The Human Estrogen Receptor-α Is a Ubiquitinated Protein Whose Stability Is Affected Differentially by Agonists, Antagonists, and Selective Estrogen Receptor Modulators

The human estrogen receptor α-isoform (ERα) is a nuclear transcription factor that displays a complex pharmacology. In addition to classical agonists and antagonists, the transcriptional activity of ERα can be regulated by selective estrogen receptor modulators, a new class of drugs whose relative agonist/antagonist activity is determined by cell context. It has been demonstrated that the binding of different ligands to ERα results in the formation of unique ERα-ligand conformations. These conformations have been shown to influence ERα-cofactor binding and, therefore, have a profound impact on ERα pharmacology. In this study, we demonstrate that the nature of the bound ligand also influences the stability of ERα, revealing an additional mechanism by which the pharmacological activity of a compound is determined. Of note we found that although all ERα-ligand complexes can be ubiquitinated and degraded by the 26 S proteasome in vivo, the mechanisms by which they are targeted for proteolysis appear to be different. Specifically, for agonist-activated ERα, an inverse relationship between transcriptional activity and receptor stability was observed. This relationship does not extend to selective estrogen receptor modulators and pure antagonists. Instead, it appears that with these compounds, the determinant of receptor stability is the ligand-induced conformation of ERα. We conclude that the different conformational states adopted by ERα in the presence of different ligands influence transcriptional activity directly by regulating cofactor binding and indirectly by modulating receptor stability. The human estrogen receptor α-isoform (ERα) is a nuclear transcription factor that displays a complex pharmacology. In addition to classical agonists and antagonists, the transcriptional activity of ERα can be regulated by selective estrogen receptor modulators, a new class of drugs whose relative agonist/antagonist activity is determined by cell context. It has been demonstrated that the binding of different ligands to ERα results in the formation of unique ERα-ligand conformations. These conformations have been shown to influence ERα-cofactor binding and, therefore, have a profound impact on ERα pharmacology. In this study, we demonstrate that the nature of the bound ligand also influences the stability of ERα, revealing an additional mechanism by which the pharmacological activity of a compound is determined. Of note we found that although all ERα-ligand complexes can be ubiquitinated and degraded by the 26 S proteasome in vivo, the mechanisms by which they are targeted for proteolysis appear to be different. Specifically, for agonist-activated ERα, an inverse relationship between transcriptional activity and receptor stability was observed. This relationship does not extend to selective estrogen receptor modulators and pure antagonists. Instead, it appears that with these compounds, the determinant of receptor stability is the ligand-induced conformation of ERα. We conclude that the different conformational states adopted by ERα in the presence of different ligands influence transcriptional activity directly by regulating cofactor binding and indirectly by modulating receptor stability. estrogen receptor α-isoform selective estrogen receptor modulator polyacrylamide gel electrophoresis ERα1 resides within the nuclei of target cells in an inactive form in the absence of hormone. Upon binding its cognate ligand estradiol, the receptor undergoes an activating conformational change permitting it to interact with specific cofactors and bind DNA response elements within target gene promoters (1Beekman J.M. Allan G.F. Tsai S.Y. Tsai M.-J. O'Malley B.W. Mol. Endocrinol. 1993; 7: 1266-1274PubMed Google Scholar, 2Horwitz K.B. Jackson T.A. Bain D.L. Richer J.K. Takimoto G.S. Tung L. Mol. Endocrinol. 1996; 10: 1167-1177Crossref PubMed Scopus (835) Google Scholar). The DNA-bound receptor-ligand complex is then capable of either activating or repressing target gene transcription, depending on both the cell and the promoter context. The classical model of ERα action suggests that the role of agonists, such as estradiol, is that of a switch converting the receptor from an inactive to an active form. It now appears that ERα pharmacology is more complex, since it has been observed that different ERα-ligands induce different changes in receptor conformation and that target cells can distinguish between these complexes (3Paige L.A. Christensen D.J. Gron H. Norris J.D. Gottlin E.B. Padilla K.M. Chang C. Ballas L.M. Hamilton P.T. McDonnell D.P. Fowlkes D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3999-4004Crossref PubMed Scopus (396) Google Scholar, 4McDonnell D.P. Clemm D.L. Herman T. Goldman M.E. Pike J.W. Mol. Endocrinol. 