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Title:Analysis of Estrogen and Antiestrogen Receptor Complexes
Author(s):Elliston, Jonathan Franklin
Doctoral Committee Chair(s):Katzenellenbogen, Benita S.
Department / Program:Physiology and Biophysics
Discipline:Physiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biology, Animal Physiology
Abstract:I have used affinity labeling ligands for the estrogen receptor (ER) to examine the structure and relatedness of ER in different target tissues and species. In addition, I have characterized the first efficient and selective affinity labeling estrogen, ketononestrol aziridine (KNA).
KNA has an apparent relative binding affinity of 8% that of estradiol and shows time-dependent irreversible binding to the ER. The agonistic activity of KNA is evident in MCF-7 cells in culture, where it increases cell growth rate and elevates the level of progesterone receptor. Labeling with ($\sp3$H) KNA proceeds in a time-, concentration-, and temperature-dependent manner; labeling is efficient and selective and, by competition studies, was shown to be estrogen specific. ER covalently labeled with ($\sp3$H) KNA sediments as a 4S species on high salt sucrose gradients, and its sedimentation position is shifted by treatment with monoclonal antibodies. On SDS-polyacrylamide gels, the labeled species migrates with a molecular weight (M$\sb{\rm r}$) of 66,000 daltons.
ER covalently labeled with the estrogen affinity label ($\sp3$H) ketononestrol aziridine (KNA) or with the antiestrogen affinity label ($\sp3$H) tamoxifen aziridine (TAZ) was subjected to limited proteolysis with trypsin (T), chymotrypsin (C), and V8 protease (V8) and analyzed on SDS-polyacrylamide gels. The similar M$\sb{\rm r}$ of intact receptors (66,000 daltons) and the proteolytic digest patterns indicate extensive homology among ER from breast (human), pituitary (rat) and uterus (rat) when liganded with estrogen or antiestrogen.
Each protease generated a distinctive ladder of ER fragments and the patterns were virtually identical for ER labeled with KNA or TAZ. Each protease yielded a relatively "resistant" receptor fragment of ca. 28,000-35,000 daltons. T and C at higher concentrations generated a smaller 6,000-8,000 dalton digest product that still contained the ($\sp3$H) KNA- or ($\sp3$H) TAZ-labeled binding site, indicating that the steroid-binding domain is restricted to a relatively small segment of the receptor and is similar for ER from these 3 different target cells.
Issue Date:1987
Type:Text
Description:98 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.
URI:http://hdl.handle.net/2142/71452
Other Identifier(s):(UMI)AAI8803030
Date Available in IDEALS:2014-12-16
Date Deposited:1987


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