Download Demonstration by Confocal Microscopy that Unliganded

Demonstration by Confocal
Microscopy that Unliganded
Overexpressed Glucocorticoid
Receptors are Distributed in a
Nonrandom Manner throughout All
Planes of the Nucleus
Vilma R. Martins*, William B. Pratt, Louis Terracio, Margaret A. Hirst, Gordon M. Ringold,
and Paul R. Housley
Department of Pharmacology (V.R.M, P.R.H.)
Department of Anatomy, Cell Biology, and Neurosciences (L.T.)
University of South Carolina School of Medicine
Columbia, South Carolina 29208
Department of Pharmacology
University of Michigan Medical School (W.B.P.)
Ann Arbor, Michigan 48109
Institute of Cancer and Developmental Biology
Syntex, Inc. (M.A.H., G.M.R.)
Palo Alto, California 94304
Mouse glucocorticoid receptors (GR) that are overexpressed in Chinese hamster ovary (CHO) cells
behave like progesterone receptors, in that the unliganded receptor localizes to the nucleus where it
resides in a loosely bound docking complex, probably in association with the 90-kDa heat shock protein (hsp90) and hsp70. In this paper we examine
the localization of the overexpressed GR within the
CHO cell nucleus by confocal microscopy. In hormone-free cells the receptor distributes in a mottled
pattern throughout all planes of the nucleus. The
receptor is not present in nucleoli and shows no
preferential localization in the periphery vs. the center of the nucleus. The mottled distribution in each
plane of the nucleus demonstrates clearly that there
are regions that do not contain receptor; thus, the
distribution of the GR is not random. When triamcinolone acetonide is added to the CHO cells, there is
no detectable change in receptor distribution. Overexpressed receptors that have either no hormonebinding activity or no DNA-binding activity because
of point mutations localize in the same mottled pattern as the wild-type receptor. These observations
are consistent with the proposal that the overexpressed GR can enter the nucleus in its unliganded
state and proceed to loci distributed throughout the
nucleus, where it is retained in an inactive docking
complex until the binding of hormone triggers its
0888-8809/91 /0217-0225S03.00/0
Molecular Endocrinology
Copyright © 1991 by The Endocrine Society
217
progression to high affinity sites where the primary
events in transcriptional activation occur. As there
is no detectable change in localization with the addition of ligand, we suggest that the docking complex may be located very near or possibly at the site
where the primary events in transcriptional activation occur. (Molecular Endocrinology 5: 217-225,
1991)
INTRODUCTION
Although glucocorticoid, estrogen, and progesterone
receptors are all recovered from hormone-free cells in
the cytosolic fraction as heteromolecular complexes in
association with the 90-kDa heat shock protein (hsp90)
(1), there exists a basic difference in the behavior of
unliganded glucocorticoid receptors (GR) vs. estrogen
and progesterone receptors in intact cells. Immunocytochemical studies have shown that unliganded estrogen and progesterone receptors are located in the
nucleus (2-4). This nuclear localization is supported by
the results of enucleation studies as well (5, 6). In
contrast, several laboratories have used immunofluorescence and immunocytochemical localization methods to demonstrate that unliganded GR are located
predominantly in the cytoplasm, and that the receptors
shift to the nucleus upon exposure to hormone (7-12).
It is not known how steroid receptors distribute once
they have entered the nucleus. Is their distribution
Vol 5 No. 2
MOL ENDO-1991
218
random, as indicated by an immunofluorescence or
immunohistochemical signal that is distributed as a
diffuse haze throughout the nucleus at all depths within
the nucleus? Is the distribution nonrandom, with the
existence of regions that do not contain receptors? Are
unliganded receptors that are retained in the inactive
docking state located toward the periphery of the nucleus? Are the loosely bound docking complexes located near the high affinity acceptor sites, which are
presumably the sites where the primary events leading
to transcriptional activation occur? Or does the receptor
undergo a spatial redistribution after it binds hormone
and moves from low affinity to high affinity attachments?