1995; 9: 659-669Crossref PubMed Google Scholar, 5Wijayaratne A.L. Nagel S.C. Paige L.A. Christensen D.J. Norris J.D. Fowlkes D.M. McDonnell D.P. Endocrinology. 1999; 140: 5828-5840Crossref PubMed Google Scholar). For instance, the anti-estrogen tamoxifen opposes estrogen action in the breast, whereas it manifests estrogenic activities in bone, the cardiovascular system, and the uterus. Reflecting its complex pharmacology, tamoxifen has recently been reclassified as a selective estrogen receptor modulator (SERM). Additional SERMs have been identified, such as raloxifene, GW5638, TSE424, lasofoxifene, and arzoxifene, each of which has distinct agonist/antagonist profiles (6Xiaodong L. Huebner V. Curr. Opin. Drug Discovery Dev. 2000; 3: 383-398PubMed Google Scholar). The challenge, therefore, has been to understand the mechanism(s) underlying SERM-mediated action and evaluate why these compounds distinguish themselves from pure agonists like estradiol. Analysis of the crystal structure of ERα revealed that the conformation of the agonist-receptor complex is distinct from that formed in the presence of antagonists (7Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.A. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2924) Google Scholar, 8Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2230) Google Scholar). Furthermore, we have used combinatorial phage display to identify a series of peptides whose ability to interact with ERα is regulated by the nature of the bound ligand. Using these probes, we have been able to show that, even among the SERMs, there are significant differences in the overall structure of the receptor (3Paige L.A. Christensen D.J. Gron H. Norris J.D. Gottlin E.B. Padilla K.M. Chang C. Ballas L.M. Hamilton P.T. McDonnell D.P. Fowlkes D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3999-4004Crossref PubMed Scopus (396) Google Scholar). These findings lend support to the hypothesis that conformation is a primary regulator of ERα-coactivator interactions. One of the first coactivators identified, SRC-1, has been shown to enhance estrogen-activated ERα transcriptional activity when overexpressed in target cells (9Onate Tsai S. Tsai M.-J. O'Malley B.W. 1995; PubMed Scopus Google Scholar). In it was observed that of the activity of whereas it has on the activity of Z. O'Malley B.W. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar). The of transcription in ERα action was demonstrated in that that the activity of tamoxifen be by and in cells Z. O'Malley B.W. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar, T.A. Richer J.K. Bain D.L. Takimoto G.S. Tung L. K.B. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar). tamoxifen in in has been shown to be with a in the of T. A.L. M. S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). cofactor is a primary determinant of a ability to distinguish among different of in addition to the conformation of the has been shown to influence the stability of the In it has been shown that in the absence of the of ERα is whereas binding receptor its to A. Endocrinology. PubMed Scopus Google Scholar, J.A. Endocrinology. PubMed Scopus Google Scholar, S. M.E. Endocrinology. PubMed Scopus Google Scholar, A.M. Endocrinology. PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In addition to estradiol, tamoxifen has been shown to ERα A.L. Nagel S.C. Paige L.A. Christensen D.J. Norris J.D. Fowlkes D.M. McDonnell D.P. Endocrinology. 1999; 140: 5828-5840Crossref PubMed Google whereas the has been shown to the of the ERα S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). these it that receptor be an that the of response to an activating ligand. The of transcription has been shown to be by the A. Cell. Full Text PDF PubMed Scopus Google and the of of these appears to directly with transcriptional activity M. S. 1999; PubMed Scopus Google Scholar, M. H. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, D.M. Z. O'Malley B.W. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). human ERα has been shown to be a for in the presence of Z. D.M. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google in is have demonstrated that in the ERα the 26 S proteasome A. 1999; PubMed Scopus Google Scholar, Mol. Endocrinol. 1999; PubMed Google Scholar). it has been that of coactivators like be a in this D.M. Z. O'Malley B.W. