|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 27, 19352-19360, July 2, 1999
From the Laboratory of Cell Biochemistry and Biology, NIDDK,
National Institutes of Health, Bethesda, Maryland 20892
Vitamin D receptor (VDR) acts as a transcription
factor mediating genomic actions of calcitriol. Our earlier studies
suggested that calcitriol induces translocation of cytoplasmic VDR, but the physiologic relevance of this finding remained uncertain. Previous
studies demonstrated that the activation function 2 domain (AF-2) plays
an essential role in VDR transactivation. To elucidate hormone-dependent VDR translocation and its role, we
constructed green fluorescent protein (GFP) chimeras with full-length
VDR (VDR-GFP), AF-2-truncated VDR (AF-2del-VDR-GFP), and ligand-binding domain (LBD)-truncated VDR (LBDdel-VDR-GFP). COS-7 cells were transiently transfected with these constructs. Western blot analysis, fluorescent microscopy, and transactivation assays showed that the
generated chimeras are expressed and fluoresce and that VDR-GFP is
transcriptionally active. After hormone treatment, cytoplasmic VDR-GFP
translocated to the nucleus in a concentration-, time-, temperature-,
and analog-specific manner. Hormone dose-response relationships for
translocation and for transactivation were similar. Truncation of LBD
and truncation of AF-2 each abolished hormone-dependent translocation and transactivation. Our data confirm a
hormone-dependent VDR translocation, demonstrate that an
intact AF-2 domain is required for this translocation, and indicate
that translocation is part of the receptor activation process.
The vitamin D receptor
(VDR)1 is a member of the
steroid/thyroid superfamily of transcription factors that mediates gene
expression in a calcitriol-dependent fashion. The
hormonally active form of vitamin D, calcitriol, is the principal
regulator of calcium homeostasis and also regulates hormone secretion,
immune functions, cell proliferation, and differentiation (1). After
ligand binding, VDR undergoes an activation process. The mechanism and
the regulatory steps involved in this activation process are under
intense investigation. An understanding of these activation steps
offers possibilities for selective pharmacological modulation of
steroid hormone actions.
Recent studies showed that steroid receptor activation involves
conformational changes (2, 3), phosphorylation/dephosphorylation (4),
interactions with coregulator proteins (2, 5, 6), and dimerization (7).
The activation process of the glucocorticoid receptor (GR), androgen
receptor, and progesterone receptor also includes a translocation of
these receptors from the cytoplasm to the nucleus. For many years,
however, VDR has been considered to reside exclusively in the nucleus
even without hormone (8, 9). More recently, significant amounts of
unliganded VDR was found in the cytoplasm with immunocytology (10, 11)
and with fluorescent labeled hormone in living cells (12). These
studies also showed a shift from cytoplasm into the nucleus after
calcitriol exposure. Nevertheless, subcellular distribution of VDR
remained controversial. Dynamic studies were needed in living cells to clarify the intracellular localization of VDR and to investigate the
physiologic significance of a VDR translocation.
Green fluorescent protein (GFP) chimeras with other steroid hormone
receptors were used recently to demonstrate hormone-induced translocation (13-17). This approach allows introduction of mutated steroid hormone receptors into GFP chimeras to explore the role of
particular domains in receptor trafficking and steroid hormone actions.
Recent studies showed that a segment of VDR close to the C terminus of
the ligand-binding domain (LBD), called the activation function 2 domain (AF-2) plays a primary role in conducting the hormonal signal to
the VDR-dependent genes (18, 19). Other steroid hormone
receptors have homologous AF-2 domains (20, 21). AF-2 could be involved
in many steps during the receptor activation process, such as hormone
binding (22, 23), dimerization (6), and a repositioning of the
C-terminal In the present study, we developed VDR-GFP chimeras to study
subcellular distribution of VDR, to clarify the existence of a
hormone-dependent translocation of VDR, and to investigate
the possibility that translocation is part of the activation process. A
GFP chimera with a truncated AF-2 region of VDR was used to investigate
the role of hormone-dependent translocation in VDR function. We report here that a subset of VDR is translocating from the
cytoplasm into the nucleus in a hormone analog-, dose-, time-, and
temperature-dependent fashion. This translocation requires an intact AF-2 region. The close correlation between hormone-induced translocation and transactivation further supports a model for VDR
activation that includes hormone-dependent translocation.