In this manuscript, we use the technique of confocal
scanning microscopy to answer some of these questions. Confocal microscopy is capable of quantitative
imaging of optical sections within a particular plane of
a cell and can be used to analyze the three-dimensional
organization of cellular macromolecules (13). To amplify
the receptor signal, we examine the localization of the
mouse GR in a Chinese hamster ovary (CHO) cell line
in which the receptor is expressed about 10O-fold over
the level of the endogenously expressed GR in parent
CHO cells (14). We have recently shown that the unliganded mouse GR in the overexpressing CHO cell lines
is located in the nucleus (15) in the same manner as
reported for estrogen and progesterone receptors produced from normal cellular genes (2-4). Also, as reported for progesterone receptors (16, 17), the overexpressed mouse GR is recovered in the cytosolic
fraction in association with hsp70 as well as hsp90 after
hypotonic cell rupture (15). The overexpressed mouse
receptor in CHO cells provides an excellent system for
examining the nuclear distribution of both unliganded
and hormone-bound receptors by immunofluorescence,
both because the receptor signal is amplified and because there is unequivocal genetic proof that the immunofluorescence signal represents the receptor. Here
we show that the unliganded GR distributes in a mottled
or patchy nonrandom manner throughout all planes of
the nucleus. The receptor is not located in the nucleolus, and there is no preference for the periphery versus
the center of the nucleus. The distribution of the immunofluorescent signal does not change with binding
of hormone.
RESULTS
Nonrandom Distribution of the Intranuclear GR
Figure 1A shows the absence of immunofluorescence
in CHO cells transfected with plasmid containing the
cDNA for dihydrofolate reductase but no cDNA for GR.
The WCL2 cells shown in Fig. 1B are transfected with
cDNA for the wild-type mouse GR. All of the panels in
Fig. 1 represent transverse intranuclear section scans
through the center of nuclei. The immunofluorescence
pattern throughout the nucleus is mottled, with promi-
nent nucleolar shadows being evident in both this and
subsequent photographs. This mottled pattern is seen
with either cold methanol or paraformaldehyde fixation.
The WCL2 cells are a cloned cell line, and the cultures
originally contained 100% GR-expressing cells. During
the past 1.5 yr, the WCL2 cells used in this study have
been maintained in serial culture, and approximately
30% of the cells no longer express GR, as judged by
immunofluorescence. In this work we do not present
fields containing the revertant cells.
The cells shown in Fig. 1 were grown in hormonefree medium (without phenol red and supplemented
with charcoal-stripped serum); thus, it is the unliganded
wild-type GR in Fig. 1B that is localizing to the nucleus.
As shown in Fig. 1C, the GR in NA cells produce the
same mottled nuclear distribution as the wild-type receptor. The NA cells express mouse receptors that
have a point mutation in the hormone-binding domain
and do not bind steroid (14). This provides genetic proof
that the mottled localization pattern cannot reflect a
steroid-mediated change in receptor localization. The
NB cells shown in Fig. 1D express receptors that contain a point mutation that eliminates DNA-binding activity (14). Because these cells show that the same GR
distribution as the cells containing the wild-type mouse
GR, the mottled distribution pattern does not require
that the receptor be bound to DNA.
Steroid Does not Alter GR Distribution
Figure 2 shows localization of the wild-type GR in WCL2
cells determined with polyclonal anti-hGR antibody 57
as well as with the BuGR2 monoclonal antibody. The
mottled nuclear distribution is seen with both antibodies, and in both cases the signal is immune specific.
There is no change in the mottled distribution pattern
after exposure to a glucocorticoid agonist {c.f. Fig. 2,
B and E with C and F, respectively). These are single
intranuclear sections, but it is important to note that we
see no alteration in receptor distribution with steroid
when nuclei are scanned in a series of planar views
such as those shown in Fig. 3. Additionally, the mottled
pattern exhibited by NA and NB cells is not altered by
steroid.