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google the mechanism(s) by which ERα is by the 26 S proteasome to be determined. Furthermore, the that in addition to the estradiol, the pure which when bound to ERα is to can induce a of ERα that ERα with in of transcription it appears therefore, that either transcriptional activity and ERα stability are not or that the mechanisms by which and ERα are degraded are not the in this study, we have a series of to evaluate the influence of ERα agonists, antagonists, and SERMs on the stability of the human ERα and between ligand-induced receptor stability and the relative agonist/antagonist activity of an of this nature to the mechanisms underlying the agonist/antagonist activity of ERα-ligands and be in the of for ERα-ligands with unique pharmacological and from was a from A. and was a from T. and human was a from Greene of was from and the and from and from was from was from from and from and from The was a from of The within this was a to be with the of the ERα used in this for the and and the gene have been D. McDonnell D.P. Mol. Endocrinol. 1996; 10: PubMed Scopus Google Scholar). cells in for to with ligand. cells used for the of cells as cell with ligand in for and as 7: PubMed Scopus Google Scholar). of was by to and with The of has been to in an in an of ERα relative to or the cells in for to for in and with for in the used for cells to be with or the for of or cells with and for or in the presence of or ligand in and with of of and as S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of in each was by and for of cell by with the which been with for by a with the for each of these the for the with specific primary to for The in and with in and then by cells in with in or the of to DNA was cells by of For of of and of receptor D.L. McDonnell D.P. Mol. Endocrinol. PubMed Scopus Google S. Mol. Cell. 1999; PubMed Scopus Google or The of DNA was to with the for which was with cells with ligand for or and as in to transcriptional of DNA as A.L. Nagel S.C. Paige L.A. Christensen D.J. Norris J.D. Fowlkes D.M. McDonnell D.P. Endocrinology. 1999; 140: 5828-5840Crossref PubMed Google Scholar). the cells in the presence of ligand for and and for and activity as D. C. A. J.D. McDonnell D.P. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). cells in and with of For of of receptor and of M. L.M. D. Cell. Full Text PDF PubMed Scopus Google The of DNA was to with cells with ligand for by in and as O. T. A. L. D. 1997; PubMed Scopus Google Scholar). was by The of the was and of and by for in the presence of The then with of by the addition of with which they for The the with the and with and by in and electrophoresis and by with the The of these was by a with of ERα and that all in the of the the of ubiquitinated ERα, ERα, the first ERα was for it was the that the that the of ERα have shown that the of this receptor in both and in the absence of is A. Endocrinology. PubMed Scopus Google Scholar, J.A. Endocrinology. PubMed Scopus Google Scholar, S. M.E. 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In this system, ligand we able to demonstrate that ERα are by the nature of the bound ligand It was for instance, that with either or to a in ERα with the in this In with tamoxifen appears to ERα and receptor the and and It is that the ligands influence both the stability of ERα and the activity of the ERα in to the transcriptional from of to we a of ERα stability in ERα in cells was with and in and either or ligand for the The relative of ERα by by shown in and relative to the estradiol, and the of of ERα within of whereas this of We conclude that ERα stability is by the nature of the bound ligand a that the different pharmacological activities of the ERα have for a between the relative transcriptional activity of transcription and of more activity with stability A. D. H. 1993; Full Text PDF PubMed Google Scholar, McDonnell D.P. Mol. Cell. 1996; PubMed Scopus Google Scholar). For instance, it has been shown that by the of transcriptional activity of its stability can be regulated A. D. H. 1993; Full Text PDF PubMed Google Scholar). on these we the ability of an ERα-ligand complex to transcription is to receptor stability. In we this relationship for and ERα, since these ligands have distinct ERα a first we the ligand-induced changes in the stability of and a inactive (6Xiaodong L. Huebner V. Curr. Opin. Drug Discovery Dev. 2000; 3: 383-398PubMed Google Scholar, S. M. Nature. PubMed Scopus Google Scholar). The within of the ligand binding which have been shown to the binding within the receptor The and of this are the as of the it is to transcription in the presence of S. Mol. Cell. 1999; PubMed Scopus Google Scholar, J.D. S. McDonnell D.P. