Characterization of a temperature-sensitive mutation in the hormone binding domain of the human estrogen receptor: Studies in cell extracts and intact cells and their implications for hormone-dependent transcriptional activation

Joseph C. Reese, Benita S Katzenellenbogen

Research output: Contribution to journalArticle

Abstract

A previous report from this laboratory (Reese, J. C., and Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 10880-10887) identified an estrogen receptor (ER) mutant which had a similar binding affinity for estradiol as wild-type ER but displayed a dose-response shift for estradiol in transactivation studies. In this study, we have utilized hormone binding, DNA binding, and gene transfer experiments to further characterize this mutant, which contains an alanine substitution for a cysteine at amino acid 447 in the hormone binding domain of the receptor. Hormone binding studies indicate that the C447A receptor is a temperature-sensitive mutant, whose instability is only apparent at elevated temperatures, and that ligand can stabilize the mutant receptor. Western blot analysis reveals that the temperature-sensitive loss of hormone binding is not attributable to a degradation of receptor protein, but rather is an inactivation of the receptor's hormone binding ability. In addition to the loss in the hormone binding capacity of the C447A mutant, this mutant shows a temperature-sensitive loss in the DNA binding ability of the receptor. Transactivation profiles of the mutant and wild-type receptors demonstrate that incubation of transfected cells with increasing concentrations of estradiol at more ambient temperatures shifts the mutant receptor's dose-response curves to the left, converging on the wild-type curve. Hence, these transactivation studies reveal that the dose-response shift observed for this mutant in cells reflects the measured instability of the hormone binding and DNA interaction of the C447A mutant that can be demonstrated in vitro. In addition, this temperaturesensitive ER mutant is of interest in that its DNA binding is now ligand-dependent with the result that transcriptional activation now parallels receptor occupancy by ligand, which is similar to other steroid hormone receptors.

Original languageEnglish (US)
Pages (from-to)9868-9873
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number14
StatePublished - May 15 1992

Fingerprint

Cell Extracts
Estrogen Receptors
Transcriptional Activation
Chemical activation
Hormones
Mutation
Temperature
Estradiol
DNA
Ligands
Steroid hormones
Gene transfer
Steroid Receptors
Alanine
Cysteine
Proteolysis
Substitution reactions
Cells
Western Blotting
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{690974074d364673a2215a6fb05242a4,
title = "Characterization of a temperature-sensitive mutation in the hormone binding domain of the human estrogen receptor: Studies in cell extracts and intact cells and their implications for hormone-dependent transcriptional activation",
abstract = "A previous report from this laboratory (Reese, J. C., and Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 10880-10887) identified an estrogen receptor (ER) mutant which had a similar binding affinity for estradiol as wild-type ER but displayed a dose-response shift for estradiol in transactivation studies. In this study, we have utilized hormone binding, DNA binding, and gene transfer experiments to further characterize this mutant, which contains an alanine substitution for a cysteine at amino acid 447 in the hormone binding domain of the receptor. Hormone binding studies indicate that the C447A receptor is a temperature-sensitive mutant, whose instability is only apparent at elevated temperatures, and that ligand can stabilize the mutant receptor. Western blot analysis reveals that the temperature-sensitive loss of hormone binding is not attributable to a degradation of receptor protein, but rather is an inactivation of the receptor's hormone binding ability. In addition to the loss in the hormone binding capacity of the C447A mutant, this mutant shows a temperature-sensitive loss in the DNA binding ability of the receptor. Transactivation profiles of the mutant and wild-type receptors demonstrate that incubation of transfected cells with increasing concentrations of estradiol at more ambient temperatures shifts the mutant receptor's dose-response curves to the left, converging on the wild-type curve. Hence, these transactivation studies reveal that the dose-response shift observed for this mutant in cells reflects the measured instability of the hormone binding and DNA interaction of the C447A mutant that can be demonstrated in vitro. In addition, this temperaturesensitive ER mutant is of interest in that its DNA binding is now ligand-dependent with the result that transcriptional activation now parallels receptor occupancy by ligand, which is similar to other steroid hormone receptors.",
author = "Reese, {Joseph C.} and Katzenellenbogen, {Benita S}",
year = "1992",
month = "5",
day = "15",
language = "English (US)",
volume = "267",
pages = "9868--9873",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "14",

}

TY - JOUR

T1 - Characterization of a temperature-sensitive mutation in the hormone binding domain of the human estrogen receptor

T2 - Studies in cell extracts and intact cells and their implications for hormone-dependent transcriptional activation

AU - Reese, Joseph C.