Plasmid Construction--
The GFP gene was amplified by
polymerase chain reaction using the pQBI25 plasmid (Quantum
Biotechnologies, Montreal, Quebec, Canada) as template with Pwo
proofreading DNA polymerase (Roche Molecular Biochemicals).
HindIII and KpnI restriction sites were inserted
into the pQBI25 plasmid before the GFP gene. The wild-type VDR and the
two truncated VDR sequences were also amplified by PCR, using the
pAV-hVDR plasmid as template (gift from Dr. J. Wesley Pike, University
of Cincinnati College of Medicine, Cincinnati, OH). The generated
amplicons (wild-type containing amino acids 1-427, AF-2-truncated
containing amino acids 1-421, and LBD-truncated containing amino acids
1-294) were inserted into the modified pQBI25 plasmid between the
HindIII and KpnI sites. The DNA sequences of all
generated constructs (VDR-GFP, AF-2del-VDR-GFP, and LBDdel-VDR-GFP) were verified using dye terminator cycle sequencing kit
(Perkin-Elmer).
Cell Extract Preparation and Immunoblotting--
Cells grown in
165-cm2 flasks were mock-transfected or transfected with 15 µg of VDR, VDR-GFP, AF-2del-VDR-GFP, or LBDdel-VDR-GFP plasmids by
LipofectAMINE Plus reagents according to the manufacturer's instructions (Life Technologies, Inc.). In another experiment, each
flask of cells was transfected with 30 µg of plasmids. After allowing
for receptor expression for 24, 48, or 72 h in the absence of
hormone, high salt extract was prepared as described before (25).
Briefly, cells were harvested, washed in PBS, resuspended in high salt
buffer containing 300 mM KCl, 1.5 mM EDTA, 10 mM Tris, 10 mM sodium molybdate, 1 mM dithiothreitol (Life Technologies, Inc.), and 1 trypsin
inhibitory unit/ml aprotinin (Sigma) and then sonicated. Lysates were
subjected to centrifugation at 40,000 × g for 1 h, and the resultant supernatants were desalted and concentrated using
Centricon-30 concentrators (Amicon, Beverly, MA). Concentrated
supernatants were denatured for 5 min at 95 °C in Tris-glycine
sample buffer (Novex, San Diego, CA) with 2.5% Transactivation Studies--
COS-7 cells (American Type Culture
Collection, Manassas, VA) were grown on 6-well plates in Dulbecco's
modified Eagle's medium (Biofluids, Rockville, MD) with 10% fetal
bovine serum (HyClone, Logan, UT). COS-7 cells were selected because
they are deficient in expressing endogenous VDR. Cells were transfected
by diethyl aminoethyl dextran method (Amersham Pharmacia Biotech) with
or without one of the three expression plasmids (0.05 µg/well), the luciferase reporter plasmid p24OH/LUC-23 (0.8 µg/well) (gift from Dr.
Hector F. DeLuca), and the Microscopy--
Whereas most of the microscopy experiments were
done using COS-7 cells, subcellular distribution of VDR-GFP was also
evaluated in ROS17/2.8 rat osteosarcoma cells (gift from G. A. Rodan, Merck Reasearch Laboratories), 3134 mouse adenocarcinoma cells
(gift from G. L. Hager, NCI, National Institutes of Health) (27), 293 adenovirus-transformed human embryonal kidney cells, C32 human melanoma cells, CV-1 green monkey kidney cells, and human dermal fibroblasts (American Type Culture Collection). Cells were subcultured into 2-chamber glass coverslip slides (Nalge Nunc Int., Naperville, IL)
and then transfected with 0.2 µg/well plasmid by LipofectAMINE Plus
reagents. Cells were used within 48 h after transfection. Vehicle
or hormones were added in Hanks' balanced salt solution (Life
Technologies, Inc.) with 1% bovine serum albumin and 2% dextrose. For
studies on living cells, an incubator was built around the microscope
to maintain controlled 37 °C temperature. Pictures were taken of
living cells before and after hormone treatment from multiple fields
with 10% attenuation and 8 line averaging. For real time imaging,
pictures were taken every 15 s from the same microscopy field at
30% attenuation of 50% laser power. Morphometric analysis was
performed using pictures of fixed cells. After hormone or vehicle
treatment, cells were fixed in Morphometric Analysis--
For evaluation of nuclear/cytoplasmic
signal distribution, confocal images were taken of each fluorescing
cell at the highest circumference of the nuclei. 60-80 cells were
evaluated for each experimental condition. Cells that showed clear
morphological changes due to protein overexpression were excluded from
the statistical analysis. Fluorescence intensities and area measurement
values were obtained from nuclei, cytoplasm, and an area in the
background using the Area Measure function of the laser scanning
microscope software (Carl Zeiss Inc.). Integrated fluorescence values
were calculated by multiplying mean brightness with the respective area
values. The ratios of nuclear integrated fluorescence and whole cell
integrated fluorescence were corrected for background brightness of the
same picture. Data are presented as mean ± 1 S.E.