GR Distribution in Nuclear Sections
Figure 3 shows a series of six optical sections, 2 /urn
apart, through several WCL2 cell nuclei. The GR immunofluorescence is again arranged in a mottled pattern present in all sections, showing that there is no
preferential localization in the nuclear periphery vs. the
core. The mottled distribution of the GR is particularly
clear when one views nuclear sections. Figure 4 shows
a high magnification view of sections from WCL2, NA,
and NB cells. These close-up views show no difference
in receptor distribution between the wild-type and mutant receptors. The immunofluorescence patterns in
Fig. 4 (A-C) show that there is no GR in the nucleolus
and that the receptor is excluded from multiple discrete
Intranuclear Distribution of GR
219
Fig. 1. Nonrandom Nuclear Distribution of Overexpressed Wild-Type and Mutant GR
Cells were incubated with the BuGR2 antibody and analyzed by confocal imaging, as described in Materials and Methods. The
images are transverse optical sections through the center of the cells. The cell lines shown are CHO plus dhfr (A), WCL2 (B), NA
(C), and NB (D).
patchy areas throughout each section. This type of
pattern has been previously observed for other nuclear
moieties, such as small nuclear ribonucleoprotein
(snRNP) structures (18). Figure 4D shows the immunofluorescence pattern obtained when a monoclonal
antibody to RNP is reacted with WCL2 cells. The pattern of U1-RNP nuclear immunofluorescence is similar
to that obtained with the anti-GR probes.
DISCUSSION
It is clear that the nuclear immunofluorescence shown
in Figs. 1-4 represents the localization of the mouse
GR. The nuclear immunofluorescence is both immune
specific for two antireceptor antibodies (Fig. 2) and
specific for the presence of mouse GR cDNA (Fig. 1).
The mottled nuclear localization is seen in the absence
of steroid (Figs. 1 -4), and it occurs with the NA mutant,
which is incapable of binding steroid (Figs. 1 and 4).
Thus, the overexpressed mouse GR in CHO cells is
delivered to the nucleus in its unliganded state.
It is not clear why the overexpressed receptor is
nuclear. Unfortunately, the level of endogenous hamster
GR in the CHO cell is too low (1/1 OOth of the level of
wild-type mouse GR in WCL2 cells) (15) to permit
detection with this immunofluorescence method. It is
possible that the endogenous hamster GR is distributed
predominantly in the cytoplasm in its unliganded state,
as has been reported for a variety of other cells (7-12).
If the endogenous hamster receptor were in the cytoplasm, the nuclear localization of the mouse GR in CHO
cells might be a result of receptor overexpression.
Cidlowski et al. (19) have recently shown that CHO
cells containing transfected human GR at a level that is
increased 3-fold above that of endogenous hamster GR
exhibit predominately cytoplasmic receptors, with some
nuclear receptor signal in the absence of glucocorticoid
agonist. As these transfected CHO cells contain approximately 5% the level of GR found in WCL2 cells,
the increased receptor content of WCL2 cells may be
sufficient to promote predominately nuclear localization
in the absence of steroid. Using our techniques we have
also examined the unliganded mouse GR in COS cells
transiently transfected with the pSV2Wrec expression
vector containing the mouse GR cDNA. In transfected
COS cells exhibiting immunofluoresence and presum-
MOL ENDO-1991
220
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Fig. 2. Receptors Overexpressed in CHO Cells Have the Same Nuclear Distribution in the Presence or Absence of Glucocorticoid
WCL2 cells were incubated with vehicle (A, B, D, and E) or 1 HM triamcinolone acetonide (C and F) and processed for confocal
imaging after incubation with nonimmune mouse IgG (A), BuGR2 antibody (B and C), normal rabbit serum (D), or antibody 57 (E
and F).
