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Molecular Endocrinology 12 (11): 1684-1695
Copyright © 1998 by The Endocrine Society

Mechanisms of Progesterone Receptor Export from Nuclei: Role of Nuclear Localization Signal, Nuclear Export Signal, and Ran Guanosine Triphosphate

Rakesh Kumar Tyagi1, Larbi Amazit1, Pierre Lescop, Edwin Milgrom and Anne Guiochon-Mantel

Hormones et Reproduction INSERM U135 Faculté de Médecine Paris-Sud 94275-Le Kremlin-Bicêtre Cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Steroid hormone receptors are, in most cases, mainly nuclear proteins that undergo a continuous nucleocytoplasmic shuttling. The mechanism of the nuclear export of these proteins remains largely unknown. To approach this problem experimentally in vivo, we have prepared cell lines permanently coexpressing the wild-type nuclear progesterone receptor (PR) and a cytoplasmic receptor mutant deleted of its nuclear localization signal (NLS) [({Delta}NLS)PR]. Each receptor species was deleted from the epitope recognized by a specific monoclonal antibody, thus allowing separated observation of the two receptor forms in the same cells. Administration of hormone provoked formation of heterodimers during nucleocytoplasmic shuttling and import of ({Delta}NLS)PR into the nucleus. Washing out of the hormone allowed us to follow the export of ({Delta}NLS)PR into the cytoplasm. Microinjection of BSA coupled to a NLS inhibited the export of ({Delta}NLS)PR. On the contrary, microinjection of BSA coupled to a nuclear export signal (NES) was without effect. Moreover, leptomycin B, which inhibits NES-mediated export, was also without effect. tsBN2 cells contain a thermosensitive RCC1 protein (Ran GTP exchange protein). At the nonpermissive temperature, the nuclear export of ({Delta}NLS)PR could be observed, whereas the export of NES-BSA was suppressed. Microinjection of GTP{gamma}S confirmed that the export of ({Delta}NLS)PR was not dependent on GTP hydrolysis. These experiments show that the nuclear export of PR is not NES mediated but probably involves the NLS. It does not involve Ran GTP, and it is not dependent on the hydrolysis of GTP. The nucleocytoplasmic shuttling of steroid hormone receptors thus appears to utilize mechanisms different from those previously described for some viral, regulatory, and heterogeneous ribonuclear proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors form a very large family of transcriptional regulators. These receptors interact with specific DNA sequences (hormone-responsive elements) usually located upstream from genes, thereby recruiting coactivators or corepressors and/or stabilizing interactions with basal transcription factors (1, 2). The function of many of these receptors is regulated by endogenous ligands: steroid or thyroid hormones, dihydroxyvitamin D3, retinoic acid, etc. The subcellular localization of nuclear receptors and specifically of steroid hormone receptors has been the subject of many studies (3, 4, 5, 6). Sex steroid (estrogen, androgen, progesterone) receptors are intranuclear, whereas glucocorticoid receptors (GR) are located totally or in part in the cytoplasm of cells in the absence of hormone (6, 7, 8, 9). After administration of hormone, the receptor migrates into the nucleus. These observations reflect a dynamic situation: the receptor continuously shuttles between the nucleus and the cytoplasm with, at any given time, a major fraction of the protein being present either in the nucleus (sex steroid receptors) or in the cytoplasm (unliganded GR) (10, 11, 12, 13). The signal involved in the transport of receptors from the cytoplasm to the nucleus has been extensively studied. In the case of the progesterone receptor (PR), this nuclear localization signal (NLS) is a large region (61 amino acids) extending over the hinge region and the second zinc finger. It comprises four segments formed of basic amino acids. Experimentally, it may be divided into two regions: a C-terminal signal that is constitutively active and a N-terminal (second zinc finger) region that is active only in the presence of ligand (hormone or antagonist) (10). The signal(s) involved in the outward movement of receptors from the nucleus to the cytoplasm are still debated. Initially, using heterokaryons and energy deprivation experiments, we obtained evidence that the export of receptors from the nucleus was also NLS mediated (14). However, these methods do not allow any further insight into the mechanisms involved. Furthermore, nuclear export signals (NES) comprised of stretches of hydrophobic residues have recently been described, as have sequences involved in the nucleocytoplasmic shuttling of heterogeneous nuclear ribonucleoproteins (15, 16, 17, 18). The objective of this study was to examine the possibility that such sequences could be involved in the subcellular trafficking of the receptor. We describe here an in vivo model system that can be used to study, in intact cells, the export of PR from the nucleus. We have applied this system to the analysis of the mechanisms involved in this export.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Model System to Study PR Export from the Nucleus
Initially we considered the possibility of using mutant PR{Delta}638–642. Its steroid-binding properties and biological activity are identical to those of wild-type receptor (10). It is deleted from the constitutive region of the NLS. Only the hormone-dependent region of the NLS is conserved in PR{Delta}638–642. This receptor mutant is thus cytoplasmic in the absence of hormone and is transported into the nucleus in its presence. Reversal of the reaction by washing out the hormone could possibly lead to the nuclear export of the receptor. To examine this hypothesis, we used an L cell line permanently expressing this receptor mutant (19) incubated with [3H]progesterone. Receptor transfer into the nucleus was monitored by immunocytochemistry. The hormone was then washed out, and its dissociation from receptor was followed by counting the radioactivity: 92.8% of the receptor-bound hormone was dissociated in 15 min. However, up to 20 h after hormone washing, there was no reappearance of the receptor in the cytoplasm (not shown).

