Hormone binding and transcription activation by estrogen receptors: Analyses using mammalian and yeast systems

Benita S Katzenellenbogen, Bhavna Bhardwaj, Henry Fang, B. Avery Ince, Farzad Pakde, Joseph C. Reese, David Schodin, Carol K. Wrenn

Research output: Contribution to journalArticle

Abstract

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the human estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labeling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. C530A mutant), C381 becomes the site of estrogen-competible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed mutagenesis of selected residues in the ER and region-specific chemical mutagenesis of the ER hormone binding domain with initial phenotypic screening in yeast have enabled the identification of a region near C530 important in discrimination between estrogens and antiestrogens and of other residues important in hormone-dependent transcriptional activation. Some ER mutants with alterations in the carboxy-terminal portion of the hormone binding domain are transcriptionally inactive yet bind hormone and also function as potent dominant negative ERs, suppressing the activity of wild-type ER at low concentrations. These studies reveal a separation of the hormone binding and transcription activation functions of the ER. They are also beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer. In addition, some dominant negative receptors may prove useful in examining ER mechanisms of action and in suppressing the estrogen-dependent growth of breast cancer cells.

Original languageEnglish (US)
Pages (from-to)39-48
Number of pages10
JournalJournal of Steroid Biochemistry and Molecular Biology
Volume47
Issue number1-6
DOIs
StatePublished - Dec 1993

Fingerprint

Transcription
Estrogen Receptors
Yeast
Transcriptional Activation
Yeasts
Chemical activation
Hormones
Estrogen Receptor Modulators
Mutagenesis
Estrogens
Ligands
Labeling
Breast Neoplasms
Site-Directed Mutagenesis
Cysteine
Bioactivity
Screening
Cells
Amino Acids

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Endocrinology
  • Clinical Biochemistry
  • Cell Biology

Cite this

Hormone binding and transcription activation by estrogen receptors : Analyses using mammalian and yeast systems. / Katzenellenbogen, Benita S; Bhardwaj, Bhavna; Fang, Henry; Ince, B. Avery; Pakde, Farzad; Reese, Joseph C.; Schodin, David; Wrenn, Carol K.

In: Journal of Steroid Biochemistry and Molecular Biology, Vol. 47, No. 1-6, 12.1993, p. 39-48.

Research output: Contribution to journalArticle

Katzenellenbogen, Benita S ; Bhardwaj, Bhavna ; Fang, Henry ; Ince, B. Avery ; Pakde, Farzad ; Reese, Joseph C. ; Schodin, David ; Wrenn, Carol K. / Hormone binding and transcription activation by estrogen receptors : Analyses using mammalian and yeast systems. In: Journal of Steroid Biochemistry and Molecular Biology. 1993 ; Vol. 47, No. 1-6. pp. 39-48.
@article{08d76aec65c3435c90d2366f758f4727,
title = "Hormone binding and transcription activation by estrogen receptors: Analyses using mammalian and yeast systems",
abstract = "We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the human estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labeling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. C530A mutant), C381 becomes the site of estrogen-competible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed mutagenesis of selected residues in the ER and region-specific chemical mutagenesis of the ER hormone binding domain with initial phenotypic screening in yeast have enabled the identification of a region near C530 important in discrimination between estrogens and antiestrogens and of other residues important in hormone-dependent transcriptional activation. Some ER mutants with alterations in the carboxy-terminal portion of the hormone binding domain are transcriptionally inactive yet bind hormone and also function as potent dominant negative ERs, suppressing the activity of wild-type ER at low concentrations. These studies reveal a separation of the hormone binding and transcription activation functions of the ER. They are also beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer. In addition, some dominant negative receptors may prove useful in examining ER mechanisms of action and in suppressing the estrogen-dependent growth of breast cancer cells.",
author = "Katzenellenbogen, {Benita S} and Bhavna Bhardwaj and Henry Fang and Ince, {B. Avery} and Farzad Pakde and Reese, {Joseph C.} and David Schodin and Wrenn, {Carol K.}",
year = "1993",
month = "12",
doi = "10.1016/0960-0760(93)90055-2",
language = "English (US)",
volume = "47",
pages = "39--48",
journal = "Journal of Steroid Biochemistry and Molecular Biology",
issn = "0960-0760",
publisher = "Elsevier Limited",
number = "1-6",

}

TY - JOUR

T1 - Hormone binding and transcription activation by estrogen receptors

T2 - Analyses using mammalian and yeast systems

AU - Katzenellenbogen, Benita S

AU - Bhardwaj, Bhavna

AU - Fang, Henry

AU - Ince, B. Avery

AU - Pakde, Farzad

AU - Reese, Joseph C.

AU - Schodin, David

AU - Wrenn, Carol K.

PY - 1993/12

Y1 - 1993/12

N2 - We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the human estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labeling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. C530A mutant), C381 becomes the site of estrogen-competible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed mutagenesis of selected residues in the ER and region-specific chemical mutagenesis of the ER hormone binding domain with initial phenotypic screening in yeast have enabled the identification of a region near C530 important in discrimination between estrogens and antiestrogens and of other residues important in hormone-dependent transcriptional activation. Some ER mutants with alterations in the carboxy-terminal portion of the hormone binding domain are transcriptionally inactive yet bind hormone and also function as potent dominant negative ERs, suppressing the activity of wild-type ER at low concentrations. These studies reveal a separation of the hormone binding and transcription activation functions of the ER. They are also beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer. In addition, some dominant negative receptors may prove useful in examining ER mechanisms of action and in suppressing the estrogen-dependent growth of breast cancer cells.

AB - We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the human estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labeling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. C530A mutant), C381 becomes the site of estrogen-competible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed mutagenesis of selected residues in the ER and region-specific chemical mutagenesis of the ER hormone binding domain with initial phenotypic screening in yeast have enabled the identification of a region near C530 important in discrimination between estrogens and antiestrogens and of other residues important in hormone-dependent transcriptional activation. Some ER mutants with alterations in the carboxy-terminal portion of the hormone binding domain are transcriptionally inactive yet bind hormone and also function as potent dominant negative ERs, suppressing the activity of wild-type ER at low concentrations. These studies reveal a separation of the hormone binding and transcription activation functions of the ER. They are also beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer. In addition, some dominant negative receptors may prove useful in examining ER mechanisms of action and in suppressing the estrogen-dependent growth of breast cancer cells.

UR - http://www.scopus.com/inward/record.url?scp=0027749202&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027749202&partnerID=8YFLogxK

U2 - 10.1016/0960-0760(93)90055-2

DO - 10.1016/0960-0760(93)90055-2

M3 - Article

C2 - 8274440

AN - SCOPUS:0027749202

VL - 47

SP - 39

EP - 48

JO - Journal of Steroid Biochemistry and Molecular Biology

JF - Journal of Steroid Biochemistry and Molecular Biology

SN - 0960-0760

IS - 1-6

ER -