Intranuclear Distribution of GR
221
Fig. 3. GR Innmunofiuorescence in Successive Nuclear Sections of WCL2 Cells
Optical sectioning of WCL2 cells incubated with BuGR2 antibody was carried out at 2-/xm increments, starting at a level near
the coverslip (A) and progressing toward the apical surface (B-F).
ably overexpressing the mouse GR, the receptor signal
is localized to the nucleus (data not shown). Although
several cell types containing normal amounts of endogenous GR exhibit an unliganded receptor signal that is
predominately cytoplasmic (7-12, 19), it should be
noted that it has been shown by both an immunocytochemical technique using the BuGR2 antibody (20) and
by cell enucleation techniques (6, 20) that the majority
MOL ENDO-1991
222
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Fig. 4. Magnified Images of GR and RNP Intranuclear Localization
WCL2 (A and D), NA (B), and NB (C) cells were incubated with BuGR2 (A-C) or anti-RNP (D) antibodies and scanned at high
magnification.
of the unliganded GR is located in nuclei of GH3 pituitary
tumor cells. Thus, it is possible that in some cell types
the endogenous (nontransfected) GR is localized predominantly to the nucleus.
Picard and Yamamoto (21) have identified two nuclear localization signals in the GR. One signal (NL1) is
located just to the COOH-terminal side of the DNAbinding domain, and the other (NL2) is located within
the COOH-terminal one third of the receptor that contains the hormone-binding domain. Nuclear localization
of fusion proteins containing NL2 is hormone dependent, whereas nuclear localization of fusion proteins
directed by NL1 is not (21). In that the mottled distribution of the overexpressed mouse GR shown in this
paper is independent of hormone, it is possible that the
passage of GR into the nucleus and to the sites that
make up this pattern is directed by NL1.
We have tested the simple model in which exposure
of the NL1 sequence is correlated with nuclear localization of the unliganded GR. The overexpressed mouse
GR in WCL2 cytosol and the normally expressed mouse
GR in L-cell cytosol were reacted with the AP64 antibody directed against the NL1 sequence. In both cases,
about 20% of the untransformed non-DNA-binding receptors reacted with the AP64 antibody, and this value
increased to 85-90% after receptor transformation (15).
Thus, we found no differential accessibility of the NL1
sequence on receptors in cytosol prepared from cells
in which the unliganded GR is localized entirely to the
nucleus (WCL2 cells) and in cytosol prepared from cells
where a substantial portion of the receptor is located in
the cytoplasm (L-cells) (20). This result could represent
a difference between the in vivo and in vitro accessibility
of NL1, or the ability of the NL1 sequence to interact
with the nuclear localization apparatus may not be
reflected by immunoadsorption of the GR with AP64.
Alternatively, it is possible that the NL1 sequence is not
required for the nuclear localization process we are
observing in WCL2 cells, and exposure of NL1 might
be required only for the tight nuclear association seen
after ligand binding.
Given our current primitive understanding of the systems that determine transport to and within the nucleus,
it is reasonable to propose that the attachment of the
overexpressed mouse GR via a nuclear localization
signal to some cytoskeletal-based transport system
permits its movement from its cytoplasmic site of synthesis to multiple foci located within all planes of the
nucleus. The mottled pattern of GR distribution in the
nucleus indicates that there are regions (in addition to
Intranuclear Distribution of GR
nucleoli) to which the receptor is not transported. The
wild-type mouse receptors in WCL2 cells are transcriptionally active in a hormone-dependent manner (14),
and after hypotonic cell rupture, the unliganded receptors are recovered in cytosol in a complex that contains
both hsp90 and hsp70 (15). It seems likely that unliganded GR in WCL2 cells are transported to the loci
represented by the mottled distribution within the nucleus, and once they are there, the receptors remain in
a docking complex until the binding of hormone triggers
their conversion to high affinity association with nuclear
acceptor sites. Although the elements that make up the
proposed docking complex have not been characterized, hsp90 and hsp70 are potential components.