This absence of reentry of the PR{Delta}638–642 mutant into the cytoplasm may be due to the fact that masking of the hormone-dependent NLS in the second zinc finger region probably necessitates reassociation with heat shock proteins. This reassociation may be impeded in the mutated receptor (20, 21). However, it has been proposed that heat shock proteins are necessary for hormone binding (22), and we have observed that after the washing procedure the receptor remained able to bind the hormone (not shown). Thus, mechanisms other than lack of reassociation with heat shock proteins may explain the absence of reentry of PR{Delta}638–642 into the cytoplasm.

We then considered another model system. When cells are cotransfected with a wild-type receptor and a mutant devoid of the entire NLS region [({Delta}NLS)PR], the former receptor species is present in the nucleus and the latter in the cytoplasm. Observation of both receptors in the same cells is made possible by deleting specific epitopes, recognized by defined monoclonal antibodies (10). If the cells are treated with hormone, the cytoplasmic mutant becomes localized in the nucleus. This is due to hormone- induced formation of dimers during the nucleocytoplasmic shuttling of the nuclear monomer. The dimer is then transported into the nucleus due to the presence of the NLS on one of the receptor monomers.

We thus considered the possibility that during nucleocytoplasmic shuttling dissociation of hormone from the dimer when the latter is in the cytoplasm could lead to monomer separation: the nuclear monomer, carrying a NLS, being reimported into the nucleus and the ({Delta}NLS)PR mutant, devoid of NLS, accumulating in the cytoplasm. An L cell line permanently expressing both receptor forms was treated with progesterone, which provoked, as previously shown, the transfer of the ({Delta}NLS)PR mutant into the nucleus. When hormone was washed out of the cells, an export of ({Delta}NLS)PR could be observed in 2 h and was nearly complete in 4 h (Fig. 1Go). This experimental setting could thus be used to study PR export in intact cells.



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  		Figure 1. A Model System to Study Receptor Export from the Nucleus

In an L cell line permanently coexpressing wild-type PR and ({Delta}NLS)PR, the latter was localized in the cytoplasm in the absence of hormone (-H). In the presence of progesterone (+H), it was shifted into the nucleus. After 4 h of washing (+HW), it was exported back to the cytoplasm. The Mi60 antibody was used to detect ({Delta}NLS)PR (the epitope for this antibody was deleted from the wild-type receptor). Protein localization was determined using confocal microscopy. Bar, 10 µm.

 
Putative NESs in the PR
A systematic search of PR sequence was made to identify putative NES and shuttling signals. Sequences homologous to NES were found in the N-terminal region of the receptor (amino acids 190–198 and 516–524) and in the ligand-binding domain (amino acids 681–689 and 816–824).

There was no region of receptor homologous to the ribonucleoprotein shuttling M9 and K signals (17, 18). A weak homology (29% homology in 31 residues) was found between PR amino acids 233–263 and M9 signal, but this was mainly due to the presence of glycine residues in both these regions. Furthermore, previous experiments have shown that a PR deleted of this region still exhibits nucleocytoplasmic shuttling (10).

In further experiments, we examined the possibility that PR export from the nucleus could be mediated by a NES or, as suggested previously, by a NLS (14).

Nuclear Export of PR. Competition with BSA Coupled to NLS and NES. Effect of Leptomycin B
BSA prelabeled with fluorescein isothiocyanate (FITC) was coupled to the NLS of SV40 large T antigen. BSA was also coupled to a control mutated inactive NLS (mutNLS) (23, 24). We initially verified that the NLS-coupled BSA could compete with receptor for import into nuclei. The protein was microinjected into the cytoplasm of L cells expressing the PR{Delta}638–642 mutant. Hormone administration provoked receptor entry into the nuclei in cells not microinjected or microinjected with BSA (Fig. 2Go) or with BSA coupled to mutNLS (data not shown). In cells microinjected with NLS-BSA, the hormone treatment failed to produce receptor entry into the nucleus. The same result was observed after different incubation times varying from 1–2 h (not shown).