Cloning and Expression of Fusion Proteins--
To develop a
physiologically relevant model for studying intracellular trafficking
of VDR, we generated a series of VDR-GFP chimeras with a highly
fluorescent GFP variant fused in frame to the C terminus of the human
VDR. A Gly-Ala linker was used between the VDR and GFP to provide a
flexible link between the two proteins. In addition to the chimera
containing the wild-type VDR (VDR-GFP), two other chimeras were
generated, one with a truncated AF-2 region (AF-2del-VDR-GFP), and
another with a large deletion within the ligand-binding region
(LBDdel-VDR-GFP). In the AF-2del-VDR-GFP, six amino acids were deleted
at the C terminus of VDR. This deletion severely compromise
hormone-dependent transactivation capacity (28, 29). In the
LBDdel-VDR-GFP, we generated the same mutation that was identified in
cells from one of our patients with hereditary vitamin
D-dependent rickets (30). Previous studies showed that the
resulting truncated protein does not bind hormone and does not activate
VDR target genes (31).
Fusion protein expression was monitored by both microscopy and Western
blot analysis in transiently transfected COS-7 cells. Microscopy showed
the presence of fluorescent proteins as early as 4 h after
transfection, and a maximal expression after 24 h. To verify
whether the predicted size proteins were expressed, we prepared high
salt protein extracts 48 h after transfection with 15-µg
plasmids for each flask of cells. Using Western blot analysis, fusion
proteins were detected with both the anti-GFP and the anti-VDR
antibodies (Fig. 1). We detected
immunoreactivity with both antibodies for a 77-kDa band in the VDR-GFP
extract (Fig. 1, A and B, lane 3), a 76-kDa band
in the AF-2del-VDR-GFP extract (Fig. 1, A and B, lane
4), and a 61-kDa band in the LBDdel-VDR-GFP extract (Fig. 1,
A and B, lane 5), which were the expected
molecular masses for the chimeric proteins. No significant degradation
products, incorrectly expressed proteins, 27-kDa GFP (compare with
purified GFP on Fig. 1A, lane 2), or 50-kDa VDR (compare
with extract from VDR overexpressing cells on Fig. 1B, lane
2) was detected in the extracts from cells expressing chimeric
proteins. However, in samples prepared after transfection with more
plasmids or with longer posttranscription time, degradation products
were detectable (data not shown). Based on these data, in subsequent
experiments care was taken to minimize overexpression and protein
degradation by using relatively low plasmid concentrations and by
minimizing the time for expression.
VDR-GFP Is Transcriptionally Competent--
Transcriptional
activation capacities of the full-length and truncated VDR-GFP fusion
proteins were determined by a luciferase reporter assay in COS-7 cells
as described under "Experimental Procedures." When
VDR-GFP-transfected cells were stimulated by 10 nM
calcitriol, the reporter gene was activated by 13-fold (Fig. 2). No activation above the endogenous
level was detected after 10 nM calcitriol treatment in
cells that were transfected with AF-2del-VDR-GFP or LBDdel-VDR-GFP. No
activation was detected after exposure to D3 in cells
transfected by either full-length or truncated VDR-GFP constructs (Fig.
2). These data demonstrated that the transiently expressed VDR-GFP is
transcriptionally active in COS-7 cells. Furthermore, VDR-GFP
transcriptional activity is calcitriol analog-specific and is also
contingent upon an intact ligand-binding domain.