Given a generalized mottled distribution pattern, one
cannot conclude from our failure to see any change in
GR distribution on exposure to hormone (Fig. 2) that
there is no intranuclear movement of the GR as it
proceeds from low affinity (docking) to high affinity
association with the nucleus. It is clear, however, that
no gross rearrangement (e.g. from the periphery to
more central regions) occurs. Perrot-Applanat et al. (22)
examined the effect of hormone on localization of the
rabbit progesterone receptor at the electron microscopic level using monoclonal antibody and the proteinA-gold technique. They found that the unliganded receptor was randomly scattered over clumps of condensed chromatin, but after hormone administration, it
was mainly detected in the border regions between
condensed chromatin and nucleoplasm. A very local
redistribution of this nature could probably not be detected with the confocal techniques we have employed
in this work.
The thyroid hormone receptor is a member of the
steroid receptor family (23). In contrast to glucocorticoid, estrogen, and progesterone receptors, the thyroid
hormone receptor does not bind hsp90 (24) and does
not enter a loosely bound docking association with the
nucleus (25, 26). In its unliganded state, the thyroid
hormone receptor rapidly becomes tightly associated
with the nucleus (25, 26) and can in some instances
act as a transcriptional inhibitor (27). Thus, it would
seem that the unliganded thyroid hormone receptor
makes its way directly to appropriate regions of the
genome containing thyroid hormone response elements
with which the receptor directly interacts. The absence
of any gross movement of the overexpressed GR when
hormone is added to WCL2 cells suggests that the
docking complexes containing unliganded receptor may
be located at multiple termini of the nuclear transport
pathway for the receptor. Although the unliganded GR
is presumably not bound to a glucocorticoid response
element (28) in the same manner as the unliganded
thyroid hormone receptor is probably bound to a thyroid
hormone response element, the docking complexes
may, nevertheless, be located very close to, or even at,
the loci where the primary events in transcriptional
activation occur. It is interesting to note that snRNP
particles, which exhibit an immunofluorescence pattern
similar to that of the overexpressed mouse GR (Fig. 4),
223
appear to be localized within the nucleus to a reticular
network that extends between the nucleolar surface
and the nuclear envelope (29).
Often, the nuclear localization of a steroid hormone
receptor has been thought to be determined by its
DNA-binding activity (see Ref. 30 for a review of nuclear
binding models). Some models have been based on the
assumption that hormone-mediated transformation of
a steroid receptor to the DNA-binding state results in
its passive trapping in the nucleus by binding to DNA.
As we show that the NB mutant GR, which has no
DNA-binding activity, is localized to the nucleus and
has the same mottled distribution pattern within the
nucleus as the wild-type receptor (Figs. 1 and 4), it is
clear that DNA-binding activity is irrelevant to the nuclear localization process we are observing.
MATERIALS AND METHODS
Materials
Triamcinolone acetonide, polylysine, methotrexate, nonimmune mouse immunoglobulin G (IgG), 1,4-diazabicyclo[2.2.2.]
octane, and fluorescein isothiocyanate-conjugated antirabbit
IgG were obtained from Sigma Chemical Co. (St. Louis, MO).
Dulbecco's Modified Eagle's Medium without phenol red was
purchased from Hazleton (Lenexa, KS). Iron-supplemented bovine calf serum was obtained from Hyclone Laboratories (Logan, UT). The BuGR2 monoclonal antibody (31) was kindly
provided by Drs. William J. Hendry and Robert W. Harrison
(University of Arkansas for Medical Sciences, Little Rock, AK).
