0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 265, No. 18, Issue of June 25. pp. 10740-10745,199O

Printed in U.S. A.

The Non-DNA-binding Heterooligomeric Form of Mammalian Steroid Hormone Receptors Contains a hsp90-bound 59-Kilodalton Protein*

(Received for publication, October 26, 1989)

Jack-Michel Renoir, Christine Radanyi, Lee E. Faberg, and Etienne-Emile Baulieu From the Institut National de la Sante! et de la Recherche Midicale, Unit6 de Recherche sur les Communications Hormonales KJ33), and Facultt? de M&de&e, Laboratoire Hormones, 94275 Biktre Cedex, France and the $ Department of Obstetrics/ Gynecology and Physiology, Medical College of Ohio, Toledo. Ohio 43699

Untransformed cytosol receptors for progesterone (PR), androgen (AR), estrogen (ER), and glucocorti- costeroid (GR) in rabbit tissues contain a 59-kDa pro- tein (p59) (Tai, P. K. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochem- istry 25,5269-5275) and a 90-kDa heat shock protein (hsp90). In the present study, receptors from calf uterus (PR, AR, ER, and GR) and from human breast cancer MCF7 cells (PR and GR) were also shown to be comprised of hsp90 and p59. These heterooligomer receptor complexes were stabilized both by transition metal oxyanions (molybdate and tungstate) and chem- ical cross-linking with dimethylpimelimidate. In 0.4 M

KCl, tungstate-stabilized (but not molybdate-stabi- lized) PR, AR, ER, and GR retained hsp90, but lost p59. Dimethylpimelimidate cross-linking prevented p59 dissociation from hsp90-receptor complexes. Sta- bilization with tungstate and/or cross-linking permit- ted immunoaffinity purification of untransformed rab- bit as well as calf PR and ER on EC1-Affi-Gel 10 column (an anti-p59 immunoadsorbant). Combined im- munoaffinity purification and cross-linking experi- ments indicated that p59 is bound to hsp90 in the cytosol. We propose that in the nontransformed steroid receptor, p59 interacts with hsp90 rather than with the hormone binding subunit.

Untransformed mammalian and avian steroid receptors are large molecules with sedimentation coefficients of 8-10 S in low salt density gradients (see Refs. 1 and 2 for recent re- views). These heterooligomers have been observed at physio- logical ionic strength (3), do not bind DNA-cellulose (4-6), and are stabilized by transition metal oxyanions (7, 8). Cur- rent evidence suggests that 8-10 S receptors are present in situ (9, 10). Although untransformed receptor structure is complex, two non-steroid-binding components have been characterized. The first, a 90-kDa non-hormone-binding pro- tein (3, ll), has been identified as a heat shock protein

* This work was supported in part by the Institut National de la Santa! et de la Recherche Mkdicale. the Centre National de la Re- cherche Scientifique, and the United States Public Health Service (Grant DK 41881). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(hsp90)’ (12-14). Cross-linking studies in situ (15) as well as in cytosol (16) verified the presence of hsp90 in nontrans- formed receptors. The release of hsp90 during hormone- induced transformation’ (activation) has been ascribed to be a prerequisite for receptor binding to hormone response ele- ments of target genes (reviews in Refs. 1 and 2).

A second receptor-associated protein (p59) has also been detected. This was made possible by use of the monoclonal antibody KN382/EC1 (ECI) (17, 18). p59 has been found in nontransformed rabbit e&radio1 (ER), progesterone (PR), glucocorticosteroid (GR), and androgen (AR) receptors. Al- though p59 is an abundant protein in several rabbit tissues (18), its presence in tissues of other animal species has not been well documented.

In the present study, we utilized immunoaffinity chroma- tography of covalently cross-linked and oxyanion-stabilized receptors to establish that p59 is part of the rabbit nontrans- formed PR, GR, ER, and AR. Comparable results were found with calf uterine and MCF7 receptors. We also present data suggesting that a single molecule of p59 is bound per 8-10 S heterooligomeric rabbit PR, and that p59 is bound directly to hsp90 rather than to the receptor.