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Norris J.D. Gron H. Paige L.A. Hamilton P.T. D.J. Fowlkes D. McDonnell D.P. Mol. Cell. 1999; PubMed Google Scholar). this a from which to evaluate the relationship between transcriptional activity and receptor stability. The transcriptional activity and stability of the and in an cell We to ERα the of a promoter to the influence of the ligands on the ERα The influence of ligands on the stability of these was demonstrated in we able to the of ligand-induced ERα in this as was observed in the In the cell a of transcriptional activity in the presence of these although the of the ERα, it was to induce the of inactive and These the of a between the ability to transcription and a in receptor stability. This was not when the was to In although activity on both ERα and the impact of these ligands on the was not was ERα and and a relationship between or transcriptional activity and relative impact on stability not be observed. The that the that the stability of the complex are different from that pure or ERα. This is an that suggests that in addition to the ability to transcription, are able to the of ERα. It was to the observed differences in the of and in the presence of both and are to by the in or the of this to interact with specific cofactor complexes as For this we used or in which of the transcriptional of the has been the of receptor have shown that the is able to transcription when to DNA by class of ERα ligand D.P. Clemm D.L. Herman T. Goldman M.E. Pike J.W. Mol. Endocrinol. 1995; 9: 659-669Crossref PubMed Google Scholar). of this transcriptional ERα to transcriptional although in a that does not the ERα transcriptional Using this we the inactive a transcription The is to interact with cofactors such as J.D. S. McDonnell D.P. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Norris J.D. Gron H. Paige L.A. Hamilton P.T. D.J. Fowlkes D. McDonnell D.P. Mol. Cell. 1999; PubMed Google Scholar). The of in with the of transcriptional activity and receptor stability are and the of coactivators to the by estradiol. we the transcriptional activities of and on an promoter in The activity of the on a promoter was as a S. M. Nature. PubMed Scopus Google Scholar). The of these in cells a ligand In this when bound to estradiol, both and activity and both to and and results an in which the is the of ERα, not These that the that ERα for is the overall transcriptional activity and not the of activity when bound to either or a relationship between transcriptional activity and stability was not observed. Specifically, in this tamoxifen both and and and it of and has been and This suggests that, of its to transcription, a of the receptor be in the of the stability of ERα. and degraded all of the to transcription and the structure of These results the for of the complex from complexes formed in the presence of and as as the for receptor in the presence of from and on the that transcriptional activity and receptor stability are in the presence of estradiol, we that since DNA binding transcriptional in DNA binding also influence the stability of receptor-ligand this we an ERα in which the DNA binding has been in a that it from directly with estrogen response elements we the transcriptional activity of this on both a classical estrogen response transcription is by ERα binding to and on an transcription is by ERα binding to that are to an M. Cell. Full Text PDF PubMed Scopus Google Scholar, A. D. 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The from the to and with an The of was observed when both ERα and a of ERα was able to to the ubiquitinated of ERα not in the absence of and binding of ERα to the appears to be by the within the and not the in the DNA binding the of these of ERα, that the receptor to be ubiquitinated the ERα has a it is that an role in the of ERα. was that the ERα not directly with the of formed each For instance, although the ERα in cells with are that from cells and the of the first of relative to ERα appears to be This a in the of we the of the formed it is the all to the ERα These that ERα is the ubiquitinated whereas ERα was the This with that is the of ERα and that is the The ability of to the of is to why this ligand the Furthermore, on that when bound to receptor we the of in the absence and presence of and we able to show that the of of was in the presence of the of of ERα was not different from that of the ERα. the to which ERα was ubiquitinated was not different from This the mechanism by which from that of and suggests that and transcriptional can influence the which ERα the of the classical of ERα the role of an was that of an all or it was to understand different binding the are able to distinct It is now that the model is and that the receptor undergoes different binding different ligands which cofactors bind to the In support of this we recently a on ERα that is tamoxifen binding and that peptides that bind to this tamoxifen activity not the activity of SERMs J.D. Paige L.A. Christensen