Polyclonal antibody 57 was elicited in a rabbit against a peptide
corresponding to amino acids 346-367 of the human GR (19)
and was kindly provided by Dr. John A. Cidlowski (University
of North Carolina, Chapel Hill, NC). The corresponding amino
acid sequence of the mouse GR differs by only one amino
acid, a conservative substitution of Val for lie. We have previously determined that polyclonal antibody 57 reacts with the
mouse GR on blots and in vitro. Bodipy-conjugated antimouse
IgG was from Molecular Probes (Eugene, OR). The monoclonal
antibody to U1-RNP (32) was kindly provided by Dr. Jeffrey R.
Patton (University of South Carolina, Columbia, SC).
Cell Lines
Construction of the four CHO cell lines used in this work has
been previously described (14). Essentially, CHO cells were
transfected with a plasmid containing the cDNA for dihydrofolate reductase (dhfr) and a plasmid containing wild-type or
mutant mouse GR cDNA, and then selected for varying degrees of amplification by growth in methotrexate (MTX).
The cell lines are: 1) CHO plus dhfr, CHO cells transfected
with only the cDNA for dhfr and maintained in 10 MM MTX; 2)
WCL2 cells, a CHO subline transfected with wild-type mouse
GR and maintained in 3 ^M MTX; 3) NA cells, CHO cells that
were transfected with a mutant mouse GR that does not bind
steroid because of a point mutation in the hormone-binding
domain where Glu546 has been changed to Gly (14); NA cells
were maintained in 100 ^ M MTX; and 4) NB cells, CHO cells
expressing a mutant mouse GR that binds hormone but is
unable to bind to DNA because of a point mutation in the DNAbinding domain in which Arg484 has been changed to His (14);
NB cells were maintained in 10 ^M MTX. WCL2, NA, and NB
are all cloned cell lines that contain approximately equal
amounts of immunoreactive GR protein (15). All CHO cell lines
were grown in monolayer culture in Dulbecco's modified Eagle
Vol 5 No. 2
MOL ENDO-1991
224
medium supplemented with 40
supplemented calf serum.
proline and 10% iron-
Confocal Microscopy
In preparation for confocal studies, all cells were grown on
polylysine-coated glass coverslips in medium without phenol
red containing charcoal-stripped serum for 2 days, with daily
medium changes. For the experiment shown in Fig. 2, cells
were further incubated in medium containing vehicle or 1 MM
triamcinolone acetonide for 30 min at 37 C. Coverslips were
washed with PBS, immersed in cold (-20 C) methanol for 10
min, and washed with PBS. Cells were permeabilized with
0.2% Triton X-100 in PBS at room temperature for 10 min and
washed again with PBS. Cells were then incubated for 1 h at
37 C with one of the following primary antibodies diluted in
PBS containing 0.5% BSA: BuGR2 or normal mouse IgG (60
^g/ml), antibody 57 or normal rabbit serum (1:100), or antiRNP (1:100). After washing with 0.5% BSA in PBS, cells were
subsequently incubated for 1 h at 37 C with one of the
following diluted in 0.5% BSA in PBS: Bodipy-conjugated
antimouse IgG (1:40) or fluorescein isothiocyanate-conjugated
antirabbit IgG (1:40). Coverslips were inverted onto depression
slides containing 10 mg/ml 1,4-diazabicyclo[2.2.2.]octane in
PBS-glycerol (1:3) to retard laser bleaching and submitted to
fluorescence microscopy using a Bio-Rad MRC-600 laser
scanning confocal microscope (Richmond, CA). Serial optical
sections were obtained at 0.5- to 2-^m increments.
Acknowledgments
We thank Drs. William J. Hendry III and Robert W. Harrison III
for providing the BuGR2 monoclonal antibody, Dr. John A.
Cidlowski for providing polyclonal antibody 57 and for communicating results before publication, and Dr. Jeffrey R. Patton
for providing the monoclonal antibody to U1-RNP. We are
grateful to Nancy Vinson in the Integrated Microscopic Analysis Facility at the University of South Carolina School of
Medicine for technical assistance with the confocal instrument.