Subcellular Distribution of VDR-GFP in the Absence and Presence of
the Ligand--
For microscopy, VDR-GFP was transiently expressed in
various types of cultured cells as described under "Experimental
Procedures." VDR-GFP fluorescence was easily detectable in every type
of cell tested, and the subcellular distribution was also similar
regardless of cell type. Without hormone treatment, fluorescence from
unliganded VDR-GFP was detected both in the cytoplasm and in the
nucleus (Figs. 3, A, C, and
E, and 4A). The
cytoplasmic distribution was homogeneous in most cells, but in a
subpopulation of cells VDR-GFP concentrated in the perinuclear region
(Fig. 4A). Within the nucleus, unliganded VDR-GFP
distributed homogeneously, with the exclusion of the nucleoli.
Significant cell-to-cell differences in the nuclear/cytoplasmic brightness ratios were apparent within the same cell population. Whereas some cells contained the receptors predominantly in the cytoplasm, most cells had the same brightness in the cytoplasm and in
the nucleus, and occasionally the nucleus appeared moderately brighter
than the cytoplasm. Experiments with higher plasmid concentration for
transfection or with longer time after transfection showed that
overexpressing cells often had large fluorescent clumps in the
cytoplasm and within the nucleus and that fluorescence increased disproportionately in the nucleus.
When the VDR-GFP expressing cells were treated with 100 nM
calcitriol for 30 min at 37 °C, the fluorescent signal from the cytoplasm shifted into the nucleus (Fig. 3, B, D, and
F). After hormone treatment, the cytoplasm became dark, and
the nuclear fluorescence intensity significantly increased. This
hormone dependent translocation was apparent in every cell line
that we studied. Within the nucleus after hormone treatment, the
fluorescence distribution became more heterogeneous; in some cells,
small foci were detected, but the nucleoli remained void of receptors
(Fig. 3).
Real-time Imaging of Ligand-dependent VDR-GFP
Translocation--
To demonstrate that the shift in fluorescence
intensity from the cytoplasm into the nucleus after hormone treatment
represents true translocation of receptors, real-time imaging was
carried out on single COS-7 cells by confocal microscopy during hormone treatment. Care was taken to maintain constant 37 °C temperature and
to minimize photodamage. Viability of cells after imaging was tested by
monitoring membrane integrity and subsequent cell division.
Translocation was detected as early as 5 min after hormone treatment
and completed within 25-30 min (Fig. 4). First, receptors moved from
the periphery of the cell into the perinuclear region (Fig. 4,
B and C). During this time, receptors along the
plasma membrane became visible (Fig. 4D, at P).
These receptors moved from the plasma membrane into the nucleus at
later time points (Fig. 4, G and H). Perinuclear
vacuoli, characteristic of this cell type, remained dark during
translocation. After 5 min with calcitriol, a gradual increase in
nuclear fluorescence paralleled a decrease in the cytoplasm (Fig. 4,
C-H). After 30 min, cytoplasmic fluorescence was minimal
(Fig. 4H). No additional translocation was detected when
cells were followed up to 2 h. After removal of hormone,
cytoplasmic fluorescence was restored within 30 min (data not shown).
Similar hormone-dependent translocations were recorded in
melanoma cells (data not shown). Although it is apparent in Fig. 3,
A, C, and E, and in Fig. 4A that
VDR-GFP can move into the nucleus without hormone treatment, these
experiments conclusively demonstrated that calcitriol induces the
translocation of the cytoplasmic VDR-GFP into the nucleus.
VDR-GFP Translocation Is Time- and
Temperature-dependent--
COS-7 cells were used for
quantitative characterization of hormone-dependent
translocation of VDR-GFP. Because COS-7 cells are deficient in VDR, the
motion rate of VDR-GFP was not influenced by the motion rate of
unlabeled VDR which would homodimerize with labeled VDR in other types
of cells. Images were taken from fixed cells as described under
"Experimental Procedures." We used methanol fixation because
preliminary experiments showed that this fixation method causes the
least change in cytoplasm/nucleus VDR-GFP distribution (data not
shown), but this fixation still caused changes in intranuclear distribution. The most noticeable change was that the nucleoli were no
longer void of receptors after fixation (compare Fig. 4 and Fig.
5C).
Previous studies with GFP chimeras, as well as our preliminary
experiments, showed that fluorescence intensities in cell compartments are measures of receptor number. We therefore determined the integrated brightness values from the cytoplasm and nucleus at each condition. Statistical analysis of data allowed characterization of the
translocation process, despite the inhomogeneity of nuclear/cytoplasmic
distribution. Our data show that before hormone treatment, only about
25% of total cellular VDR-GFP fluorescence is in the nucleus (Fig.
