Ligands Induce Conformational Changes in the Carboxyl-Terminus of Progesterone Receptors which are Detected by a Site-Directed Antipeptide Monoclonal Antibody
Nancy L. Weigel, Paul Prendergast, Dean P. Edwards
Candace Magda
A. Beck, Altmann,
Patricia A. Estes, Kurt Christensen,
and
Baylor College of Medicine (N.L.W.) Cell Biology Department Houston, Texas 77030 University of Colorado Health Sciences Center Department of Pathology Denver, Colorado 80262
We have prepared a monoclonal antibody, C-262, to a synthetic peptide that contains the carboxy-terminal 14 amino acids from progesterone receptors (PR). This sequence is 100% conserved in all species of PRs that have been cloned to date, suggesting that this antibody will recognize all mammalian and avian PR. The C-262 antibody recognizes both native and denatured forms of the receptor. However, it does not recognize PR when they are bound to the hormone agonists progesterone or R5020. Surprisingly the antibody does recognize PR when they are bound to the steroid antagonist RU466. This suggests that progestin agonists induce a conformational change in the receptor that occludes the C-262 epitope in the carboxyl-terminus, whereas unliganded receptors and receptors bound with RU466 assume distinct conformations that leaves the C-terminal tail accessible to the C-262 antibody. (Molecular Endocrinology 6: 1565-1597, 1992)
tors (PRs) in human breast cancer cells (4) and in chicken oviduct (5, 6) differ from other steroid hormone receptors since they contain two ligand binding polypeptides, termed A and B receptors. PR-A is a truncated form of PR-B that appears to arise as a result of alternate use of a downstream translation start site on a single PR mRNA (7-8) or through a separate mRNA produced through a second promoter in the receptor gene (9). The functional role for the A and B receptors is not known, but there is evidence that the two receptor isoforms exhibit differential target gene specificities, presumably mediated through the N-terminal domain (10). Monoclonal antibodies (MAbs) produced to the estrogen receptor (ER) (11, 12), PR (13-17) glucocorticoid receptor (GR) (18-20) and androgen receptors (21) have been instrumental in advancing our understanding of steroid receptor molecules. These MAbs have been valuable for analyzing structural properties of receptors, for immunocytochemical localization of receptors, and clinically for immunoassay of ER/PR in breast tumors (see reviews, Refs. 22-24). Most MAbs produced so far have been generated against purified native receptors. For the most part antibodies produced in this manner recognize epitopes in the N-terminal domain, since this is the most variable antigenic portion of receptors (25). The failure to produce antibodies with high frequency to DNA and steroid binding domains is likely due to a high degree of conservation in these regions. To produce antibodies to specific functional regions, second generation epitope-specific antibodies have been raised by use of synthetic peptides as antigens. Most of these are rabbit polyclonal serum antibodies (26-31) however, antipeptide monoclonal antibodies have also been produced (32, 33). The antipeptide antibodies produced so far have been raised
INTRODUCTION Receptors for steroid hormones, vitamin D3, thyroid hormone, and retinoic acid represent a superfamily of ligand-activated transcription factors that share a common modular structure. This includes a variable Nterminal domain (A/B region), and highly conserved domains for DNA binding (region C) and steroid binding (region E/F). Region D is hydrophilic and is believed to provide a hinge between the steroid and DNA binding domains (see reviews, Refs. l-3). Progesterone recep0888-8809/92/l 585-i 597$03.00/O Molecular Endocrinology Copyright 0 1992 by The Endocrine
Society
1585 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 November 2015. at 02:55 For personal use only. No other uses without permission. . All rights reserved.
MOL 1586
ENDO.
against sequences from the amino-terminal side of the DNA binding domain (30, 32) within the DNA binding domain (26, 28, 29, 33) or within the hinge region (27, 29). Various antibodies that recognize sequences in the DNA binding domain or the hinge region have the interesting property that their binding sites are occluded in the inactive 8-10s oligomeric complex. Antibody recognition has been reported to require receptor activation and dissociation from the oligomeric complex. Antibodies directed to the DNA binding region of various receptors have also been reported to inhibit receptorDNA binding. The C-terminal steroid binding domain is a structurally complex region that is responsible for multiple functions. In addition to providing structural features for the hormone binding site, this domain in various receptors has been shown to contain sequences required for receptor dimerization and for binding of heat shock protein 90. Receptor-associated heat shock protein 90 is thought to inhibit DNA-binding activity in the absence of hormone. The C-terminus also harbors a liganddependent transcriptional activation domain that presumably provides a surface for receptor interaction with other transcription factors (see reviews, Refs. l-3, 9). Deletion of only 42 carboxyl-terminal amino acids from PR causes the loss of progesterone binding, suggesting that the C-terminal tail of PR is important for ligand binding (34). Several variant truncated mRNAs for steroid receptors have been detected in some breast cancers, suggesting that aberrant forms of receptors may play a role in progression of some breast tumors (3536). However, it is not yet known whether truncated receptors are actually expressed. In order to provide a new probe for functional studies of the complex Cterminal domain and for detection of truncated receptors in breast cancer, we have prepared a monoclonal antibody, C-262, to the extreme carboxyl-terminal sequence of PR. Because this region is 100% conserved in all PRs whose sequences are known (9, 37-39) this antibody should react with PRs across all species. The present study describes some interesting and unexpected properties of this C-terminal-specific antibody. The MAb recognizes PR in solution or when complexed to DNA only when receptors are unliganded or bound with the progesterone antagonist RU486, but not when they are bound with the hormone agonists progesterone or R5020. The C-262 MAb also blocks binding of R5020 to human PR (hPR) but not binding of RU486. The ability of C-262 to discriminate between agonist and antagonist bound PR suggests that progesterone and RU486 induce distinct conformational changes in the C-terminus of receptor. The C-262 MAb therefore is a useful reagent not only to detect Nterminally truncated receptors but also to probe for ligand-induced conformational changes in the C-terminus of PRs.