AU - Katzenellenbogen, Benita S

PY - 1992/5/15

Y1 - 1992/5/15

N2 - A previous report from this laboratory (Reese, J. C., and Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 10880-10887) identified an estrogen receptor (ER) mutant which had a similar binding affinity for estradiol as wild-type ER but displayed a dose-response shift for estradiol in transactivation studies. In this study, we have utilized hormone binding, DNA binding, and gene transfer experiments to further characterize this mutant, which contains an alanine substitution for a cysteine at amino acid 447 in the hormone binding domain of the receptor. Hormone binding studies indicate that the C447A receptor is a temperature-sensitive mutant, whose instability is only apparent at elevated temperatures, and that ligand can stabilize the mutant receptor. Western blot analysis reveals that the temperature-sensitive loss of hormone binding is not attributable to a degradation of receptor protein, but rather is an inactivation of the receptor's hormone binding ability. In addition to the loss in the hormone binding capacity of the C447A mutant, this mutant shows a temperature-sensitive loss in the DNA binding ability of the receptor. Transactivation profiles of the mutant and wild-type receptors demonstrate that incubation of transfected cells with increasing concentrations of estradiol at more ambient temperatures shifts the mutant receptor's dose-response curves to the left, converging on the wild-type curve. Hence, these transactivation studies reveal that the dose-response shift observed for this mutant in cells reflects the measured instability of the hormone binding and DNA interaction of the C447A mutant that can be demonstrated in vitro. In addition, this temperaturesensitive ER mutant is of interest in that its DNA binding is now ligand-dependent with the result that transcriptional activation now parallels receptor occupancy by ligand, which is similar to other steroid hormone receptors.

AB - A previous report from this laboratory (Reese, J. C., and Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 10880-10887) identified an estrogen receptor (ER) mutant which had a similar binding affinity for estradiol as wild-type ER but displayed a dose-response shift for estradiol in transactivation studies. In this study, we have utilized hormone binding, DNA binding, and gene transfer experiments to further characterize this mutant, which contains an alanine substitution for a cysteine at amino acid 447 in the hormone binding domain of the receptor. Hormone binding studies indicate that the C447A receptor is a temperature-sensitive mutant, whose instability is only apparent at elevated temperatures, and that ligand can stabilize the mutant receptor. Western blot analysis reveals that the temperature-sensitive loss of hormone binding is not attributable to a degradation of receptor protein, but rather is an inactivation of the receptor's hormone binding ability. In addition to the loss in the hormone binding capacity of the C447A mutant, this mutant shows a temperature-sensitive loss in the DNA binding ability of the receptor. Transactivation profiles of the mutant and wild-type receptors demonstrate that incubation of transfected cells with increasing concentrations of estradiol at more ambient temperatures shifts the mutant receptor's dose-response curves to the left, converging on the wild-type curve. Hence, these transactivation studies reveal that the dose-response shift observed for this mutant in cells reflects the measured instability of the hormone binding and DNA interaction of the C447A mutant that can be demonstrated in vitro. In addition, this temperaturesensitive ER mutant is of interest in that its DNA binding is now ligand-dependent with the result that transcriptional activation now parallels receptor occupancy by ligand, which is similar to other steroid hormone receptors.

UR - http://www.scopus.com/inward/record.url?scp=0026767470&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026767470&partnerID=8YFLogxK

M3 - Article

C2 - 1577818

AN - SCOPUS:0026767470

VL - 267

SP - 9868

EP - 9873

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 14

ER -