5A). After hormone treatment
for 30 min, the nuclear fluorescence increased to about 70% of the
total cellular fluorescence. This change reflects a
hormone-dependent movement of half the VDR-GFP into the
nucleus. Analysis of the time course of VDR-GFP translocation shows a
half-maximal response 15 min after calcitriol and slightly earlier
(~13 min) after the same dose of the more potent analog, KH1060.
Temperature sensitivity was tested to verify whether hormone
dependent translocation of VDR-GFP is an active process.
Transfected cells were treated with hormone at 4 °C, at room
temperature, and at 37 °C. After 30 min with hormone, translocation
was detected only at 37 °C (Fig. 5, B and C).
If translocation were driven by diffusion, we would detect a shift of
receptors from the cytoplasm into the nucleus at room temperature.
However, we did not find translocation at 20 °C or lower
temperatures, suggesting that motion is driven by an
energy-dependent mechanism.
Hormone Dose Dependence of Cytoplasm-to-Nucleus
Translocation--
To characterize concentration dependence of VDR-GFP
translocation, images were taken from fixed cells before and 30 min
after exposure to graded doses of hormones. Fig.
6 shows that a small shift of
fluorescence from the cytoplasm into the nucleus is detectable with 100 pM of calcitriol or 10 pM of KH1060.
Half-maximal translocation was at 5 nM or 10 pM
of calcitriol or KH1060, respectively (Fig. 6). When hormone-dose
dependence of translocation is compared with hormone dose dependence of
transactivation, the similarities of these two functions become clear.
Half-maximally effective dose for calcitriol to induce transcription
was also 5 nM, and for KH1060, it was 10 pM.
These data show that KH1060 is more potent than calcitriol in inducing
both translocation and transactivation by the full-length VDR-GFP and
suggest that translocation is part of the receptor activation
process.
Specificity of Cytoplasm-to-Nucleus
Translocation--
Hormone-induced changes in receptor functions are
characterized by hormone analog specificity and by a dependence on the
structural integrity of the receptor protein.
To test the hormone-dependent translocation of VDR-GFP, we
compared the effects of different hormone analogs on VDR-GFP
translocation. Saturating doses of calcitriol induced complete receptor
translocation (Fig. 7). The same degree
of translocation could be achieved by saturating doses of KH1060 (Fig.
6). On the other hand, at the same concentration, D3 a
hormonally inactive analog was unable to induce any translocation of
VDR-GFP (Fig. 7).
To further explore the specificity of hormone action on translocation,
we monitored nuclear/cytoplasmic distribution of a LBDdel-VDR-GFP after
hormone treatment. Before hormone treatment, the nuclear/cytoplasmic
distribution of this truncated receptor was indistinguishable from the
distribution of wild-type VDR-GFP. No translocation of this mutant was
detected at 100 nM calcitriol (Fig. 7). These experiments
prove that hormone-dependent translocation of VDR-GFP is a
specific hormone action. For an effective translocation, the ligand has
to be hormonally active, and the ligand-binding domain has to be intact.
Cytoplasm-to-Nuclear Translocation of VDR Is Dependent on an Intact
AF-2 Region--
Whereas intact nuclear localization signals are
needed for hormone-independent translocation of VDR (11), we assumed
that a distinct receptor domain is responsible for the
hormone-dependent translocation. Because an intact AF-2
region of VDR is essential for hormone-dependent
transactivation, we tested whether the AF-2 region is involved in the
hormone-dependent translocation of VDR. COS-7 cells were
transfected with AF-2del-VDR-GFP, and we measured hormone-dependent translocation by microscopy and
morphometric analysis. Truncation of the AF-2 region did not affect
hormone-independent translocation, but the calcitriol-induced
translocation of AF-2del-VDR-GFP was completely abolished (Fig.
8). The deletion of the AF-2 region interferes not only with coactivator protein binding and
transactivation but also with calcitriol binding. In contrast, the
synthetic analog KH1060 is known to bind with high affinity to an AF-2
deleted VDR (3, 22). Thus, we tested AF-2del-VDR-GFP translocation after incubation with KH1060. At physiologically relevant
concentrations, KH1060 failed to induce any translocation or
transactivation (data not shown). However, an extremely high
concentration of KH060 (100 nM) induced a slight
translocation and a small increase in reporter activity in
cotransfection experiments (Fig. 8). This effect further supported the
idea that a connection exists between translocation and
transactivation. Most importantly, these experiments suggested that
beyond allowing for hormone binding, intact functioning of the AF-2
region is required for hormone-dependent translocation of
VDR-GFP.
