Mutagenesis of cysteines in the hormone binding domain of the human estrogen receptor: Alterations in binding and transcriptional activation by covalently and reversibly attaching ligands

Research output: Contribution to journalArticle

Abstract

We have carried out experiments to determine the role of the cysteines in the hormone-binding domain (HBD) of the human estrogen receptor (ER) in receptor function. In each mutant receptor, 1 of the 4 cysteines in the HBD (cysteines 381, 417, 447, and 530) was changed by in vitro mutagenesis of the ER cDNA (containing Gly400) from cysteine to alanine; Cys530 was also mutated to a serine. The mutant and wild-type receptor cDNAs were expressed in Chinese hamster ovary cells using an expression vector containing the Rous sarcoma virus promoter. The mutant and wild-type receptors were assayed for hormone binding and for their ability to activate estrogen-responsive reporter plasmids. All ER mutants bound estradiol (E2) with affinity similar to wild-type ER, displaying a K(d) between 0.3 and 0.8 nM (wild-type ER K(d) = 0.45 ± 0.10 nM). All were capable of covalent labeling by the affinity ligands ketononestrol aziridine, an estrogen agonist, and tamoxifen aziridine, an antagonist. Since in previous work we identified Cys530 as the site of covalent attachment of these ligands (Harlow, K.W., Smith, D.N., Katzenellenbogen, J.A., Greene, G.L., and Katzenellenbogen, B.S. (1989) J. Biol. Chem. 264, 17476-17485) it appears that an alternate residue(s) can be labeled in the absence of a cysteine at position 530; studies with methyl methanethiosulfonate, a cysteine-specific reagent, suggest that this residue is probably another cysteine in the HBD. The C381A, C417A, C530A and C530S ERs showed E2-stimulated transcriptional activation profiles similar to wild-type ER whereas the dose response for E2 for the C447A mutant was shifted to the right, requiring 50 x higher E2 concentrations to achieve half-maximal response. Tamoxifen aziridine inhibited E2-stimulated transcription, and ketononestrol aziridine stimulated transcription by wild-type, C530A, and C530S ER, but the effectiveness of these covalently attaching ligands was altered in the C530A and C530S mutants. Thus, these two mutant receptors are altered in their transactivation response to agonist and antagonist affinity labeling ligands but are unaltered in their response to reversibly binding estrogens and antiestrogens. In addition, we show that a mutant ER (C447A) can have an affinity for E2 similar to that of wild-type ER but differs in its ability to activate transcription in response to E2, indicating a decoupling of the hormone binding and transcriptional activation functions in this receptor.

Original languageEnglish (US)
Pages (from-to)10880-10887
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number17
StatePublished - Sep 6 1991

Fingerprint

Mutagenesis
Estrogen Receptors
Transcriptional Activation
Cysteine
Chemical activation
Hormones
Ligands
Transcription
Estrogens
Labeling
Complementary DNA
Rous sarcoma virus
Estrogen Receptor Modulators
Cricetulus
Viruses
Alanine
Serine
Ovary
Estradiol
Plasmids

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{78bd4139ac644d33a8f4fbace7ad815e,
title = "Mutagenesis of cysteines in the hormone binding domain of the human estrogen receptor: Alterations in binding and transcriptional activation by covalently and reversibly attaching ligands",
abstract = "We have carried out experiments to determine the role of the cysteines in the hormone-binding domain (HBD) of the human estrogen receptor (ER) in receptor function. In each mutant receptor, 1 of the 4 cysteines in the HBD (cysteines 381, 417, 447, and 530) was changed by in vitro mutagenesis of the ER cDNA (containing Gly400) from cysteine to alanine; Cys530 was also mutated to a serine. The mutant and wild-type receptor cDNAs were expressed in Chinese hamster ovary cells using an expression vector containing the Rous sarcoma virus promoter. The mutant and wild-type receptors were assayed for hormone binding and for their ability to activate estrogen-responsive reporter plasmids. All ER mutants bound estradiol (E2) with affinity similar to wild-type ER, displaying a K(d) between 0.3 and 0.8 nM (wild-type ER K(d) = 0.45 ± 0.10 nM). All were capable of covalent labeling by the affinity ligands ketononestrol aziridine, an estrogen agonist, and tamoxifen aziridine, an antagonist. Since in previous work we identified Cys530 as the site of covalent attachment of these ligands (Harlow, K.W., Smith, D.N., Katzenellenbogen, J.A., Greene, G.L., and Katzenellenbogen, B.S. (1989) J. Biol. Chem. 264, 17476-17485) it appears that an alternate residue(s) can be labeled in the absence of a cysteine at position 530; studies with methyl methanethiosulfonate, a cysteine-specific reagent, suggest that this residue is probably another cysteine in the HBD. The C381A, C417A, C530A and C530S ERs showed E2-stimulated transcriptional activation profiles similar to wild-type ER whereas the dose response for E2 for the C447A mutant was shifted to the right, requiring 50 x higher E2 concentrations to achieve half-maximal response. Tamoxifen aziridine inhibited E2-stimulated transcription, and ketononestrol aziridine stimulated transcription by wild-type, C530A, and C530S ER, but the effectiveness of these covalently attaching ligands was altered in the C530A and C530S mutants. Thus, these two mutant receptors are altered in their transactivation response to agonist and antagonist affinity labeling ligands but are unaltered in their response to reversibly binding estrogens and antiestrogens. In addition, we show that a mutant ER (C447A) can have an affinity for E2 similar to that of wild-type ER but differs in its ability to activate transcription in response to E2, indicating a decoupling of the hormone binding and transcriptional activation functions in this receptor.",
author = "Reese, {J. C.} and Katzenellenbogen, {B. S.}",
year = "1991",
month = "9",
day = "6",
language = "English (US)",
volume = "266",
pages = "10880--10887",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "17",

}

TY - JOUR

T1 - Mutagenesis of cysteines in the hormone binding domain of the human estrogen receptor

T2 - Alterations in binding and transcriptional activation by covalently and reversibly attaching ligands

AU - Reese, J. C.