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  		Figure 2. Microinjection of NLS-BSA Inhibits the Import of PR{Delta}638–642

In an L cell line permanently expressing PR{Delta}638–642, the mutant was detected in the cytoplasm in the absence of hormone (-H). Either NLS-BSA or BSA was microinjected into the cytoplasm, and the cells were incubated with progesterone for 2 h (+H). PR{Delta}638–642 was immunodetected (left) and BSA-FITC was monitored (right). In the cells microinjected with NLS-BSA, an inhibition of the import of PR{Delta}638–642 was observed (left, arrows). In the cells microinjected with BSA, no significant inhibition of the import of PR{Delta}638–642 was observed (left, arrows). Bar, 25 µm.

 
Similar results were obtained in cells coexpressing wild-type receptor and NLS-deleted cytoplasmic ({Delta}NLS)PR. Hormone treatment provoked the transfer of the latter into the nucleus. This effect was not observed when the cells were microinjected with NLS-BSA.

Having thus established the suitability of our conditions of microinjection of NLS-BSA to compete out receptor entry into the nucleus, we used the same microinjection conditions to study receptor export from the nucleus. Cells coexpressing wild-type and ({Delta}NLS) receptors were treated with progesterone. The latter receptor mutant migrated into the nucleus. Washing out of hormone provoked its exit from the nucleus. This export of the ({Delta}NLS)PR from the nucleus into the cytoplasm was inhibited if, before hormone withdrawal, the cells were microinjected with NLS-BSA (Fig. 3Go). The wild-type receptor was retained in the nucleus showing that it was receptor export that was inhibited and not the reentry of the dimer into the nucleus (data not shown). There was no inhibition of ({Delta}NLS)PR export if BSA (Fig. 3Go) or mutNLS-BSA (data not shown) was microinjected into the nucleus. Similar results were observed in COS-7 cells transiently transfected with wild-type and ({Delta}NLS)PR (not shown). Microinjection of NES-BSA either into the cytoplasm or into the nucleus did not, on the contrary, inhibit receptor export from the nucleus into the cytoplasm (Fig. 3Go).



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  		Figure 3. Microinjection of NLS-BSA Inhibits the Export of ({Delta}NLS)PR

A, The L cell line permanently coexpressing wild-type PR and ({Delta}NLS)PR was treated with progesterone. Cell nuclei were microinjected with either NLS-BSA, BSA, or NES-BSA, and hormone was washed out. ({Delta}NLS)PR was immunodetected (left) and BSA-FITC was monitored (right). Protein localization was determined using confocal microscopy. In the cells microinjected with NLS-BSA, an inhibition of the export of ({Delta}NLS)PR was observed (left, arrows). In the cells microinjected with BSA, no inhibition of the export of ({Delta}NLS)PR was observed (left, arrows). In the cells microinjected with NES-BSA, there was no inhibition of ({Delta}NLS)PR export (left, arrows). Bar, 10 µm. B, The subcellular localization of ({Delta}NLS)PR was determined in at least 100 cells for each experimental condition: -H, untreated cells; +H, progesterone-treated cells; +HW, cells after hormone withdrawal.

 
It has recently been shown that leptomycin B inhibits NES-mediated protein export (25). Leptomycin B interacts with crm1, which is involved in this export (26, 27, 28, 29). We first verified that leptomycin B had no effect on the import of PR (data not shown). We then studied its effect on the export of PR. L cells coexpressing wild-type and ({Delta}NLS)PR mutant were incubated with progesterone, provoking entry of ({Delta}NLS)PR into the nucleus. Leptomycin B, added before and maintained during the course of hormone withdrawal, did not prevent receptor export from the nucleus (Fig. 4Go). In the same conditions leptomycin B completely inhibited the export of NES-BSA microinjected into the nucleus (Fig. 4Go). This experiment further confirmed that receptor export does not follow the same pathway as NES-mediated export.



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  		Figure 4. Leptomycin B Inhibits NES-Mediated Protein Export but Not PR Export

A (upper panel), The permanent L cell line coexpressing wild-type receptor and ({Delta}NLS)PR was treated with progesterone. ({Delta}NLS)PR was shifted into the nucleus (+H). Hormone was washed out in the absence (+HW) or in the presence of 20 nM leptomycin B (+LB, +HW). No inhibition of export was observed in the presence of leptomycin B (+LB, +HW). Lower panel, NES-BSA was microinjected into the nucleus of L cells. The site of microinjection was monitored by the coinjection of a TRITC-conjugated IgG (right). Cells were preincubated (+LB) or not (-LB) with 20 nM leptomycin B for 1 h. An inhibition of the export of NES-BSA was observed in the presence of leptomycin B (left, +LB). Protein localization was determined using confocal microscopy. Bar, 10 µm. B, The subcellular localization of ({Delta}NLS)PR was observed in at least 200 cells in each experimental condition. In the same parallel experiment, the export of NES-BSA was completely inhibited (not shown).

 
Ran GTP and the Nuclear Export of PR
Previous experiments have suggested that steroid hormone nuclear export is not energy dependent (12, 19). Since recent studies have stressed the importance of Ran GTP in the nuclear transport mechanisms (30, 31), we analyzed its role in the nuclear export of PR. GTP hydrolysis by Ran is necessary for NLS-mediated protein import (32, 33, 34). Ran is submitted to a cycle of activation-inactivation (35). GTP is hydrolyzed in the cytoplasm by a RanGAP (36, 37). In the nucleus, the exchange factor RCC1 replaces GDP by GTP (38, 39).