We constructed and characterized a VDR-GFP chimera to study VDR
distribution and intracellular motion in living cells. With this
transcriptionally active chimera, we showed that VDR partitions between
the cytoplasm and nucleus. Our studies revealed that VDR-GFP translocates into the nucleus both by a hormone-independent and a
hormone-dependent mechanism. Using deletion mutants we
found that the AF-2 region of VDR is involved in the
hormone-dependent translocation.
We decided to use a GFP chimera of VDR for these studies because other
methods to visualize VDR gave controversial results about subcellular
distribution and were not suitable to detect receptor motion in real
time. We were also encouraged by our previous success using a GFP
chimera of GR to study hormone-dependent translocation in
real time (15). Our laboratory became interested in studies of VDR
translocation almost 10 years ago when, using immunocytology with
microwave fixation, we found that about half of the unliganded VDR
resides in the cytoplasm and translocates into the nucleus after
hormone addition, whereas the accepted dogma was that VDR is
exclusively nuclear (10). Since then, several studies supported our
findings (11, 12, 32, 33), but a direct demonstration of receptor
movement was awaited to clarify the mechanisms and role of VDR
translocation. Although it is relatively easy to make plasmids encoding
chimeric proteins, the resulting protein must retain the functions of
both proteins to be used as a model. The protein also has to be stable,
as degradation products could prevent interpretation of experimental
results. Here, we showed that both the GFP and the VDR parts of the
chimeric protein remain functional in cultured cells; the GFP-tagged
VDR remains transcriptionally active (Figs. 2 and 6), and the GFP
remains fluorescent (Figs. 3-5 and 7). Western blot analysis
demonstrated that VDR-GFP is expressed and relatively stable, without
significant degradation in the first 48 h after transfection (Fig.
1). These data demonstrate that VDR-GFP is suitable for studying
receptor distribution and motion.
Using VDR-GFP, we clarified that unliganded VDR resides both in the
cytoplasm and in the nucleus. Within the same cell population, we found
great heterogeneity in the nuclear/cytoplasmic ratio of fluorescence.
Frequently, the nuclei looked brighter than the cytoplasm, but because
of the larger volume of the cytoplasm, statistical analysis of the
integrated brightness values revealed that only about 25% of the
receptors were in the nucleus without hormone. Hence, the distribution
of VDR is similar to the distribution of thyroid receptor Most importantly, however, the VDR-GFP could be observed in real time,
to reveal a rapid hormone-induced translocation of cytoplasmic
receptors into the nucleus. Earlier studies with immunocytology and
with a fluorescent calcitriol derivative, BODIPY-calcitriol, suggested
that a time-dependent shift of VDR takes place from the
cytoplasm to the nucleus. Here, with the use of VDR-GFP, we were able
to record the translocation of the receptor itself in real time (Fig.
4). Morphometric analysis of digital images allowed for detailed
characterization of this hormone-dependent translocation. Our experiments showed that this process, like nuclear VDR functions, is time-dependent (Figs. 4 and 5A),
temperature-sensitive (Fig. 5, B and C), is
hormone dose-dependent (Fig. 6), shows calcitriol analog
specificity (Fig. 7), and shows dependence on an intact ligand binding
domain (Fig. 7). These results demonstrate conclusively for the first
time that hormone induces VDR translocation, much as hormone induces
translocation of GR (15), androgen receptor (14), mineralocorticoid
receptor (13), and thyroid receptor (16).
Distribution of VDR-GFP within the nucleus also changed with addition
of calcitriol. After hormone treatment, the intranuclear VDR-GFP
pattern became less homogeneous, and small intranuclear foci developed.
The availability of functional VDR-GFP should advance our ability to
explore the significance of these intranuclear changes and investigate
the architecture of VDR target sites in a physiologically relevant context.
The ease of tracking VDR movement with this GFP chimera invites its use
to investigate mechanisms of both hormone-independent and
hormone-dependent VDR movement.
