Nuclear progesterone receptor is mainly heat shock protein 90-free in vivo.

Tuohimaa, Pentti; Pekki, Anu; Bläuer, Merja; Joensuu, Timo; Vilja, Pekka; Ylikomi, Timo

doi:10.1073/pnas.90.12.5848

Cited by 28 publications

(27 citation statements)

References 41 publications

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“…In fact, a hsp90-containing 8S progesterone receptor complex has been obtained from purified nuclei of rabbit uterus in the absence of the cognate hormone (23). On the other hand, the reported conclusion that the nuclear progesterone receptor is mainly hsp90-free (24) seems to be due to the inability to detect the small fraction of the abundant cytoplasmic hsp9O that would be colocalized in nuclei with PR. Here we were able to demonstrate that some 9OWT, when cotransfected with rPR, is localized in the nucleus.…”

Section: Several Important Deductions Stem From These Results (I)mentioning

confidence: 95%

In vivo functional protein-protein interaction: nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus.

Kang

Devin

Cadepond

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

123

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In target tissue extracts, heat shock protein hsp9O has been found associated to aDl unganded steroid receptors. Modulation ofimportant functions ofthese receptors, including prevention of DNA binding and optimiztion of transcriptional activity, has been attributed to hsp9O. However no unequivocal in vivo demonstration of interaction between receptors and hsp9O has been presented. We targeted chicken hsp9O, a mainly cytoplasmic protein, with the nucleoplamin nuclear lozation signal (90NLS). After transfection into COS-7 ceDls, 9ONLS was found in the nucleus with specific immunofluorescence and confocal microscopy techniques. A human glucocorticosteroid receptor mutant devoid of NLS sequence was also expressed in COS-7 cells and found exclusively cytoplasmic. Coexpression of 9ONLS and of the cytoplasmic human glucocorticosteroid receptor mutant led to complete nuclear localization ofthe receptor, indicating its piggyback transport by 9ONLS and thus physical and functional interaction between the two proteins in the absence of hormone. The same nuclear localization was obtained after cotransfection of 9ONLS and a cytoplasmic rabbit progesterone receptor mutant. Finally, coexpression of wild-type rabbit progesterone receptor (nuclear) and wildtpe hsp9O (cytoplasmic) into COS-7 cells provoked partial relocalization of hsp9O into the nucleus. These experiments lay the groundwork on which to study hsp9O as a chaperone, regulating activities of steroid receptors and possibly participating in their nuclear-cytoplasmic shuttling.Steroid hormone receptors are hormone-dependent transcriptional activators bearing hormone-independent nuclear localization signals (NLSs) whose efficiency is the primary determinant of their subcellular localization in the absence of hormone; thus the glucocorticosteroid receptor is predominantly located in the cytoplasm, whereas estrogen and progesterone receptors are essentially nuclear (1-3). Despite this, however, a puzzling observation is that all unliganded steroid receptors form "8S" complexes with 90-kDa heat shock protein (hsp90) in the cytosol of target cell homogenates (4-7), hsp90 being an abundant and ubiquitous protein described as essentially cytoplasmic. Two functions have been ascribed to receptor-bound hsp90: masking of the receptor DNA binding domain and maintenance of the ligand binding domain in a functional hormone-binding conformation, at least for gluco-and mineralocorticosteroid receptors (reviewed in refs. 8 and 9). The in vivo requirement of appropriate levels of hsp90 for efficient hormonal activation of steroid receptors in yeast has also been reported (10). The lack of direct evidence for the in vivo interaction between the two proteins has, however, led to the suspicion that hsp90-steroid receptor complexes represent an artifactual association occurring during cell homogenization or that theseThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 ...

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Section: Several Important Deductions Stem From These Results (I)mentioning

confidence: 95%

In vivo functional protein-protein interaction: nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus.

Kang

Devin

Cadepond

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

123

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“…The direct interaction between hMR and Factin cannot be extended to other steroid receptors. Indeed, GR interacts with F-actin via hsp90, while both progesterone [27,28] and estrogen receptors [29,30] are nuclear, and F-actin is mainly cytoplasmic.…”

Section: Discussionmentioning

confidence: 99%

Human mineralocorticoid receptor interacts with actin under mineralocorticoid ligand modulation

et al. 1996

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The human mineralocorticoid receptor of the steroid receptor family contains a modular structure with domain E which is considered to be a hormone binding domain. Recombinant protein approaches enabled us to clearly determine that this domain is also able to interact with F-actin (Kd about 2 ~M) and G-actin. Moreover, it was revealed that this mineralocorticoid receptor domain/actin interaction was modulated by specific mineralocorticoid ligands. Agonist (aldosterone) steroid binding almost totally (91%) abolished the interaction with F-actin, while antagonist (progesterone) binding allowed more than 30% of this binding. Steroid modulation of the interaction between domain E and actin indicated that this actin binding is specific and could be essential for cellular mineralocorticoid receptor activity.