A cell line derived from BHK21 cells and called tsBN2 contains a thermosensitive RCC1 (regulator of chromosome condensation 1) (40, 41). The inactivation of RCC1 at the nonpermissive temperature (39.5 C) induces an intranuclear depletion of Ran GTP and the inhibition of nuclear protein import (42). We prepared a tsBN2 cell line permanently expressing the PR{Delta}638–642 mutant to study receptor import. We then cotransfected tsBN2 cells with plasmids encoding wild-type PR and ({Delta}NLS)PR to study receptor nuclear entry and export by methods described above. We also cotransfected BHK21 cells with the same plasmids as a control.

In BHK21 cells, at both 33.5 C and 39.5 C, ({Delta}NLS)PR entered the nucleus under the effect of hormone and returned into the cytoplasm after washing out of hormone as previously described for L or COS-7 cells. The same results were obtained in tsBN2 cells at the permissive temperature (33.5 C). After 6 h incubation at the nonpermissive temperature (39.5 C), we observed the disappearance of RCC1 (data not shown). There was also redistribution of Ran from the nucleus into the cytoplasm as previously described (43) (data not shown).

At 39.5 C, the hormone-induced transfer of PR{Delta}638–642 (Fig. 5Go) or ({Delta}NLS)PR into the nucleus was impeded. Furthermore, partial exit of wild-type receptor from the nucleus was observed (Fig. 5Go). This result is very similar to that previously observed with inhibitors of energy synthesis (19). It is consistent with receptor exit from the nucleus being nondependent on the integrity of RCC1, whereas receptor reentry into the nucleus is inhibited in the absence of RCC1. This leads, during receptor nucleocytoplasmic shuttling, to a progressive accumulation of wild-type receptor in the cytoplasm.



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  		Figure 5. Effect of RCC1 Depletion on the Import of PR in tsBN2 Cells

A (upper panel), In a synchronized tsBN2 cell line permanently expressing PR{Delta}638–642, the mutant PR was cytoplasmic in the absence of hormone (-H). It was shifted into the nucleus after hormone treatment (+H). If cells were preincubated for 6 h at the nonpermissive temperature, the shift to the nucleus was impeded (39.5 C, +H). Lower panel, In a synchronized tsBN2 permanent cell line expressing wild-type PR, PR was nuclear at the permissive temperature (33.5 C). After 6 h incubation at the nonpermissive temperature, a fraction of the receptor was shifted into the cytoplasm (39.5 C). Protein localization was determined using confocal microscopy. Bar, 10 µm. B, The subcellular localization of wild-type PR was observed in at least 200 cells at the permissive and nonpermissive temperatures.

 
Receptor export from the nucleus was studied as described above by washing out progesterone after having exposed the cells to the nonpermissive temperature. ({Delta}NLS)PR was transferred in part into the cytoplasm (Fig. 6AGo). The partial character of the transport was probably due to the inhibition of wild-type receptor reentry into the nucleus. Formation of dimers between ({Delta}NLS)PR and wild-type PR is necessary for the NLS-mediated export of ({Delta}NLS)PR (10).



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  		Figure 6. Effect of RCC1 Depletion on PR Export and on the NES-Mediated Protein Export

A, tsBN2 and BHK21 cell lines were cotransfected with plasmids encoding wild-type PR and ({Delta}NLS)PR and synchronized. They were treated with progesterone at the permissive temperature (+H). Cells were then incubated for 6 h at the nonpermissive temperature, and hormone was washed out at the nonpermissive temperature (39.5 C, +HW). ({Delta}NLS)PR was immunodetected and scored in 200 cells for each experimental condition. In tsBN2 cells, a shift to the cytoplasm of the ({Delta}NLS)PR was observed in most of the cells after withdrawal of the hormone at the nonpermissive temperature. B, NES-BSA was microinjected into the nuclei of synchronized tsBN2 and BHK21 cells. Cells were either maintained at the permissive temperature (33.5 C) or preincubated for 6 h at the nonpermissive temperature (39.5 C). After microinjection, they were further incubated for 1 h at the same temperature. The export of NES-BSA was markedly inhibited at the nonpermissive temperature in tsBN2 cells.

 
We next analyzed NES-mediated protein export in tsBN2 cells. The cells were incubated at the nonpermissive temperature and microinjected with NES-BSA. A marked inhibition of nuclear export was observed (Fig. 6BGo). A normal nuclear export was observed in the same cells kept at the permissive temperature, as well as in BHK21 control cells (Fig. 6BGo). These experiments thus showed further differences in the mechanisms of PR export and those of NES-mediated protein export. Whereas nuclear Ran GTP is necessary for NES-mediated export, it is not necessary for receptor export.

