Characterization of high affinity and low affinity dexamethasone binding sites on male rat liver nuclear envelopes

Gillian M. Howell, Yvonne A. Lefebvre

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Steroids must traverse the nuclear envelope before exerting their action at the chromatin. However, few studies have been done to elucidate the mechanism by which steroids traverse this membrane barrier. As first steps towards investigating the mechanism, we have characterized the binding sites for dexamethasone on male rat liver nuclear envelopes. The nuclear envelopes, prepared in the presence of dithiothreitol, were isolated from purified nuclei after treatment with DNase 1 at high pH. Binding of dexamethasone to the nuclear envelopes was measured after 16h of incubation at 0-4°C. At pH 7.4, only a single high capacity, low affinity binding site for dexamethasone was identified. However, at pH 8.6, two sites were identified; a low capacity, high affinity site and a high capacity, low affinity site. Adrenalectomy of the animal before preparation of the membranes caused loss of the high affinity site and reduction in the number of the lower affinity sites. Acute dexamethasone treatment of adrenalectomized rats resulted in the reappearance of the high affinity site but long term treatment with dexamethasone was required for complete restoration of the high affinity sites and reappearance of any of the low affinity sites. The steroid specificity of these nuclear envelope binding sites was different from that of the cytosolic glucocorticoid receptor, generally showing broader specificity. However, triamcinolone acetonide, which is a potent competitor for binding to the glucocorticoid receptor, did not compete effectively. The binding sites were sensitive to protease treatment and salt extraction studies revealed that the dexamethasone binding sites do not represent proteins non-specifically bound to the nuclear envelope. The affinity and the hormone responsiveness of the high affinity site are similar to those of the nuclear glucocorticoid receptor. Therefore, the nuclear envelope may be a site of action of glucocorticoids.

Original languageEnglish (US)
Pages (from-to)977-986
Number of pages10
JournalJournal of Steroid Biochemistry
Volume33
Issue number5
DOIs
StatePublished - Nov 1989

Fingerprint

Nuclear Envelope
Glucocorticoid Receptors
Liver
Dexamethasone
Rats
Steroids
Binding Sites
Membranes
Triamcinolone Acetonide
Deoxyribonucleases
Dithiothreitol
Glucocorticoids
Restoration
Chromatin
Animals
Peptide Hydrolases
Salts
Hormones
Adrenalectomy
Cytoplasmic and Nuclear Receptors

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Cite this

Characterization of high affinity and low affinity dexamethasone binding sites on male rat liver nuclear envelopes. / Howell, Gillian M.; Lefebvre, Yvonne A.

In: Journal of Steroid Biochemistry, Vol. 33, No. 5, 11.1989, p. 977-986.

Research output: Contribution to journalArticle

@article{484ef6a780e74319a1a484acb6be2dd2,
title = "Characterization of high affinity and low affinity dexamethasone binding sites on male rat liver nuclear envelopes",
abstract = "Steroids must traverse the nuclear envelope before exerting their action at the chromatin. However, few studies have been done to elucidate the mechanism by which steroids traverse this membrane barrier. As first steps towards investigating the mechanism, we have characterized the binding sites for dexamethasone on male rat liver nuclear envelopes. The nuclear envelopes, prepared in the presence of dithiothreitol, were isolated from purified nuclei after treatment with DNase 1 at high pH. Binding of dexamethasone to the nuclear envelopes was measured after 16h of incubation at 0-4°C. At pH 7.4, only a single high capacity, low affinity binding site for dexamethasone was identified. However, at pH 8.6, two sites were identified; a low capacity, high affinity site and a high capacity, low affinity site. Adrenalectomy of the animal before preparation of the membranes caused loss of the high affinity site and reduction in the number of the lower affinity sites. Acute dexamethasone treatment of adrenalectomized rats resulted in the reappearance of the high affinity site but long term treatment with dexamethasone was required for complete restoration of the high affinity sites and reappearance of any of the low affinity sites. The steroid specificity of these nuclear envelope binding sites was different from that of the cytosolic glucocorticoid receptor, generally showing broader specificity. However, triamcinolone acetonide, which is a potent competitor for binding to the glucocorticoid receptor, did not compete effectively. The binding sites were sensitive to protease treatment and salt extraction studies revealed that the dexamethasone binding sites do not represent proteins non-specifically bound to the nuclear envelope. The affinity and the hormone responsiveness of the high affinity site are similar to those of the nuclear glucocorticoid receptor. Therefore, the nuclear envelope may be a site of action of glucocorticoids.",
author = "Howell, {Gillian M.} and Lefebvre, {Yvonne A.}",
year = "1989",
month = "11",
doi = "10.1016/0022-4731(89)90249-5",
language = "English (US)",
volume = "33",
pages = "977--986",
journal = "Journal of Steroid Biochemistry and Molecular Biology",
issn = "0960-0760",
publisher = "Elsevier Limited",
number = "5",

}

TY - JOUR

T1 - Characterization of high affinity and low affinity dexamethasone binding sites on male rat liver nuclear envelopes

AU - Howell, Gillian M.

