisher
Oxford University Press
Copyright
Copyright © 1985 by The Endocrine Society
ISSN
0013-7227
eISSN
1945-7170
DOI
10.1210/endo-117-2-515
Publisher site
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Abstract

We have used chemical cross-linking and dense amino acid labeling of estrogen receptors to characterize the subunit nature and rate of turnover of nuclear 5S estrogenreceptor complexes. When MCF-7 human breast cancer cells are incubated with [3H]estradiol or [3H]antiestrogen [a-[4-pyrrolidinoethoxy] phenyl-4-hydroxy-a-nitrostilbene (CI628M) or (Z)- l-[4-(2-[iV-aziridinyl]ethoxy)phenyl]l,2-diphenyl-l-butene (tamoxifen aziridine)] and nuclear estrogen-receptor complexes are extracted with 0.6 M KC1 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 mol wt, while the noncross-linked or the cross-linked but mercaptan- cleaved receptor is 65,000 mol wt. Both receptor species are also detectable by interaction with an immunoadsorbent column containing antireceptor monoclonal antibody. For analyses of receptor turnover rate, cells exposed for different time periods to medium containing dense (16N, 13C, and 2H) amino acids were labeled with [3H]antiestrogen [l-[4-(2- dimethylaminoethoxy) phenyl ] 1-[ 4-hydroxypheny 1 ]2-pheny 1 - but-l-(2)ene (trans-hydroxytamoxifen) or CI628M] 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 (t½∼4h), suggesting that the 5.4S nuclear receptor is composed of two species which turn over at the same rate. We conclude from these cross-linking and density shift experiments that the nuclear 5S receptor complex consists of two similarly sized units, which turn over with similar half-lives. These data provide strong evidence that the 5S nuclear receptor complex is a homodimer of two 4S, 65,000 mol wt monomers. (Endocrinology117: 515–522,1985) This content is only available as a PDF. Author notes * This work was supported by NIH Grants CA-18119 and CA-31870 (to B.S.K.) and CA-02897, American Cancer Society Grant BC86, and Abbott Laboratories (to G.L.G.). A preliminary report of portions of this work was presented at the Seventh International Congress of Endocrinology, Quebec, July 1984 (Ref. 46). Copyright © 1985 by The Endocrine Society

Journal

Endocrinology – Oxford University Press

Published: Aug 1, 1985

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