To analyze the role of GTP in receptor export by another approach, we microinjected cells with GTP{gamma}S, a nonhydrolyzable competitive inhibitor of GTP. It has been shown previously that it can inhibit NLS-mediated protein import (32, 33). The L cells coexpressing wild-type and ({Delta}NLS)PR were used. We initially established the conditions, especially for GTP{gamma}S concentration, in which hormone-mediated nuclear transfer of PR{Delta}638–642 mutant and of ({Delta}NLS)PR was blocked. We then analyzed the effect of GTP{gamma}S on PR export. We treated cells with progesterone, microinjected their nuclei with GTP{gamma}S, and washed out the hormone. There was no inhibition of receptor transfer into the cytoplasm (Fig. 7AGo). We performed the same experiment in the L cell line expressing wild-type PR. A partial export of wild type receptor from the nucleus was observed after microinjection of GTP{gamma}S into the nucleus (Fig. 7BGo). This result suggests that during the shuttling process receptor reentry, but not receptor exit, from the nucleus requires GTP hydrolysis.



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  		Figure 7. Effect of GTP{gamma}S on the Export of ({Delta}NLS)PR

A, The L cell line permanently coexpressing wild-type and ({Delta}NLS)PR was incubated with progesterone. Cells were microinjected with GTP{gamma}S, and hormone was then washed out. +H, Progesterone-treated cells; +HW, cells after hormone withdrawal; GTP{gamma}S, +HW, cells microinjected with GTP{gamma}S. ({Delta}NLS)PR was immunodetected and scored in at least 100 cells for each experimental condition. There was no inhibition of the export of ({Delta}NLS)PR in the microinjected cells. B, The wild-type PR was immunodetected in the same conditions in 100 cells. A shift to the cytoplasm of the wild-type PR was observed in the microinjected cells.

 
Similar experiments were also performed with the nonhydrolyzable analog of ATP:ATP{gamma}S. There was partial inhibition of receptor entry into the nucleus and no effect on PR export (not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The signal(s) involved in the nuclear export that occurs during the nucleocytoplasmic shuttling of steroid hormone receptors remain to be identified. In the PR, NLS have been precisely mapped (10, 44) and we have proposed that they play a role in both the nuclear import and the nuclear export of the protein (14). Regions of homology to NES are also present in PR. Proteins carrying such signals have recently been shown to interact with specific proteins of the importin ß family, such as crm1 or exportin1 and CAS (cellular apoptosis susceptibility gene) (27, 28, 29, 45, 46). However, microinjection of NES-BSA into cells did not inhibit PR export. Furthermore, experiments probing the Ran GTP cycle and the effect of leptomycin B gave different results when NES-mediated export was compared with the export of the PR. These experiments show not only that PR export is not mediated by a NES carried by the receptor but that it also does not occur through an interaction of the receptor with a NES-bearing protein. In contrast, the involvement of the NLS in PR nuclear export was suggested by inhibition after microinjection of NLS-BSA.

NES-mediated export of proteins is an energy- and temperature-dependent phenomenon (15). It is inhibited by agents blocking the production of ATP, such as sodium azide or apyrase (29, 47). However, whether the source of energy is GTP or ATP is unknown. Ran GTP plays a role in this export. Ran GTP is present in the complex NES-protein-exportin, and a depletion of intranuclear Ran GTP inhibits NES-mediated export (27, 45, 46, 48, 49, 50, 51). However, it has also been shown that Ran-dependent GTP hydrolysis is not required in the NES-mediated export of proteins (49, 51).

Our data also show that the nuclear export of NES-BSA is inhibited in tsBN2 cells at the nonpermissive temperature, where the exchange factor RCC1 is destroyed, thus disrupting the nucleocytoplasmic gradient of Ran GTP. In contrast, PR export is only very slightly affected in the same conditions. It remains possible that low concentrations of GTP are preserved in the absence of RCC1 (51). Such concentrations would be sufficient to promote PR export but not NES-mediated export. In vitro experiments, using ran mutants or ran preloaded with different nucleotides, will be necessary to further analyze this mechanism.

The role of ATP in PR nucleocytoplasmic exchanges is not clear. The previously shown energy dependence of nuclear accumulation of PR may be indirect, with ATP being necessary for the resynthesis of GTP, or may be related to receptor retention in the nucleus (19). For GR, a nuclear subtrafficking has been described (52). GR is cytoplasmic in the absence of hormone and is shifted into the nucleus in its presence. The GR can only be extracted from nuclei by high ionic strength. When the hormone is washed out, GR remains nuclear but becomes easily extractable even at low ionic strength. A shift of GR into the cytoplasm is seen only after several hours of incubation in hormone-free medium (53).