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“…Since p23 and the immunphilins are though to be associated with the complex through the Hsp90, it is most probably the latter which is responsible for masking the αD epitope. In a previous immunohistochemical study, we showed that αD can recognize endogenous PR in the nucleus of chicken oviduct cells, indicating that the epitope is readily accessible, and furthermore, that most of the PR molecules in the cell nucleus are in dissociated form [Tuohimaa et al 1993]. In addition, we have previously shown that fixation does not cause artificial exposure of the epitope [Pekki and Tuohimaa 1989].…”

Section: Discussionmentioning

confidence: 91%

“…There is evidence that Hsp90 is a cytoplasmic protein and that there is not a sufficient amount of Hsp90 molecules in the nucleus to form complexes with nuclear steroid receptors [Tuohimaa et al 1993].There is, however, evidence that the glucocorticoid receptor (GR), which is predominantly cytoplasmic in its unoccupied form, can be crosslinked in vivo with Hsp90, which would imply that steroid receptors may be associated with Hsp90 in the cytoplasmic compartment. [Rexin et al 1991].…”

confidence: 99%

“…They were incubated with serum-free Optimem (Life Technologies) containing the DNA-liposome-complexes for 24h at 37°C and thereafter in medium with 10% charcoal-stripped fetal bovine serum for 2h; the medium was changed and the cells further incubated for 24h. Wild-type chicken progesterone receptor (cPR21) and wild-type chicken Hsp90 expression vectors were used and have been described elsewhere [Tuohimaa et al 1993,Turcotte et al 1990]. …”

Section: Cell Culture Transfection and Expression Vectorsmentioning

confidence: 99%

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Evidence for the existence of an oligomeric, non-DNA-binding complex of the progesterone receptor in the cytoplasm

Passinen

Ylikomi²

2009

Eur J Histochem

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Steroid receptors are found as a hetero-oligomeric complex in cell extracts. Due to the dynamic interaction between receptor-associated proteins and receptors, it is difficult to study the oligomeric complex in living cells. Here this was attempted in cells in which the interaction was stabilized by introducing molybdate into the cells or by incubating the cells at low temperature. The complex was studied with an antibody (αD) recognizing only the dissociated form of the chicken progesterone receptor (PR) and with antibodies (PR22, PR6). Recognizing also oligomeric forms of the receptor. When wild-type chicken PR was transfected, all antibodies showed nuclear staining. Molybdate or cold treatment of cells resulted in cytoplasmic accumulation of the PR as detected with PR22/PR6. αD, however, stained predominantly the nuclear PR in treated cells. These findings suggest that when the oligomeric complex of the PR is stabilized in intact cells in vivo and then crosslinked with paraformaldehyde, a portion of the cytoplasmic receptor is seen as an oligomeric complex, whereas, in the nucleus, most, if not all receptor molecules are in dissociated form. Steroid receptors belong to a large family of nuclear receptors functioning as ligand-regulated transcription factors. Receptor function is mediated by receptor-interacting proteins; co-activators interact with agonist-occupied receptors and mediate transcription activation while co-repressors interact with non-liganded or antagonist-occupied and mediate transcription repression. Ligand binding induces a considerable change in the structure of the ligandbinding domain (LBD), affecting the surface exposition of the receptor which dictates the interaction of the cofactors [Moras and Gronemeyer 1998, Renaud et al. 1995, Wurtz et al. 1996. The earliest known receptor-interacting protein is Hsp90 [Dougherty et al. 1984]. It is known to interact in vitro with the LBD of the steroid receptor in a ligand-dependent manner: it binds to non-liganded steroid receptor but not to agonist-occupied receptor. Non-liganded steroid receptor LBDs act as intramolecular repressors of the activation functions of steroid receptors as well as of heterologous proteins when covalently linked to them [Picard et al. 1988]. Deletion of the LBD generates a constitutively active steroid receptor which does not form a stable oligomeric complex with Hsp90 in vitro. In view of these data, it has been proposed that the Hsp90 interaction is responsible for the repressor function of non-liganded LBDs [Scherrer et al. 1993]. Binding of Hsp90 interferes with the DNA binding of the receptor, which has been regarded as one possible mechanism by which Hsp90 functions as a corepressor. Hsp90 is also required for proper ligand binding of the glucocorticoid receptor [Bresnick et al. 1989]. It has been proposed that the oligomeric form of steroid receptors represents a poised conformation, inactive but ready to interact with the ligand. Ligand binding is thought to dissociate the complex and allow receptor binding to ...

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Nuclear progesterone receptor is mainly heat shock protein 90-free in vivo.

Cited by 28 publications

References 41 publications

In vivo functional protein-protein interaction: nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus.

In vivo functional protein-protein interaction: nuclear targeted hsp90 shifts cytoplasmic steroid receptor mutants into the nucleus.

Human mineralocorticoid receptor interacts with actin under mineralocorticoid ligand modulation

Evidence for the existence of an oligomeric, non-DNA-binding complex of the progesterone receptor in the cytoplasm

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