EXPERIMENTAL PROCEDURES3

RESULTS AND DISCUSSION

Stabilization of the Rabbit Uterus PR by Oxyanions and Chemical Cross-linking-In the absence of KCl, the rabbit uterus cytosolic PR prepared in PG or in PGM buffer migrates at 9.5 S. As shown in Fig. la, the 9.5 S rabbit uterine receptor sedimented at -11 S after incubation with ECI. This occurred irrespective of the presence (or absence) of molybdate or tungstate. Addition of a second antibody (anti-mouse IgG) partially pelleted the receptor (Fig. la). The steroid had no

1 The abbreviations and trivial names used are: hsp90, heat shock protein of M, = 90,000; R5020,17,21-[17-methyl-3H]dimethylpregna- 4,9 (lo)-diene-3,20-dione; RU486,[6,7-3H1-17fl-hydroxy-llfi-(4-di- methylaminophenyl)-l70c-(l-propyn~l)-oes~~a-4,9-~ien-3~one;R1881, 1780H,17a-methyl. estra. 4,9,11,triene-3-one; Ez, estradiol: PR, pro- gesterone receptor;’ ER, e&radio1 receptor; GR, glucocorticosteioid receptor: AR. androeen receptor: DMP, dimethvlpimelimidate; u59, pro&in of M; = 59,0‘60; AC88, minoclonal antidohy raised against a orotein of M. = 88.000 from Achlva ambisexualis: EC, KN382/EC,. . monoclonal antibody raised against nontransformed progesterone receptor from rabbit uterus (16).

* The term transformation will be used throughout this paper to describe the process whereby the receptor is dissociated from a -9 S to a -4-6 S form.

3 Portions of this paper (including “Experimental Procedures,” Figs. 1,2,3, and 5, and Tables I and II) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

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p59 in Nontransformed Steroid Receptors

effect on this assembly because similar results were obtained with the use of either the agonist (R5020) or the antagonist (RU486). We suggest that the incomplete shift of the 9.5 S PR may be best explained by a partial loss of p59 during cytosol preparation.

In high salt medium, molybdate-stabilized R5020-PR is recovered as a 4.5 S species in contrast to tungstate-stabilized PR, which remains 8.5 S (Fig. lb). This argues in favor of a greater resistance of the latter to the dissociating effect of high ionic strength as already observed with the mineralocor- ticosteroid receptor (27). This effect is still observed when the gradients also contain 20 mM tungstate ions as opposed to the situation when molybdate ions are present (see Table I). None of the 4.5 or 8.5 S forms seen in Fig. lb is recognized by EC,, suggesting the absence of p59.

In addition, it should be noted that the RU486-bound, molybdate-stabilized PR migrates as a 6-7 S form in the presence of high salt (Fig. lb), but the tungstate-stabilized PR, regardless of whether it is bound to R5020 or RU486, is still recovered as an 8.5 S species. Again, none of these forms is displaced by EC,. The agonist-bound 6-7 S PR form had already been observed in the rabbit uterus (28), and, in human breast cancer MCF7 (29) and T-47D (30) cells, antagonist- bound 6 S PR forms were also detected. These forms could represent a dimer of PR, with reference to recent data on dimers of ER (31) and GR (32), and is not further studied in this paper.

To attain almost complete stabilization of the heterooligo- merit structure, we found it was necessary to cross-link the receptor with dimethylpimelimidate (DMP). The sedimenta- tion coefficient in this instance (rabbit PR) was 8.5 S in 0.4 M KC1 (Fig. lc). After incubation with ECI, it sedimented at 9.6 S, thus demonstrating that DMP cross-linking did not denature the EC1 epitope (Fig. lc). Addition of a second antibody further increased the molecular mass (Fig. lc). This adds further credence to the notion that the loss of EC, binding to the high salt tungstate-stabilized PR (Fig. lb and Table I) was due to the release of p59.

We can conclude from the experiments summarized in Table I that, 1) contrary to molybdate ions, tungstate ions inhibit the 0.4 M KCl-induced transformation of the cytosol receptor and the release of hsp90 (6), 2) molybdate and tungstate ions are unable to prevent p59 dissociation from the heterooligomer, and 3) RU486 seems to be more efficient than R5020 in maintaining the molybdate-stabilized PR in a larger form (Fig. lb), as already observed (33). A similar stabilizing effect was also reported for RU486 binding to the heterooligomeric GR (34).

Molecular Weight Estimates of Untransformed Rabbit PR- M, values of both native and cross-linked PR are summarized in Table II. High salt treatment of tungstate-stabilized native PR reduced the size of the receptor from 317 kDa to 250 kDa. This difference, in concert with the loss of EC1 binding, likely reflects p59 dissociation and suggests the presence of a single molecule of p59 in the 9.5 S PR. Parallel experiments with cross-linked PR gave an apparent molecular weight of 319,000 and 290,000. Knowing that the high salt cross-linked PR binds EC1, we suggest that this decrease of the calculated M, is a direct effect of the salt on the three-dimensional confor- mation of the complex rather than dissociation of subunits. Calculation of M, based on hydrodynamic parameters after KCl-induced release of p59 from the tungstate-stabilized het- eromeric rabbit uterus PR also suggests the presence of a single molecule of p59 in the 9.5 S PR.