The mechanism of nuclear export of steroid hormone receptors is presently not known but may involve karyopherin {alpha},2 as has been shown previously for the reexport of CBP20 (54). During nuclear import, proteins carrying NLS are bound to karyopherin {alpha}. The latter interacts with karyopherin ß, which is an adaptor for nucleoporin-mediated import. In the nucleus, karyopherin ß interacts with Ran GTP, and this leads to dissociation from karyopherin {alpha} and nucleoporins. The NLS-carrying proteins then dissociate from karyopherin {alpha} that is exported from the nucleus by interaction with protein CAS, which belongs to the karyopherin ß family (45). The affinity of CAS is approximately 10-fold higher for free karyopherin {alpha} than it is for karyopherin {alpha} complexed with a NLS-carrying protein (45). The export of free karyopherin {alpha} is thus favored, but it remains possible that a small fraction of karyopherin {alpha} complexed to the NLS protein is also reexported from the nucleus. The competition between NLS-BSA and hormone-dependent import of PR{Delta}638–642 favors this hypothesis. The PR{Delta}638–642 mutant import being much slower than the NLS-BSA import implies that NLS-BSA is still partially bound to karyopherin {alpha} during its shuttling, competing with PR{Delta}638–642 binding. It has been shown previously that CAS-mediated karyopherin {alpha} export uses a different pathway from crm1-mediated export (49). Such a mechanism would be compatible with the predominantly nuclear localization of PR and its relatively slow nucleocytoplasmic shuttling.

Another possibility arises from the properties of the NLS of PR. This NLS is not a canonical NLS, like the NLS of SV40 large T antigen. Rather, it is a complex signal, composed of four clusters of basic amino acids, interspersed with hydrophobic amino acids. It is thus possible that this NLS contains different signals interacting with different types of receptor during import, export, or both processes. Indeed, at least five types of karyopherin {alpha}, belonging to two families, have been cloned (55). These two families of karyopherins do not display the same specificities (56, 57). Competition between NLS-BSA and hormone-dependent import of PR{Delta}638–642 mutant could also be explained by the binding of NLS-BSA to karyopherin {alpha}, preventing its recycling to the cytoplasm. The export of PR is mediated by the interaction of the NLS region with another protein. In vitro experiments will be necessary to address this question.

Protein nucleocytoplasmic shuttling may be related to three different mechanisms. In the first type, a group of proteins enter the nucleus where they interact with the cargo which they will subsequently deliver into the cytoplasm. In the case of HIV-1 Rev, HTLV-1 Rex, and E4, the cargo is a viral RNA (premessengers of HIV-1 or of HTLV-1, and adenovirus messenger RNA, respectively). The cargo may also be a regulatory protein: export of the catalytic subunit of cAMP-dependent protein kinase by the heat-stable protein kinase inhibitor or of NF{kappa}B by I{kappa}B (47, 58). The nuclear export is related to the presence in the protein of a NES, which allows a rapid, energy- dependent accumulation of the protein in the cytoplasm. It confers to the cell the ability to respond rapidly to changing conditions such as those occurring at different stages of the cell cycle or during modulation by external stimuli (16). A second type of nucleocytoplasmic shuttling is observed for the heterogeneous nuclear ribonucleoproteins A1 and K. In the nucleus, they interact with the pre mRNAs, which undergo maturation and are then exported from the nucleus. It is a rapid nucleocytoplasmic shuttling already observed by 1 h after formation of heterokaryons (17). The same signals are involved in the nuclear export and import. The import pathway is different from the classic NLS pathway and involves transportin instead of the importin {alpha}-ß complex (59, 60). The export pathway is energy dependent. Nucleocytoplasmic shuttling of steroid receptors, and specifically of PR, seems to correspond to a third type. These proteins reside mainly in the nucleus where they exert their main biological function. Their nuclear export is markedly slower, requiring 4–12 h to be observed in heterokaryons (10, 12, 14). Both entry and exit from the nucleus seem to be NLS dependent. The import is energy dependent, whereas export is not. During the shuttling, the receptor may interact with cytoplasmic proteins or exert a biological activity in the cytoplasm (61, 62, 63, 64). Some antihormones, such as ICI 182780, exert their activity, at least in part, by inhibiting this type of nucleocytoplasmic shuttling (12).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
Nomenclature
Derivatives denoted with a {Delta} lack the receptor segment delineated by the numbered amino acids. Plasmids encoding the wild-type rabbit PR (pKSV-rPR), and various mutants (PR{Delta}638–642, PR{Delta}373–546, and PR{Delta}25–103, 547–662) have been previously described (10, 19, 65). The epitope for antibody Mi60 was deleted from the wild-type receptor ({Delta}373–546). This mutant is nuclear and displays a biological activity similar to that of the wild-type receptor (65). In the following text it will therefore be referred to as wild-type PR. The epitope for the antibody Let126 ({Delta}25–103) was also deleted from the mutant PR{Delta}547–662, which lacks both the constitutive and the hormone-inducible NLS. It will be referred to as ({Delta}NLS)PR.

Cell Cultures and DNA Transfection
Simian COS-7 cells (66) and mouse L cells were grown in DMEM supplemented with antibiotics and 10% FCS under 5% CO2-95% air at 37 C.