The hormone-independent translocation likely results from receptor
association with proteins of the nuclear import machinery. Previous
studies determined that the nuclear import of steroid receptors
requires binding of their NLS sequences to the NLS receptor importin
We explored the mechanisms of the hormone-dependent VDR
translocation as well. Previous papers (5, 42) reported that a highly
conserved region at the extreme C terminus of nuclear receptors,
including the vitamin D receptor, the AF-2 domain, mediates
hormone-dependent transactivation and interaction with coactivators (5, 10). Recently, it became clear that hormone binding
results in the repositioning of the helix 12 that contains the AF-2
domain and that this conformational change presents an interface for
binding to coactivating proteins, such as CBP and SRC-1 (2, 3, 21, 22,
28, 42). Our results indicate that the AF-2 region also mediates
hormone-dependent translocation of VDR (Fig. 8). We found
that truncation of the AF-2 domain did not affect hormone-independent
translocation but completely abolished the ability of calcitriol to
initiate VDR-GFP translocation. Because truncation of the AF-2 domain
interferes with calcitriol binding we also tested the ability of a
synthetic calcitriol analog, KH1060, to induce translocation. This
analog has been shown to bind the AF-2-truncated receptor with high
affinity but was ineffective in inducing transactivation of a
VDR-responsive reporter. KH1060 did not induce translocation of
AF-2del-VDR-GFP, suggesting that binding of coactivator proteins
through the AF-2 region is involved in hormone-dependent
VDR translocation. The close correlation between the dose-response of
transactivation and translocation also suggested that
hormone-dependent translocation is part of the receptor
activation process.
It is not yet clear how VDR translocation contributes to
transactivation. A function of this translocation could be to carry the
hormone into the nucleus (43). This suggestion is supported by in
vivo findings; specific accumulation of calcitriol within the
nuclei of intestinal villus cells has been detected 0.5 h after
the administration of labeled hormone (44).
Hormone-dependent VDR translocation can also function to
rapidly increase the number of transcriptionally competent receptors
within the nucleus. This is consistent with the observation that an
increase in intestinal calcium absorption and in 9-kDa calcium-binding
protein mRNA level can be detected 1 h after calcitriol
injection in vitamin D deficient rats (44). Because regulation of
intestinal calcium absorption is the main function of vitamin D, this
rapid hormone-dependent translocation of VDR could have
high physiological relevance.
We developed the first transcriptionally active highly fluorescent
VDR-GFP chimera, allowing us to easily see and study VDR movement in
real time. The use of VDR-GFP yielded new insights into the mechanisms
of VDR activation. Our experiments clarified that VDR translocates into
the nucleus by both a hormone-independent and a
hormone-dependent mechanism. Using AF-2del-VDR-GFP, we
identified that the AF-2 region mediates hormone-dependent
translocation. Further details of the mechanisms of
hormone-dependent translocation, including the
identification of transport proteins, remain to be determined. This new
model permits us for the first time to search for ligands and
regulatory mechanisms that differentially affect receptor motion.
We thank Drs. J. W. Pike (University of
Cincinnati College of Medicine, Cincinnati, OH) for the pAVhVDR
plasmid, H. F. DeLuca (University of Wisconsin, Madison, WI) for
the p24OH/LUC-23 reporter plasmid and the VD2F12 anti-VDR antibody,
G. L. Hager (NCI, National Institutes of Health) for the 3134 cells, G. A. Rodan (Merck Research Laboratories, West Point, PA)
for the ROS 17/2.8 cells, D. Love (NIDDK, National Institutes of
Health) for useful suggestions about cloning, G. Poy (NIDDK, National
Institutes of Health) for the oligonucleotide synthesis and DNA
sequencing, and Leo Pharmaceuticals (Ballerup, Denmark) for the KH1060.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Bldg. 8, Rm. 422, NIDDK, National Institutes of Health, 8 Center Dr., Bethesda, MD 20892. Tel.: 301-402-2868; Fax: 301-496-9431; E-mail:
jul{at}helix.nih.gov.
The abbreviations used are:
VDR, vitamin D
receptor;
AF-2, activation function 2 domain;
D3, cholecalciferol;
GFP, green fluorescent protein;
GR, glucocorticoid
receptor;
LBD, ligand-binding domain;
NLS, nuclear localization
signal.
