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
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ABSTRACT
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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) [(
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 (
NLS)PR into the nucleus.
Washing out of the hormone allowed us to follow the export of
(
NLS)PR into the cytoplasm. Microinjection of BSA coupled to a NLS
inhibited the export of (
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 (
NLS)PR could be observed, whereas the export of NES-BSA
was suppressed. Microinjection of GTP
S confirmed that the export of
(
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.
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INTRODUCTION
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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.
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RESULTS
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Model System to Study PR Export from the Nucleus
Initially we considered the possibility of using mutant
PR
638642. 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
638642. 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
638642 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
638642 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
[(
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 (
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
(
NLS)PR mutant into the nucleus. When hormone was washed out of the
cells, an export of (
NLS)PR could be observed in 2 h and was
nearly complete in 4 h (Fig. 1
). 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 ( 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 ( NLS)PR (the epitope
for this antibody was deleted from the wild-type receptor). Protein
localization was determined using confocal microscopy.
Bar, 10 µm.
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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 190198 and
516524) and in the ligand-binding domain (amino acids 681689 and
816824).
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 233263 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
638642 mutant. Hormone administration
provoked receptor entry into the nuclei in cells not microinjected or
microinjected with BSA (Fig. 2
) 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 12 h (not shown).
Similar results were obtained in cells coexpressing wild-type receptor
and NLS-deleted cytoplasmic (
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 (
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 (
NLS)PR from the nucleus into
the cytoplasm was inhibited if, before hormone withdrawal, the cells
were microinjected with NLS-BSA (Fig. 3
).
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
(
NLS)PR export if BSA (Fig. 3
) 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 (
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. 3
).

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Figure 3. Microinjection of NLS-BSA Inhibits the Export of
( NLS)PR
A, The L cell line permanently coexpressing wild-type PR and ( NLS)PR
was treated with progesterone. Cell nuclei were microinjected with
either NLS-BSA, BSA, or NES-BSA, and hormone was washed out. ( 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 ( NLS)PR was observed (left,
arrows). In the cells microinjected with BSA, no inhibition of
the export of ( NLS)PR was observed (left,
arrows). In the cells microinjected with NES-BSA, there was no
inhibition of ( NLS)PR export (left, arrows).
Bar, 10 µm. B, The subcellular localization of
( 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.
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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
(
NLS)PR mutant were incubated with progesterone, provoking entry of
(
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. 4
). In the same
conditions leptomycin B completely inhibited the export of NES-BSA
microinjected into the nucleus (Fig. 4
). This experiment further
confirmed that receptor export does not follow the same pathway as
NES-mediated export.
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
638642 mutant to study receptor import. We then
cotransfected tsBN2 cells with plasmids encoding wild-type PR and
(
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, (
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
638642 (Fig. 5
) or (
NLS)PR into the nucleus was
impeded. Furthermore, partial exit of wild-type receptor from the
nucleus was observed (Fig. 5
). 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 638642, 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.
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Receptor export from the nucleus was studied as described above by
washing out progesterone after having exposed the cells to the
nonpermissive temperature. (
NLS)PR was transferred in part into the
cytoplasm (Fig. 6A
). The partial
character of the transport was probably due to the inhibition of
wild-type receptor reentry into the nucleus. Formation of dimers
between (
NLS)PR and wild-type PR is necessary for the NLS-mediated
export of (
NLS)PR (10).
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. 6B
).
A normal nuclear export was observed in the same cells kept at the
permissive temperature, as well as in BHK21 control cells (Fig. 6B
).
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
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 (
NLS)PR were used. We initially established the
conditions, especially for GTP
S concentration, in which
hormone-mediated nuclear transfer of PR
638642 mutant and of
(
NLS)PR was blocked. We then analyzed the effect of GTP
S on PR
export. We treated cells with progesterone, microinjected their nuclei
with GTP
S, and washed out the hormone. There was no inhibition of
receptor transfer into the cytoplasm (Fig. 7A
). 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
S
into the nucleus (Fig. 7B
). This result suggests that during the
shuttling process receptor reentry, but not receptor exit, from the
nucleus requires GTP hydrolysis.
Similar experiments were also performed with the nonhydrolyzable analog
of ATP:ATP
S. There was partial inhibition of receptor entry into the
nucleus and no effect on PR export (not shown).
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DISCUSSION
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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
,2 as has been
shown previously for the reexport of CBP20 (54). During nuclear import,
proteins carrying NLS are bound to karyopherin
. 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
and
nucleoporins. The NLS-carrying proteins then dissociate from
karyopherin
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
than it is for karyopherin
complexed with a NLS-carrying
protein (45). The export of free karyopherin
is thus favored, but
it remains possible that a small fraction of karyopherin
complexed
to the NLS protein is also reexported from the nucleus. The competition
between NLS-BSA and hormone-dependent import of PR
638642 favors
this hypothesis. The PR
638642 mutant import being much slower than
the NLS-BSA import implies that NLS-BSA is still partially bound to
karyopherin
during its shuttling, competing with PR
638642
binding. It has been shown previously that CAS-mediated karyopherin
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
, 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
638642 mutant could also
be explained by the binding of NLS-BSA to karyopherin
, 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
B
by I
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
-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 412 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).
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MATERIALS AND METHODS
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Plasmids
Nomenclature
Derivatives denoted with a
lack the receptor segment delineated by
the numbered amino acids. Plasmids encoding the wild-type rabbit PR
(pKSV-rPR), and various mutants (PR
638642, PR
373546, and
PR
25103, 547662) have been previously described (10, 19, 65).
The epitope for antibody Mi60 was deleted from the wild-type receptor
(
373546). 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 (
25103) was also deleted from the
mutant PR
547662, which lacks both the constitutive and the
hormone-inducible NLS. It will be referred to as (
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 3648 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-)(
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 (
NLS)PR
was selected to achieve optimum conditions for nuclear import and
export assays.
A L cell line permanently expressing PR
638642 has previously been
described (19).
tsBN2 cell lines permanently expressing wild-type (Mi60-)PR
and PR
638642 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
638642 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 manufacturers instructions. The number of peptides conjugated per
BSA molecule was estimated by mobility shift assays on SDS
polyacrylamide gels. Coupling was an average of 710 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
S and ATP
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 lEnseignement
Supérieur.
1 R. K. Tyagi and L. Amazit contributed equally to this work. 
2 Karyopherin and importin are synonyms to
designate the same protein. 
Received for publication May 8, 1998.
Revision received July 22, 1998.
Accepted for publication August 10, 1998.
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