Immunoaffinity Purification of the Rabbit PR on EC1-Affi- Gel IO-Both native and DMP-cross-linked rabbit uterine PR

12345676

-200

-- -92

* -69

*, m .

-46

FIG. 4. Immunoblots of samples from different sources with EC,. Aliquots of cytosol, prepared in PG buffer, were subjected to SDS-PAGE (7.5% acrylamide) prior to immunoblotting. Cytosol sam- ples included: lane 1, chick oviduct (20 fig of protein); lane 2, rabbit uterus (15 hg of protein); lane 3, calf uterus (25 pg of protein); lane 4, rat liver (20 pg of protein); lane 5, rabbit kidney (12 pg of protein); lane 6, COLT-7 cells (25 pg of protein); lane 7, 0.4 M KC1 nuclear extract from rabbit uterus (40 pg of protein); lane 8, human liver cytosol (100 fig of protein). Position of markers (kDa) are indicated on the right of the figure.

were eluted from EC1-Affi-Gel 10 immunoadsorbant as 9.5 S complexes (Fig. 2a). EC1 displaced the [3H] RU486-bound receptors to -11 S, indicating the retention of p59. DMP cross-linking of the immunoaffinity eluate demonstrated that this material also contained p59. Again EC1 increased the size of the PR (Fig. 2b).

Stabilization of Other Steroid Receptors by Tungstate and DMP Cross-linking-As shown earlier with the PR, p59 was also a KCl-dissociable component of the untransformed uter- ine androgen receptor (Fig. 3, a and b). Although tungstate maintained an -8 S structure, it was ineffective in retaining p59 in the untransformed complex. This would suggest that tungstate functions to hold hsp90 to the receptor. Similar results were obtained with rabbit uterine ER and GR (data not shown). In contrast, cross-linked AR migrating at -9 S in 0.4 M KC1 gradients (no oxyanions) contained p59, since EC, displaced the radioactive peak to -10 S (Fig. 3~). Alto- gether, these results let us suggest that both stabilizing effects of tungstate and cross-linking could be general for all untrans- formed steroid hormone receptors.

Species and Organ Distribution of p59-Immunoblotting with EC, revealed a single 59-kDa protein in extracts of either rabbit uterine cytosol or nuclei (Fig. 4, lanes 2 and 7). In addition to p59, EC1 recognized several other proteins in rabbit kidney cytosol (lane 5). We found a single 59-kDa protein in the cytosol of human liver (lane 8), calf uterus (lane 3), and mink CCL4 lung cells4 (not shown). A doublet of 57- 59 kDa was observed in monkey kidney COS-7 cells (lane 6) as has been reported for human lymphoblast cytosol (35). No signal was noted with either chicken oviduct (lane 1) or rat liver cytosol (lane 4). Protein bands less than 59 kDa may have resulted from proteolysis of p59. In some instances, signals at -75 and -130 kDa were seen. We believe this to be due to nonspecific characteristics of the system because they can be seen in a sample devoid of immunoreactive p59 (lane 4).

Although p59 is found in the cytosol fraction of homoge- nates, it is also present in the 0.4 M KC1 nuclear extract of rabbit uterus cells (lane 7) as already reported (36). No lactate dehydrogenase activity was measured in the nuclear extract arguing in favor of the absence of cytoplasmic contamination. Moreover, immunohistochemical data (37) have indicated that p59 is a nuclear protein. This implies that, as unoccupied steroid receptors, p59 would leak from the nucleus into the cytosol compartment upon tissue fractionation. Recent results

4 J.-M. Renoir and A. Zimiecki, unpublished data.

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10742 p59 in Nontransformed Steroid Receptors

suggest the presence of a nontransformed form of PR in the nucleus in absence of hormone, thus implying that a part of p59 could leak in association with both PR and hsp90 already located in the nucleus (36).

p59 in Untransformed Calf Receptors-EC, increased the sedimentation coefficient of the nontransformed calf uterine ER from 8.9 S to 11 S (Fig. 5) suggesting the presence of p59 in the complex. Although 0.4 M KC1 dissociated the unstabil- ized receptor (data not shown), its effect upon cross-linked ER was minimal, reducing the sedimentation coefficient from -8.9 S to -8.2 S (Fig. 5b). However, p59 was present in the complex because EC, increased the size of the cross-linked untransformed ER (Fig. 5b). Addition of a second antibody displaced the ER.ECi complexes to the bottom of the tube (Fig. 5, a and b). Unrelated second antibody (goat anti-rabbit IgG) had no effect on the sedimentation behavior of the receptor (data not shown). The amount of 4-5 S ER is higher after DMP treatment of the calf uterus cytosol (Fig. 5b) than before (Fig. 5a); this could be due to partial release of ER from the heterooligomer during the cross-linking procedure.