BHK21 (67) and tsBN2 cells (68), a temperature-sensitive mutant derived from BHK21, were grown in DMEM supplemented with antibiotics and 10% FCS (Life Technologies, Gaithersburg, MD) under 10% CO2-90% air at 33.5 C or 39.5 C (restrictive temperature for tsBN2). For synchronization in G1 phase, the cells were cultured for 36–48 h in isoleucine-free MEM containing 5% charcoal- stripped dialyzed FCS as described (69).

Petri plates (35 mm) (Nunc, Roskilde, Denmark) were precoated with human fibronectin (Life Technologies) at 1 µg/ml PBS for 2 h at 37 C. Cells were plated and transfected by the lipofectAMINE method (Life Technologies) according to the instructions from the manufacturer.

Progesterone was used at a concentration of 10 nM for 4 h unless stated otherwise. Hormone withdrawal under optimized conditions was performed by replacement of the hormone-containing medium with the medium supplemented with 10% charcoal-stripped FCS. After the first three rinses, the cells were further rinsed every hour for 4 h using 3 ml medium per rinse. The rinses were always performed in the presence of cycloheximide (10 µg/ml) to inhibit neosynthesis of PR.

Lyophilized leptomycin B (a generous gift from B. Wolff, Novartis, Austria) was solubilized to a stock concentration of 10 mM in dimethylsulfoxide and stored in aliquots at -20 C. Leptomycin B (20 nM) was prepared in culture medium. It was added to the cells 1 h before the experiment and remained active in the medium for at least 24 h. Control cells were treated with the same concentration of dimethylsulfoxide alone.

Permanent Cell Lines
Mouse L cells were cotransfected with the plasmids encoding wild-type (Mi60-)PR, (Let126-)({Delta}NLS)PR, and the plasmid pSV-neo conferring resistance to G418 (Geneticin, Life Technologies) using the calcium phosphate precipitate method (70). Different ratios of plasmid DNA (varying from 1:1 to 10:1) were tested on PR import and export. A ratio of 4:1 between the wild-type PR and the cytoplasmic ({Delta}NLS)PR was selected to achieve optimum conditions for nuclear import and export assays.

A L cell line permanently expressing PR{Delta}638–642 has previously been described (19).

tsBN2 cell lines permanently expressing wild-type (Mi60-)PR and PR{Delta}638–642 were obtained by cotransfection of the corresponding plasmids with the plasmid pSV-neo using the lipofectAMINE transfection method. Clones resistant to G418 were selected and screened for expression of PR by immunocytochemistry. These permanent cell lines were routinely cultured in DMEM supplemented with antibiotics and 10% charcoal-stripped FCS in the presence of G418. These clones have now been studied for more than 20 passages and stably express the corresponding forms of receptor.

Hormone Withdrawal Experiment
The L cell line permanently expressing PR{Delta}638–642 was used. This mutant is cytoplasmic in the absence of hormone and shifts into the nucleus in its presence. It thus allows the simple monitoring of the effect of the hormone. Its steroid-binding properties are identical to those of the wild-type receptor (10). It was cultured in the presence of either 10 nM [3H]progesterone or 10 nM [3H]progesterone and 1 µM unlabeled progesterone for 4 h. The cells were rinsed three times during 5 min at 37 C in DMEM containing 10% charcoal-stripped FCS. The cells were then harvested at varying time points up to 20 h by trypsinization. Whole-cell extracts were prepared at a cell-buffer ratio of 1:3 in TEG buffer (10 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 10% glycerol) containing 0.6 M KCl. Resuspended cells were lysed by freeze/thawing (71). The Centricon 30 system (Amicon, Beverly, MA) was used to lower the ionic strength of the supernatant to 20 mM KCl.

The total amount of PR present was determined by a dot-blot assay using Mi60 antibody. An aliquot of the supernatant was dotted on a nitrocellulose membrane. Receptor was probed with the monoclonal anti-PR antibody Mi60 (15 µg/ml in PBS containing 1% BSA and 0.5% NP40) for 1 h at 23 C. Mouse antibodies were detected with 125I-labeled antimouse antibody (Amersham International, Little Chalfont, UK) for 1 h at 23 C and autoradiographed. The dots were counted and compared with those obtained with known amounts of PR.

Receptor-bound hormone was determined by the dextran-coated charcoal method (72). Correction was performed for nonspecific binding (incubation in presence of an excess of unlabeled hormone). Binding capacity of the cells was determined by incubation for 4 h with 10 nM [3H]R5020 or with 10 nM [3H]R5020 and 1 µM unlabeled R5020 (nonspecific binding).