Received October 16, 1990. Revision received November
26,1990. Accepted November 29,1990.
Address requests for reprints to: Paul R. Housley, Department of Pharmacology, University of South Carolina School of
Medicine, Room D-6, Building 1, Columbia, South Carolina
29208.
This work was supported by a fellowship grant from the
Interamerican Development Bank and Universidade de Sao
Paulo (to V.R.M.), American Cancer Society Grant IN-107 (to
P.R.H.), and NIH Grants DK-36905 (to P.R.H.), CA-28010 (to
W.B.P.), HL-40424 (to L.T.), and GM-25821 (to G.M.R.).
* Present address: Faculdade de Medicina, Disciplina de
Oncologia, Universidade de Sao Paulo, Sao Paulo, Brazil.
REFERENCES
1. Pratt WB 1987 Transformation of glucocorticoid and progesterone receptors to the DNA-binding state. J Cell
Biochem 35:51-68
2. King WJ, Greene GL 1984 Monoclonal antibodies localize
oestrogen receptor in the nuclei of target cells. Nature
307:745-747
3. Perrot-Applanat M, Logeat F, Groyer-Picard MT, Milgrom
E 1985 Immunocytochemical study of mammalian progesterone receptor using monoclonal antibodies. Endocrinology 116:1473-1484
4. Gasc JM, Delahaye F, Baulieu EE 1989 Compared intracellular localization of the glucocorticosteroid and progesterone receptors: an immunocytochemical study. Exp Cell
Res 181:492-504
5. Welshons WV, Lieberman ME, Gorski J 1984 Nuclear
localization of unoccupied oestrogen receptors. Nature
307:747-749
6. Welshons WV, Krummel BM, Gorski J 1985 Nuclear localization of unoccupied receptors for glucocorticoids,
estrogens, and progesterone in GH3 cells. Endocrinology
117:2140-2147
7. Papamichail M, Tsokos G, Tsawdaroglou N, Sekeris CE
1980 Immunocytochemical demonstration of glucocorticoid receptors in different cell types and their translocation
from the cytoplasm to the cell nucleus in the presence of
dexamethasone. Exp Cell Res 125:490-493
8. Govindan MV 1980 Immunofluorescence microscopy of
the intracellular translocation of glucocorticoid-receptor
complexes in rat hepatoma (HTC) cells. Exp Cell Res
127:293-297
9. Antakly T, Eisen HJ 1984 Immunocytochemical localization of glucocorticoid receptor in target cells. Endocrinology 115:1984-1989
10. Fuxe K, Wikstrom AC, Okret S, Agnati LF, Harfstrand A,
Yu ZY, Granholm L, Zoli M, Vale W, Gustafsson JA 1985
Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and diencephalon using a monoclonal
antibody against rat liver glucocorticoid receptor. Endocrinology 117:1803-1812
11. Wikstrom AC, Bakke O, Okret S, Bronnegard M, Gustafsson JA 1987 Intracellular localization of the glucocorticoid
receptor: evidence for cytoplasmic and nuclear localization. Endocrinology 120:1232-1242
12. Qi M, Hamilton BJ, DeFranco D 1989 V-mos oncoproteins
affect the nuclear retention and reutilization of glucocorticoid receptors. Mol Endocrinol 3:1279-1288
13. Shotton D, White N 1989 Confocal scanning microscopy:
three-dimensional biological imaging. Trends Biol Sci
14:435-440
14. Hirst MA, Northrop JP, Danielsen M, Ringold GM 1990
High level expression of wild type and variant mouse
glucocorticoid receptors in Chinese hamster ovary cells.