Sucrose gradient fractions of Fig. 5 were subjected to elec- trophoresis and immunoblotting to locate the three compo- nents (receptor, hsp90, and p59). Under conditions when the calf uterine ER migrates as an 8.9 S complex (Fig. 5a), the majority of p59 was found at -6.5 S (Fig. 6A). When EC1 increased the sedimentation coefficient to -11 S (Fig. 5a), a portion of the p59 shifted to the 9-11 S region (Fig. 6B). Addition of a second antibody sedimented both the receptor and p59 to the bottom of the tube (Figs. 5a and 6C). In panels B and C, signal at -53 kDa represents EC, heavy chain. In panel A, signals at -50 kDa and 45 kDa were due, in part, to nonspecific interaction between the 3.6 S internal marker peroxidase and a component of the Vectastain Kit, and per- haps in part, to proteolysis. Similar results were found with calf uterine PR (data not shown).

-zoo

-92

p59- -69

-L-r-

--=-*yg= -46

FK. 6. Immunoblots with EC, of calf uterine cytosol frac- tionated by sucrose density gradient centrifugation. Aliquots (50 ~1) of the sucrose gradient fractions of Fig. 5 were subjected to SDS-PAGE (5-15% gradient) and immunoblotting. The number of the fractions is indicat.ed at the top of the gels. Panel A, cytosol alone. Panel R, cytosol incubated with EC, prior to centrifugation. Panel C, cytosol incubated with EC, plus goat anti-mouse IgG. Molecular weight of standards (rainhoru markers) are indicated on the ri&t. Migration position of ER and the standards GO and PO are indicated by arrow at both the top and the bottom.

-200

hsp90- -92

-69

-46

I3 hsp90-

t t b I 3 5 7 9 II I.? 15 II 19

C' .' : . .

. . hsp90- .e .~ _..s-

t t EA ER

FIG. 7. Immunoblots with AC88 of calf uterine cytosol frac- tionated by sucrose density gradient centrifugation. Same ex- periment as that described in Fig. 6 except that AC88 was used instead of EC,. Molecular weight standards are indicated on the right.

We also followed the distribution of hsp90 in the samples of calf uterine ER of Fig. 5. hsp90 sedimented between 6.5 and 11 S (Fig. 7A) when the receptor migrated as an 8.9 S complex. After incubation of the cytosol with EC, a portion of the hsp90 shifted to a heavier region of the gradient (Fig. 7B). After addition of the second antibody, we found a signif- icant displacement of hsp90 to the bottom of the tube (Fig. 7C). These data provide compelling evidence for the associa- tion of hsp90, p59, and the steroid binding unit in a common complex. Hsp90, has already been found complexed with other proteins such as the transforming tyrosine kinase of a number of retroviruses (38-40), the heme-regulated eukaryotic initi- ation factor 2a kinase (41), and also can bind to F actin (42, 43) or tubulin (44). In addition, hsp90 is a component of numerous untransformed steroid hormone (45-49) and dioxin (50, 51) receptors. Its widespread distribution in sucrose gra- dient may represent such a large heterogeneity. Recently, two groups (52, 53) have proposed that the so-called 8-10 S nontransformed receptors could be core units derived from a larger heteromeric complex. hsp90 and p59 are some of the proteins associated in this complex. The major part of p59 migrates at -6.5 S in calf uterus (Fig. 6) and rabbit uterus cytosol (not shown). Treatment of cytosol by NaSCN, urea, and KC1 provokes a change in the migration of p59 which is mainly found in -3 S regions5 (not shown). Thus, p59 could be originally present as a homo- or a heterooligomer, but results exclude disulfide linkage between the subunits.

The interaction of EC1 with calf uterus PR, GR, and AR has also been analyzed by density gradient, before or after DMP cross-linking (not shown). The results are similar to those obtained with calf ER (Fig. 5) and led to the conclusion that p59 is also a component of the large untransformed form of these receptors in this system. Similarly, 8-9 S PR and GR of human breast cancer MCF7 cells also contain p59. Immu- noaffinity purification of calf ER and PR gave results similar to those shown in Fig. 2 for the rabbit PR (not shown).

Isolation of p59. hsp90 Complexes after Cross-linking-In the experiment of Fig. 8, native (untreated) or DMP-cross-

’ J.-M. Renoir, unpublished results.

