Structural Analysis of Covalently Labeled Estrogen Receptors by Limited Proteolysis and Monoclonal Antibody Reactivity

Benita S Katzenellenbogen, Geoffrey L. Greene, Jonathan F. Elliston, Frederick J. Monsma, Penelope A. Springer, Yvonne S. Ziegler

Research output: Contribution to journalArticle

Abstract

We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine ([3H]TAZ) were treated with trypsin (T), a-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66 000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between p/ 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P37 revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms <47K and T-generated forms <3IK) were no longer immunoreactive. In contrast, use of the antibody H222Spγ revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.

Original languageEnglish (US)
Pages (from-to)2364-2373
Number of pages10
JournalBiochemistry
Volume26
Issue number8
DOIs
StatePublished - Jan 1 1987

Fingerprint

Proteolysis
Structural analysis
Estrogen Receptors
Monoclonal Antibodies
Rats
Antibodies
Cytoplasmic and Nuclear Receptors
Photofluorography
Gels
Hormones
Breast Neoplasms
Protein Sequence Analysis
Ladders
Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural Analysis of Covalently Labeled Estrogen Receptors by Limited Proteolysis and Monoclonal Antibody Reactivity. / Katzenellenbogen, Benita S; Greene, Geoffrey L.; Elliston, Jonathan F.; Monsma, Frederick J.; Springer, Penelope A.; Ziegler, Yvonne S.

In: Biochemistry, Vol. 26, No. 8, 01.01.1987, p. 2364-2373.

Research output: Contribution to journalArticle

Katzenellenbogen, Benita S ; Greene, Geoffrey L. ; Elliston, Jonathan F. ; Monsma, Frederick J. ; Springer, Penelope A. ; Ziegler, Yvonne S. / Structural Analysis of Covalently Labeled Estrogen Receptors by Limited Proteolysis and Monoclonal Antibody Reactivity. In: Biochemistry. 1987 ; Vol. 26, No. 8. pp. 2364-2373.
@article{af788db80c804be68cb5bc6963936d2c,
title = "Structural Analysis of Covalently Labeled Estrogen Receptors by Limited Proteolysis and Monoclonal Antibody Reactivity",
abstract = "We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine ([3H]TAZ) were treated with trypsin (T), a-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66 000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between p/ 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P37 revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms <47K and T-generated forms <3IK) were no longer immunoreactive. In contrast, use of the antibody H222Spγ revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.",
author = "Katzenellenbogen, {Benita S} and Greene, {Geoffrey L.} and Elliston, {Jonathan F.} and Monsma, {Frederick J.} and Springer, {Penelope A.} and Ziegler, {Yvonne S.}",
year = "1987",
month = "1",
day = "1",
doi = "10.1021/bi00382a043",
language = "English (US)",
volume = "26",
pages = "2364--2373",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "8",

}

TY - JOUR

T1 - Structural Analysis of Covalently Labeled Estrogen Receptors by Limited Proteolysis and Monoclonal Antibody Reactivity

AU - Katzenellenbogen, Benita S

AU - Greene, Geoffrey L.

AU - Elliston, Jonathan F.

AU - Monsma, Frederick J.

AU - Springer, Penelope A.

AU - Ziegler, Yvonne S.

PY - 1987/1/1

Y1 - 1987/1/1

N2 - We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine ([3H]TAZ) were treated with trypsin (T), a-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66 000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between p/ 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P37 revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms <47K and T-generated forms <3IK) were no longer immunoreactive. In contrast, use of the antibody H222Spγ revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.

AB - We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine ([3H]TAZ) were treated with trypsin (T), a-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66 000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between p/ 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P37 revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms <47K and T-generated forms <3IK) were no longer immunoreactive. In contrast, use of the antibody H222Spγ revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.

UR - http://www.scopus.com/inward/record.url?scp=0023255738&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023255738&partnerID=8YFLogxK

U2 - 10.1021/bi00382a043

DO - 10.1021/bi00382a043

M3 - Article

C2 - 3620450

AN - SCOPUS:0023255738

VL - 26

SP - 2364

EP - 2373

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 8

ER -