Vo16No.10
RESULTS MAbs Raised Against a C-Terminal Synthetic Peptide Recognize Authentic PR Under Native and Denaturing Conditions MAbs were produced against a synthetic peptide containing the C-terminal 1Camino acid sequences from PR. Screening of hybridomas was based on the ability of secreted MAbs to recognize the peptide antigen by enzyme-linked immunoabsorption assay (ELISA) and to also react with authentic hPR by Western blot assay. This resulted in isolation of two murine immunoglobulin G, (IgG,) MAbs, designated C-262 and C-l 612. Although the two MAbs were derived from different parental hybridoma cultures, their reactivities with hPR were indistinguishable and are thus likely to be the same or similar antibodies. Subsequent studies described were conducted with C-262, but identical results were obtained with C-1612. Since this antibody should recognize PR from different species, we chose two substantially different PRs, chicken PR (cPR) and hPR for further characterization. Both cPR and hPR are expressed as A and B isoforms (4-g) but the cPR isoforms [PR-B = 86 kilodaltons (kDa) and PR-A = 72 kDa) are smaller than the corresponding isoforms of human receptors (PR-B = 120 kDa and PR-A = 94 kDa). Figure 1 is a Western blot of chicken oviduct cytosol with the C-262 MAb and another MAb, PR-22, that was prepared against native cPR by Sullivan and coworkers (14). Comparisons of lane 1 (C-262) and lane 4 (PR-22) show that the C-262 MAb is highly specific and recognizes the same PR-A and PR-B bands as those recognized by the PR-22 MAb (14). Preincubation of C-262 with excess C-terminal peptide used as antigen (lane 2) inhibits Western blot binding of C-262 to
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Nancy L. Weigel, Paul Prendergast, Dean P. Edwards
Candace Magda
A. Beck, Altmann,
Patricia A. Estes, Kurt Christensen,
and
Baylor College of Medicine (N.L.W.) Cell Biology Department Houston, Texas 77030 University of Colorado Health Sciences Center Department of Pathology Denver, Colorado 80262
We have prepared a monoclonal antibody, C-262, to a synthetic peptide that contains the carboxy-terminal 14 amino acids from progesterone receptors (PR). This sequence is 100% conserved in all species of PRs that have been cloned to date, suggesting that this antibody will recognize all mammalian and avian PR. The C-262 antibody recognizes both native and denatured forms of the receptor. However, it does not recognize PR when they are bound to the hormone agonists progesterone or R5020. Surprisingly the antibody does recognize PR when they are bound to the steroid antagonist RU466. This suggests that progestin agonists induce a conformational change in the receptor that occludes the C-262 epitope in the carboxyl-terminus, whereas unliganded receptors and receptors bound with RU466 assume distinct conformations that leaves the C-terminal tail accessible to the C-262 antibody. (Molecular Endocrinology 6: 1565-1597, 1992)
tors (PRs) in human breast cancer cells (4) and in chicken oviduct (5, 6) differ from other steroid hormone receptors since they contain two ligand binding polypeptides, termed A and B receptors. PR-A is a truncated form of PR-B that appears to arise as a result of alternate use of a downstream translation start site on a single PR mRNA (7-8) or through a separate mRNA produced through a second promoter in the receptor gene (9). The functional role for the A and B receptors is not known, but there is evidence that the two receptor isoforms exhibit differential target gene specificities, presumably mediated through the N-terminal domain (10). Monoclonal antibodies (MAbs) produced to the estrogen receptor (ER) (11, 12), PR (13-17) glucocorticoid receptor (GR) (18-20) and androgen receptors (21) have been instrumental in advancing our understanding of steroid receptor molecules. These MAbs have been valuable for analyzing structural properties of receptors, for immunocytochemical localization of receptors, and clinically for immunoassay of ER/PR in breast tumors (see reviews, Refs. 22-24). Most MAbs produced so far have been generated against purified native receptors. For the most part antibodies produced in this manner recognize epitopes in the N-terminal domain, since this is the most variable antigenic portion of receptors (25). The failure to produce antibodies with high frequency to DNA and steroid binding domains is likely due to a high degree of conservation in these regions. To produce antibodies to specific functional regions, second generation epitope-specific antibodies have been raised by use of synthetic peptides as antigens. Most of these are rabbit polyclonal serum antibodies (26-31) however, antipeptide monoclonal antibodies have also been produced (32, 33). The antipeptide antibodies produced so far have been raised
INTRODUCTION Receptors for steroid hormones, vitamin D3, thyroid hormone, and retinoic acid represent a superfamily of ligand-activated transcription factors that share a common modular structure. This includes a variable Nterminal domain (A/B region), and highly conserved domains for DNA binding (region C) and steroid binding (region E/F). Region D is hydrophilic and is believed to provide a hinge between the steroid and DNA binding domains (see reviews, Refs. l-3). Progesterone recep0888-8809/92/l 585-i 597$03.00/O Molecular Endocrinology Copyright 0 1992 by The Endocrine
Society
1585 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 03 November 2015. at 02:55 For personal use only. No other uses without permission. . All rights reserved.