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p59 in Nontransformed Steroid Receptors 10743

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FIG. 8. Immunoblots with EC, and AC88 of immunopurified calf and rabbit uterine cytosols. Non-cross-linked and DMP- cross-linked rabbit and calf uterine cytosols were fractionated by EC,-Affi-Gel 10 immunoaffinity column. Following purification, sam- ples were subjected to SDS-PAGE (5-15% gradient) and EC1 (Panel A) and AC88 (Panel B) immunoblotting. Lane 1, calf uterine cytosol (95 pg of protein); lane 2, cross-linked calf uterine cytosol (450 pg of protein); lane 3, rabbit uterine cytosol (100 pg of protein); lane 4, cross-linked rabbit uterine cytosol (550 pg of protein). Immunopuri- fied cytosols were also cross-linked with DMP after elution and analyzed in lanes 5 (calf: 400 pg of protein) and 6 (rabbit: 500 pg of protein). A control experiment with nonspecific immunoadsorption of rabbit cytosol on m&se nonimmune IgGs Affi-Gel-10 is presented in lane 7 (250 pg of protein). The molecular weight of the standards is indicated on the right and left sides of the figure.

linked rabbit and calf uterine cytosols were immunoadsorbed on columns of Affi-Gel-lo-EC. Column eluates were electro- phoresed and immunoblotted with either EC1 (Fig. 8A) or AC88 (Fig. 8B). A strong signal at 59 kDa is observed with non-cross-linked material (Fig. 8A, lanes 1 and 3). After cross- linking, p59 is found in a broad band near the top of the gel (Fig. 8A, lanes 2 and 4). The additional band at 59 kDa suggests incomplete cross-linking or dissociation of p59 prior to cross-linking. Another explanation might be loss of p59 during boiling in electrophoresis sample buffer. We believe the lower M, bands are due to proteolysis of p59 over the course of the analysis.

Immunoblotting of the native immunopurified material with AC88 revealed a band at 90 kDa (Fig. 8B, lanes 1 and 3). Cross-linked samples, on the other hand, gave a faint band at the top of the gel (Fig. 8B, lanes 2 and 4). Complete disap- pearance of the 90-kDa protein indicates that even in the case of incomplete cross-linking and subsequent hsp90 dissocia- tion, the immunoadsorbent did not bind free hsp90. Thus, hsp90 is not retained by the immunoaffinity column but rather it is bound to p59 in a complex that can be stabilized by covalent cross-linking.

Immunoblots of calf and rabbit cytosols cross-linked follow- ing immunopurification yielded high M, bands (Fig. 8, A and B, lanes 5 and 6). These blots are identical with those obtained from samples cross-linked prior to chromatography. Again, the absence of any 90-kDa signal suggests that hsp90 is retained via p59. Taken in concert, these data support the notion that hsp90 and p59 are bound to each other in the cell. Immunoblotting analyses of the cross-linked samples with anti-receptor antibodies have been unsuccessful. We have ascribed this to the very low concentration of receptors.

In summary we have several important points from these studies. They are 1) tungstate inhibits completely the 0.4 M KCl-induced dissociation of hsp90 from cytosol receptor, but not that of p59; 2) cross-linking is necessary to maintain the association of p59 with the receptor because neither molyb- date nor tungstate is able to block salt-induced p59 dissocia- tion; 3) RU486 seems to be more efficient than R5020 in maintaining the large molybdate-stabilized PR; 4) p59 is

common to many species and receptors; 5) p59 appears to be associated with hsp90, rather than directly binding to the receptor. Further work is obviously necessary to elucidate the physiological role of the association of p59 with steroid hor- mone receptors. Cloning and sequencing of p59 are currently in progress in our laboratory and will be reported soon.

Acknowledgments-We thank D. Toft for the gift of AC88, C. Mercier-Bodard and I. Jung-Testas for MCF7 and COS cells, and D. Philibert (Roussel-Uclaf) for the gift of radioactive and cold R5020 and RU486. The assistance of F. Boussac, C. Legris, M. Bahloul, and J. C. Lambert for preparation of the manuscript and the figures is gratefully acknowledged.

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S”PPLEMENTARY MATERIAL

THE NON-DNA B~NDWG,%!AROCIL~GOMER~C FORM OF MAMMALIAN STRROID HORMONE RECEWORS CONTAINS

A HSPSO-BOUND 59 kDa PROTEIN

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J M Renoir, C Radanyi, L E Faber and E E Baulieureceptors contains a hsp90-bound 59-kilodalton protein.

The non-DNA-binding heterooligomeric form of mammalian steroid hormone

1990, 265:10740-10745.J. Biol. Chem. 

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