AU - Katzenellenbogen, B. S.

PY - 1991/9/6

Y1 - 1991/9/6

N2 - We have carried out experiments to determine the role of the cysteines in the hormone-binding domain (HBD) of the human estrogen receptor (ER) in receptor function. In each mutant receptor, 1 of the 4 cysteines in the HBD (cysteines 381, 417, 447, and 530) was changed by in vitro mutagenesis of the ER cDNA (containing Gly400) from cysteine to alanine; Cys530 was also mutated to a serine. The mutant and wild-type receptor cDNAs were expressed in Chinese hamster ovary cells using an expression vector containing the Rous sarcoma virus promoter. The mutant and wild-type receptors were assayed for hormone binding and for their ability to activate estrogen-responsive reporter plasmids. All ER mutants bound estradiol (E2) with affinity similar to wild-type ER, displaying a K(d) between 0.3 and 0.8 nM (wild-type ER K(d) = 0.45 ± 0.10 nM). All were capable of covalent labeling by the affinity ligands ketononestrol aziridine, an estrogen agonist, and tamoxifen aziridine, an antagonist. Since in previous work we identified Cys530 as the site of covalent attachment of these ligands (Harlow, K.W., Smith, D.N., Katzenellenbogen, J.A., Greene, G.L., and Katzenellenbogen, B.S. (1989) J. Biol. Chem. 264, 17476-17485) it appears that an alternate residue(s) can be labeled in the absence of a cysteine at position 530; studies with methyl methanethiosulfonate, a cysteine-specific reagent, suggest that this residue is probably another cysteine in the HBD. The C381A, C417A, C530A and C530S ERs showed E2-stimulated transcriptional activation profiles similar to wild-type ER whereas the dose response for E2 for the C447A mutant was shifted to the right, requiring 50 x higher E2 concentrations to achieve half-maximal response. Tamoxifen aziridine inhibited E2-stimulated transcription, and ketononestrol aziridine stimulated transcription by wild-type, C530A, and C530S ER, but the effectiveness of these covalently attaching ligands was altered in the C530A and C530S mutants. Thus, these two mutant receptors are altered in their transactivation response to agonist and antagonist affinity labeling ligands but are unaltered in their response to reversibly binding estrogens and antiestrogens. In addition, we show that a mutant ER (C447A) can have an affinity for E2 similar to that of wild-type ER but differs in its ability to activate transcription in response to E2, indicating a decoupling of the hormone binding and transcriptional activation functions in this receptor.

AB - We have carried out experiments to determine the role of the cysteines in the hormone-binding domain (HBD) of the human estrogen receptor (ER) in receptor function. In each mutant receptor, 1 of the 4 cysteines in the HBD (cysteines 381, 417, 447, and 530) was changed by in vitro mutagenesis of the ER cDNA (containing Gly400) from cysteine to alanine; Cys530 was also mutated to a serine. The mutant and wild-type receptor cDNAs were expressed in Chinese hamster ovary cells using an expression vector containing the Rous sarcoma virus promoter. The mutant and wild-type receptors were assayed for hormone binding and for their ability to activate estrogen-responsive reporter plasmids. All ER mutants bound estradiol (E2) with affinity similar to wild-type ER, displaying a K(d) between 0.3 and 0.8 nM (wild-type ER K(d) = 0.45 ± 0.10 nM). All were capable of covalent labeling by the affinity ligands ketononestrol aziridine, an estrogen agonist, and tamoxifen aziridine, an antagonist. Since in previous work we identified Cys530 as the site of covalent attachment of these ligands (Harlow, K.W., Smith, D.N., Katzenellenbogen, J.A., Greene, G.L., and Katzenellenbogen, B.S. (1989) J. Biol. Chem. 264, 17476-17485) it appears that an alternate residue(s) can be labeled in the absence of a cysteine at position 530; studies with methyl methanethiosulfonate, a cysteine-specific reagent, suggest that this residue is probably another cysteine in the HBD. The C381A, C417A, C530A and C530S ERs showed E2-stimulated transcriptional activation profiles similar to wild-type ER whereas the dose response for E2 for the C447A mutant was shifted to the right, requiring 50 x higher E2 concentrations to achieve half-maximal response. Tamoxifen aziridine inhibited E2-stimulated transcription, and ketononestrol aziridine stimulated transcription by wild-type, C530A, and C530S ER, but the effectiveness of these covalently attaching ligands was altered in the C530A and C530S mutants. Thus, these two mutant receptors are altered in their transactivation response to agonist and antagonist affinity labeling ligands but are unaltered in their response to reversibly binding estrogens and antiestrogens. In addition, we show that a mutant ER (C447A) can have an affinity for E2 similar to that of wild-type ER but differs in its ability to activate transcription in response to E2, indicating a decoupling of the hormone binding and transcriptional activation functions in this receptor.

UR - http://www.scopus.com/inward/record.url?scp=0025797584&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025797584&partnerID=8YFLogxK

M3 - Article

C2 - 2040605

AN - SCOPUS:0025797584

VL - 266

SP - 10880

EP - 10887

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 17

ER -