Comparative analysis of estrogen receptors covalently labeled with an estrogen and an antiestrogen in several estrogen target cells as studied by limited proteolysis

Jonathan F. Elliston, Benita S. Katzenellenbogen

Research output: Contribution to journalArticle

Abstract

Estrogen receptors covalently labeled with the estrogen affinity label [3H]ketononestrol aziridine (KNA) or with the antiestrogen affinity label [3H]tamoxifen aziridine (TAZ) were subjected to limited proteolysis with trypsin, α-chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on 10-20% sodium dodecyl sulfate-polyacrylamide gradient gels followed by fluorography. The similar molecular weights of intact receptors (Mr 66,000 daltons) and the proteolytic digest patterns indicate extensive homology among estrogen receptors from MCF-7 human breast cancer cells, GH4 rat pituitary cells and rat uterus when liganded with estrogen or antiestrogen. Each protease generated a distinctive ladder of estrogen receptor fragments, and the fragmentation patterns were virtually identical for estrogen receptors labeled with estrogen (KNA) or antiestrogen (TAZ). Each protease yielded a relatively "resistant" receptor fragment of about 28,000-35,000 daltons. Trypsin and chymotrypsin at higher concentrations generated a much smaller 6,000-8,000 dalton digest product that still contained the [3H]KNA- or [3H]TAZ-labeled receptor binding site. Moreover, the receptor digest patterns were similar for estrogen receptors from the three different target cells. Our studies suggest considerable structural relatedness among these three estrogen receptors and also indicate that these two affinity labels bind to a similar, perhaps identical, region of the receptor molecule.

Original languageEnglish (US)
Pages (from-to)559-569
Number of pages11
JournalJournal of Steroid Biochemistry
Volume29
Issue number6
DOIs
StatePublished - Jun 1988

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Proteolysis
Estrogen Receptor Modulators
Estrogen Receptors
Estrogens
Affinity Labels
Rats
Photofluorography
Peptide Hydrolases
Ladders
Sodium Dodecyl Sulfate
Uterus
Staphylococcus aureus
Molecular Weight
Gels
Molecular weight
Binding Sites
Cells
Breast Neoplasms
Molecules
ketononestrol aziridine

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Cite this

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abstract = "Estrogen receptors covalently labeled with the estrogen affinity label [3H]ketononestrol aziridine (KNA) or with the antiestrogen affinity label [3H]tamoxifen aziridine (TAZ) were subjected to limited proteolysis with trypsin, α-chymotrypsin, and Staphylococcus aureus V8 protease and then analyzed on 10-20{\%} sodium dodecyl sulfate-polyacrylamide gradient gels followed by fluorography. The similar molecular weights of intact receptors (Mr 66,000 daltons) and the proteolytic digest patterns indicate extensive homology among estrogen receptors from MCF-7 human breast cancer cells, GH4 rat pituitary cells and rat uterus when liganded with estrogen or antiestrogen. Each protease generated a distinctive ladder of estrogen receptor fragments, and the fragmentation patterns were virtually identical for estrogen receptors labeled with estrogen (KNA) or antiestrogen (TAZ). Each protease yielded a relatively {"}resistant{"} receptor fragment of about 28,000-35,000 daltons. Trypsin and chymotrypsin at higher concentrations generated a much smaller 6,000-8,000 dalton digest product that still contained the [3H]KNA- or [3H]TAZ-labeled receptor binding site. Moreover, the receptor digest patterns were similar for estrogen receptors from the three different target cells. Our studies suggest considerable structural relatedness among these three estrogen receptors and also indicate that these two affinity labels bind to a similar, perhaps identical, region of the receptor molecule.",
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