MOL 1586
ENDO.
against sequences from the amino-terminal side of the DNA binding domain (30, 32) within the DNA binding domain (26, 28, 29, 33) or within the hinge region (27, 29). Various antibodies that recognize sequences in the DNA binding domain or the hinge region have the interesting property that their binding sites are occluded in the inactive 8-10s oligomeric complex. Antibody recognition has been reported to require receptor activation and dissociation from the oligomeric complex. Antibodies directed to the DNA binding region of various receptors have also been reported to inhibit receptorDNA binding. The C-terminal steroid binding domain is a structurally complex region that is responsible for multiple functions. In addition to providing structural features for the hormone binding site, this domain in various receptors has been shown to contain sequences required for receptor dimerization and for binding of heat shock protein 90. Receptor-associated heat shock protein 90 is thought to inhibit DNA-binding activity in the absence of hormone. The C-terminus also harbors a liganddependent transcriptional activation domain that presumably provides a surface for receptor interaction with other transcription factors (see reviews, Refs. l-3, 9). Deletion of only 42 carboxyl-terminal amino acids from PR causes the loss of progesterone binding, suggesting that the C-terminal tail of PR is important for ligand binding (34). Several variant truncated mRNAs for steroid receptors have been detected in some breast cancers, suggesting that aberrant forms of receptors may play a role in progression of some breast tumors (3536). However, it is not yet known whether truncated receptors are actually expressed. In order to provide a new probe for functional studies of the complex Cterminal domain and for detection of truncated receptors in breast cancer, we have prepared a monoclonal antibody, C-262, to the extreme carboxyl-terminal sequence of PR. Because this region is 100% conserved in all PRs whose sequences are known (9, 37-39) this antibody should react with PRs across all species. The present study describes some interesting and unexpected properties of this C-terminal-specific antibody. The MAb recognizes PR in solution or when complexed to DNA only when receptors are unliganded or bound with the progesterone antagonist RU486, but not when they are bound with the hormone agonists progesterone or R5020. The C-262 MAb also blocks binding of R5020 to human PR (hPR) but not binding of RU486. The ability of C-262 to discriminate between agonist and antagonist bound PR suggests that progesterone and RU486 induce distinct conformational changes in the C-terminus of receptor. The C-262 MAb therefore is a useful reagent not only to detect Nterminally truncated receptors but also to probe for ligand-induced conformational changes in the C-terminus of PRs.
Vo16No.10
RESULTS MAbs Raised Against a C-Terminal Synthetic Peptide Recognize Authentic PR Under Native and Denaturing Conditions MAbs were produced against a synthetic peptide containing the C-terminal 1Camino acid sequences from PR. Screening of hybridomas was based on the ability of secreted MAbs to recognize the peptide antigen by enzyme-linked immunoabsorption assay (ELISA) and to also react with authentic hPR by Western blot assay. This resulted in isolation of two murine immunoglobulin G, (IgG,) MAbs, designated C-262 and C-l 612. Although the two MAbs were derived from different parental hybridoma cultures, their reactivities with hPR were indistinguishable and are thus likely to be the same or similar antibodies. Subsequent studies described were conducted with C-262, but identical results were obtained with C-1612. Since this antibody should recognize PR from different species, we chose two substantially different PRs, chicken PR (cPR) and hPR for further characterization. Both cPR and hPR are expressed as A and B isoforms (4-g) but the cPR isoforms [PR-B = 86 kilodaltons (kDa) and PR-A = 72 kDa) are smaller than the corresponding isoforms of human receptors (PR-B = 120 kDa and PR-A = 94 kDa). Figure 1 is a Western blot of chicken oviduct cytosol with the C-262 MAb and another MAb, PR-22, that was prepared against native cPR by Sullivan and coworkers (14). Comparisons of lane 1 (C-262) and lane 4 (PR-22) show that the C-262 MAb is highly specific and recognizes the same PR-A and PR-B bands as those recognized by the PR-22 MAb (14). Preincubation of C-262 with excess C-terminal peptide used as antigen (lane 2) inhibits Western blot binding of C-262 to
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