Antibodies
Primary anti-PR monoclonal antibodies Mi60 and Let126 were used for immunofluorescence studies at a concentration of 8 µg/ml and 5 µg/ml, respectively (73, 74). Anti-Ran monoclonal antibody (Transduction Laboratories, Lexington, KY) was used at a dilution of 1:300. Tetramethyl rhodamine isothiocyanate (TRITC)-conjugated rabbit antimouse antibody (Dakopatts, Glostrup, Denmark) or CY3-conjugated sheep antimouse antibody (Sigma Chemical Co., St. Louis, MO) were used as secondary antibodies at a dilution of 1:40 and 1:150, respectively. Anti-RCC1 goat polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a concentration of 1 µg/ml and visualized with a TRITC-conjugated rabbit antigoat antibody (Sigma) at a dilution of 1:40.

Indirect Immunofluorescence Studies
Cells were fixed and treated as described previously (10). Hoechst 33258 (Sigma) was used (1 µg/ml) during the incubation with the secondary antibody as a DNA-specific dye to visualize the nuclei. Confocal images were recorded using the LSM410 system on an Axiovert 135 M Zeiss microscope (Carl Zeiss, Thornwood, NY). The subcellular localization of the mutants was determined in at least 100 cells in each experimental condition. Staining was considered as nuclear when it was exclusively nuclear or stronger in the nucleus than in the cytoplasm. In all other cases, it was considered cytoplasmic.

Synthetic Peptides
The following peptides were used: the NLS of simian virus 40 T antigen PKKKRKVEDPYGGC (NLS), the mutated nonfunctional peptide PKTKRKVEDPYGGC (mutNLS), and the NES of the heat-stable protein kinase inhibitor GSNELALKLAGLDINKTGGC (NES), each with a C-terminal cysteine added for coupling to BSA (16, 75). The transport substrates (NLS-BSA, mutNLS-BSA, and NES-BSA) were prepared by coupling the peptides to FITC-labeled BSA (BSA-FITC). BSA-FITC was prepared using the FITC1 kit (Sigma) according to the instructions from the manufacturer. Five milligrams of synthetic peptide were mixed with 4 mg of BSA-FITC activated with succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Pierce, Cheshire, UK) according to the manufacturer’s instructions. The number of peptides conjugated per BSA molecule was estimated by mobility shift assays on SDS polyacrylamide gels. Coupling was an average of 7–10 peptides per BSA molecule.

The search for putative nuclear export or nucleocytoplasmic shuttling sequences on PR was performed with the ExPASy SIM-alignment system for protein sequences (76).

Microinjection Experiments
Cells were plated on glass coverslips precoated with human fibronectin in 35-mm dishes (Nunc). BSA and peptide-BSA conjugates were used at a concentration of 8 mg/ml. Neutralized GTP{gamma}S and ATP{gamma}S (Boehringer, Mannheim, Germany) were used at a concentration of 15 mM and 50 mM, respectively. When required, BSA-FITC (Sigma) or TRITC- conjugated rabbit antimouse antibody (Dakopatts) were used to monitor the cellular site of injection. When import was studied, the competitors were microinjected into the cell cytoplasm. When export was studied, they were microinjected into the cell nucleus. The injection buffer used was 2 mM 1,4-piperazine diethane sulfonic acid, 140 mM KCl (pH 7.4). Eppendorf Femtotips were used for microinjection. Injection pressure was generated by an Eppendorf microinjector 5242, and the microinjection was monitored under a Zeiss inverted microscope Axiovert 35.


   ACKNOWLEDGMENTS
 
We thank Ph. Leclerc (IFR21, Le Kremlin-Bicêtre, France) for technical help with the confocal microscope and J. P. Levillain and Dr. F. Troalen (Département de Biologie clinique, Institut Gustave Roussy, France) for synthesis of the peptides. We thank N. Ghinea for helpful discussion in labeling and coupling the peptides. We thank A. Jolivet for help with the monoclonal antibodies and M. Quesne for the dot-blot experiments. We also thank C. Carreaud for technical assistance. BHK21 and tsBN2 cell lines were a kind gift from Dr. A. Dickmanns (Vanderbilt University, Nashville, TN). We are grateful to Dr. B. Wolff (Novartis, Vienna, Austria) for the generous gift of Leptomycin B. The manuscript was prepared with the assistance of Dr. R. F. Casper and typed by V. Coquendeau and A. D. Dakhlia.


   FOOTNOTES
 
Address requests for reprints to: Dr. Edwin Milgrom, Hormones et Reproduction, INSERM U135, Faculté de Médecine Paris-Sud, 78, rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France. E-mail: milgrom{at}infobiogen.fr

This work was supported by the INSERM, the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer, the Faculté de Médecine Paris-Sud, and the Fondation pour la Recherche Médicale. R.K.T. received a fellowship from the INSERM and the Fondation pour la Recherche Médicale. L.A. received a fellowship from the Ministère de la Recherche et de l’Enseignement Supérieur.

1 R. K. Tyagi and L. Amazit contributed equally to this work. Back

2 Karyopherin and importin are synonyms to designate the same protein. Back

Received for publication May 8, 1998. Revision received July 22, 1998. Accepted for publication August 10, 1998.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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