Mol Endocrinol 4:162-170
15. Sanchez ER, Hirst M, Scherrer LC, Tang HY, Welsh MJ,
Harmon JM, Simons SS, Ringold GM, Pratt WB 1990
Hormone-free mouse glucocorticoid receptors overexpressed in Chinese hamster ovary cells are localized to
the nucleus and are associated with both hsp70 and
hsp90. J Biol Chem 265:20123-20130
16. Kost SL, Smith DF, Sullivan W, Welch WJ, Toft DO 1989
Binding of heat shock proteins to the avian progesterone
receptor. Mol Cell Biol 9:3829-3838
17. Smith DF, Faber LE, Toft DO 1990 Purification of unactivated progesterone receptor and identification of novel
receptor-associated proteins. J Biol Chem 265:39964003
18. Verheijen R, Kuijpers H, Vooijs P, Van Venrooij W, Ramaekers F 1986 Distribution of the 70K U1 RNA-associated protein during interphase and mitosis: correlation
with other U RNP particles and proteins of the nuclear
matrix. J Cell Sci 86:173-190
19. Cidlowski JA, Bellingham DL, Powell-Oliver FE, Lubahn
DB, Sar M 1990 Novel antipeptide antibodies to the
human glucocorticoid receptor: recognition of multiple
receptor forms in vitro and distinct localization of cytoplasmic and nuclear receptors. Mol Endocrinol 4:14271437
20. LaFond RE, Kennedy SW, Harrison RW, Villee CA 1988
Immunocytochemical localization of glucocorticoid receptors in cells, cytoplasts, and nucleoplasts. Exp Cell Res
175:52-62
21. Picard D, Yamamoto KR 1987 Two signals mediate hormone-dependent nuclear localization of the glucocorticoid
receptor. EMBO J 6:3333-3340
225
Intranuclear Distribution of GR
22. Perrot-Applanat M, Groyer-Picard MT, Logeat F, Milgrom
E 1986 Ultrastructural localization of the progesterone
receptor by an immunogold method: effect of hormone
administration. J Cell Biol 102:1191-1199
23. Evans RM 1988 The steroid and thyroid hormone receptor
superfamily. Science 240:889-895
24. Dalman FC, Koenig RJ, Perdew GH, Massa E, Pratt WB
1990 In contrast to the glucocorticoid receptor, the thyroid
hormone receptor is translated in the DNA binding state
and is not associated with hsp90. J Biol Chem 265:36153618
25. Samuels HH, Tsai JS, Casanova J, Stanley F1974 Thyroid
hormone action: in vitro characterization of solubilized
nuclear receptors from rat liver and cultured GH, cells. J
Clin Invest 54:853-865
26. Casanova J, Horowitz ZD, Copp RP, Mclntyre WR, Pascual A, Samuels HH 1984 Photoaffinity labeling of thyroid
hormone nuclear receptors: influence of n-butyrate and
analysis of the half-lives of the 57,000 and 47,000 molec-
27.
28.
29.
30.
31.
32.
ular weight receptor forms. J Biol Chem 259:1208412901
Koenig RJ, Lazar MA, Hodin RA, Brent GA, Larson PR,
Chin WW, Moore DD 1989 Inhibition of thyroid hormone
action by a non-hormone binding c-erbA protein generated
by alternative mRNA splicing. Nature 337:659-661
Becker PB, Gloss B, Schmid W, Strahle U, Schutz G 1986
In vivo protein-DNA interactions in a glucocorticoid response element require the presence of hormone. Nature
324:686-688
Spector DL1990 Higher order nuclear organization: threedimensional distribution of small nuclear ribonucleoprotein
particles Proc Natl Acad Sci USA 87:147-151
Walters MR 1985 Steroid hormone receptors and the
nucleus. Endocr Rev 6:512-543
Gametchu B, Harrison RW 1984 Characterization of a
monoclonal antibody to the rat liver glucocorticoid receptor. Endocrinology 114:274-279
Billings PB, Allen RW, Jensen FC, Hoch SO 1982 AntiRNP monoclonal antibodies derived from a mouse strain
with lupus-like autoimmunity. J Immunol 128:1176-1180