D.J. Chang D. Hamilton P.T. Fowlkes D.M. McDonnell D.P. 1999; PubMed Scopus Google Scholar). therefore, receptor conformation its cofactor an activity that the relative agonist/antagonist activity of Of has been on the cofactors that interact with ERα in the presence of different In this study, we demonstrate that ERα stability is by the nature of the bound an activity that in receptor One of the findings of this was that the of of ERα to directly with transcriptional this relationship was not for the SERMs or pure antagonists We conclude from this that the of and complexes not in the of these revealed that ERα is a ubiquitinated in the cell and that the to which it is ubiquitinated is not the in the presence of all SERMs in a we observed that the complex formed in the presence of tamoxifen was the and that this a of ERα. the complex formed in the presence of was the a of the It therefore, with to tamoxifen and that receptor stability and its of are the influence of these ligands on the receptor stability is not directly to transcriptional it is that it is the ligand-induced conformational changes in ERα that influence its by modulating its with of the The differences in the stability of the and complexes are in of the of these This of from which that differences in the structure of a ligand can to profound differences in ERα pharmacology. The ability of these ligands to of ERα in a is to be For instance, the that binding ERα have Specifically, of with tamoxifen to the of switch from by tamoxifen to tamoxifen for Google Scholar). the mechanisms for this change are not it is that of tamoxifen to the of a of within the that tamoxifen as an ERα interact in an with a transcription that is not in converting it from an to an in the J.D. Paige L.A. Christensen D.J. Chang D. Hamilton P.T. Fowlkes D.M. McDonnell D.P. 1999; PubMed Scopus Google Scholar). We that the ability of tamoxifen to ERα in cells is to be For instance, in the of have been shown to be to ERα in the absence of ligand or in the presence of D.J. Mol. 1995; PubMed Scopus Google Scholar). of ligand the presence of receptor is for target gene not in it is that of ERα be to changes in of the it has been shown that can be with either A. D. 1995; PubMed Scopus Google or both of which ERα J.D. McDonnell D.P. Google Scholar). We have demonstrated that the of ERα in the presence of directly with transcriptional In are to of D.M. Z. O'Malley B.W. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google with the that we have been able to show that it is overall transcriptional activity of ERα, and not the of a in the that ERα stability. This was demonstrated by that within transcriptional of the which transcriptional in the presence of estradiol, the stability of the when a was on these transcriptional activity was and the receptor stability the This suggests that it is not that is by the that it is transcriptional activity as a that Furthermore, the that the of of and receptor ERα is suggests that is not to of ERα. on these we mechanisms by which transcriptional activity influence receptor the complex of the the with the receptor is to active transcription and of the proteasome that are in the proteasome such as McDonnell D.P. Mol. Cell. 1996; PubMed Scopus Google Dev. 1998; PubMed Scopus Google 26 S proteasome J.W. Nature. 1995; PubMed Scopus Google Scholar, C. D. J.M. V. H. 1995; and H. U. 1999; Full Text Full Text PDF PubMed Scopus Google have been shown to interact with or is to it is the formation of a transcriptional complex, or the of transcription that the of ERα when bound to estradiol. The differences in the of an ERα-ligand complex to be in by receptor have demonstrated that ERα is in a within the nuclei in the absence of ligand H. D. G.L. Mol. Cell. 1999; 10: PubMed Scopus Google Scholar). The addition of either or results in a and of ERα a whereas the addition of results in of ERα in the S. Sci. 1993; PubMed Google Scholar, H. D. G.L. Mol. Cell. 1999; 10: PubMed Scopus Google Scholar). the of proteasome within the and the is distinct J.A. Full Text PDF PubMed Google Scholar, A.M. 1997; PubMed Scopus Google it is that these distinct ERα-ligand complexes interact with distinct proteasome complexes that different In we have demonstrated that ERα binding the are able to the stability of the is with transcriptional are for regulating the stability of and pure We that the differences in stability observed in the presence of different SERMs and the pure differences in ERα conformation by these which the of the receptor with different of the It is that ERα by different mechanisms in different cells depending on the of that this to be the then this activity be in the of new of ERα We Greene for the of for this Chang and Norris for and for