AU - Lefebvre, Yvonne A.

PY - 1989/11

Y1 - 1989/11

N2 - Steroids must traverse the nuclear envelope before exerting their action at the chromatin. However, few studies have been done to elucidate the mechanism by which steroids traverse this membrane barrier. As first steps towards investigating the mechanism, we have characterized the binding sites for dexamethasone on male rat liver nuclear envelopes. The nuclear envelopes, prepared in the presence of dithiothreitol, were isolated from purified nuclei after treatment with DNase 1 at high pH. Binding of dexamethasone to the nuclear envelopes was measured after 16h of incubation at 0-4°C. At pH 7.4, only a single high capacity, low affinity binding site for dexamethasone was identified. However, at pH 8.6, two sites were identified; a low capacity, high affinity site and a high capacity, low affinity site. Adrenalectomy of the animal before preparation of the membranes caused loss of the high affinity site and reduction in the number of the lower affinity sites. Acute dexamethasone treatment of adrenalectomized rats resulted in the reappearance of the high affinity site but long term treatment with dexamethasone was required for complete restoration of the high affinity sites and reappearance of any of the low affinity sites. The steroid specificity of these nuclear envelope binding sites was different from that of the cytosolic glucocorticoid receptor, generally showing broader specificity. However, triamcinolone acetonide, which is a potent competitor for binding to the glucocorticoid receptor, did not compete effectively. The binding sites were sensitive to protease treatment and salt extraction studies revealed that the dexamethasone binding sites do not represent proteins non-specifically bound to the nuclear envelope. The affinity and the hormone responsiveness of the high affinity site are similar to those of the nuclear glucocorticoid receptor. Therefore, the nuclear envelope may be a site of action of glucocorticoids.

AB - Steroids must traverse the nuclear envelope before exerting their action at the chromatin. However, few studies have been done to elucidate the mechanism by which steroids traverse this membrane barrier. As first steps towards investigating the mechanism, we have characterized the binding sites for dexamethasone on male rat liver nuclear envelopes. The nuclear envelopes, prepared in the presence of dithiothreitol, were isolated from purified nuclei after treatment with DNase 1 at high pH. Binding of dexamethasone to the nuclear envelopes was measured after 16h of incubation at 0-4°C. At pH 7.4, only a single high capacity, low affinity binding site for dexamethasone was identified. However, at pH 8.6, two sites were identified; a low capacity, high affinity site and a high capacity, low affinity site. Adrenalectomy of the animal before preparation of the membranes caused loss of the high affinity site and reduction in the number of the lower affinity sites. Acute dexamethasone treatment of adrenalectomized rats resulted in the reappearance of the high affinity site but long term treatment with dexamethasone was required for complete restoration of the high affinity sites and reappearance of any of the low affinity sites. The steroid specificity of these nuclear envelope binding sites was different from that of the cytosolic glucocorticoid receptor, generally showing broader specificity. However, triamcinolone acetonide, which is a potent competitor for binding to the glucocorticoid receptor, did not compete effectively. The binding sites were sensitive to protease treatment and salt extraction studies revealed that the dexamethasone binding sites do not represent proteins non-specifically bound to the nuclear envelope. The affinity and the hormone responsiveness of the high affinity site are similar to those of the nuclear glucocorticoid receptor. Therefore, the nuclear envelope may be a site of action of glucocorticoids.

UR - http://www.scopus.com/inward/record.url?scp=0024819432&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024819432&partnerID=8YFLogxK

U2 - 10.1016/0022-4731(89)90249-5

DO - 10.1016/0022-4731(89)90249-5

M3 - Article

C2 - 2689794

AN - SCOPUS:0024819432

VL - 33

SP - 977

EP - 986

JO - Journal of Steroid Biochemistry and Molecular Biology

JF - Journal of Steroid Biochemistry and Molecular Biology

SN - 0960-0760

IS - 5

ER -