Hormone-dependent Translocation of Vitamin D
Receptors Is Linked to Transactivation*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helix of the ligand-binding domain to form an
interaction surface with coregulators (2, 3, 24) and with proteins of
the transcriptional machinery (5). The role of AF-2 domain in receptor
trafficking has not been investigated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol and
kept at 
70 °C. For controls, purified GFP (CLONTECH Laboratories, Palo Alto, CA) and a high
salt extract from VDR overexpressing 293 cells (25) were used. Samples
containing 5 µg of protein were loaded onto polyacrylamide gels
(Novex, San Diego, CA), and after electrophoretic fractionation,
proteins were electrotransferred to nitrocellulose membranes. Membranes were blocked with 5% milk and then with avidin/biotin blocking kit
reagents (Vector Laboratories, Burlingame, CA) and then subjected to
Western blot analysis using VD2F12 anti-VDR (a gift from Dr. Hector F. DeLuca, University of Wisconsin, Madison, WI) or polyclonal anti-GFP
(CLONTECH) antibodies. Secondary antibodies were
from Sigma and Kirkegaard & Perry Laboratories (Gaithersburg, MD). Horseradish peroxidase-labeled streptavidin was from Amersham Pharmacia
Biotech. Blots were developed with enhanced chemiluminescence kit
reagents and protocol (Amersham Pharmacia Biotech).
-galactosidase standardization plasmid
pGL3 (1.2 µg/well) (Promega, Madison, WI). Total DNA amount was
equalized by adding sheared herring DNA. The p24OH/LUC-23 plasmid
encodes a luciferase that is under the control of the 25-hydroxyvitamin
D3 24-hydroxylase promoter (26). Transfected cells were
incubated with graded doses of either calcitriol (Solvay Duphar, Weesp,
The Netherlands),
20-epi-22-oxa-24a,26a,27a-trihomo-1,25-dihydroxyvitamin D3 (KH1060) (gift of Leo Pharmaceuticals, Ballerup,
Denmark), cholecalciferol (D3) (Sigma), or vehicle for
48 h. Cells were lysed on the plates, and both luciferase and
-galactosidase activities were determined. Luciferase detection
reagents were from Promega. -Galactosidase activity was measured by
spectrophotometry using reagents from Sigma. Luminescence data were
normalized to -galactosidase values and expressed as fold induction
relative to ethanol-treated controls. Experiments were done at least
twice, with triplicate samples. Data are presented as means ± 1 S.E.
20 °C methanol for 1 min. Images
were collected with a Zeiss Axiovert 100 fluorescent microscope
equipped with an LSM410 laser scanning unit (Carl Zeiss Inc.,
Thornwood, NY). The 488 nm line of a krypton-argon laser was used for
excitation with a bandpass 510-525 nm emission filter. 50 images were
collected from each sample, using a 100 × 1.4 numerical aperture
objective with constant acquisition parameters.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
[in a new window]
Fig. 1.
Wild-type and truncated VDR-GFP chimeras are
expressed in COS-7 cells. High salt extracts were prepared from
COS-7 cells 48 h after transfection, and proteins were separated
on a 6% (A) and a 12% (B) polyacrylamide gel by
electrophoresis. Western blot analysis with anti-GFP (A) and
anti-VDR (B) antibodies were then carried out as described
under "Experimental Procedures." Lanes contain extracts from cells
that were mock-transfected (lane 1) or transfected with GFP
(A, lane 2), VDR (B, lane 2), VDR-GFP (lane
3), AF-2del-VDR-GFP (lane 4), and LBDdel-VDR-GFP
(lane 5). In lane 3 (A and
B), a 77-kDa protein was detected by both antibodies,
showing the integrity of VDR-GFP chimera. Similarly, 76-kDa
AF-2del-VDR-GFP protein was detected in lane 4 (A
and B), and 61 kDa LBDdel-VDR-GFP was detected in lane
5 (A and B). The 27-kDa GFP protein
(A, lane 2) and the 50-kDa VDR protein (B, lane
2) were not detected in lanes 3-5.
View larger version (24K):
[in a new window]
Fig. 2.
Transactivation by VDR-GFP is calcitriol
analog-specific and contingent upon intact ligand-binding domain of the
receptor. COS-7 cells were transfected with 0.05 µg/well of each
construct. Cells were treated with 10 nM of calcitriol,
D3, or vehicle. After 48 h of hormone treatment, cells
were harvested, and transactivation assay was carried out as described
under "Experimental Procedures." Data are expressed as ratio of
luciferase activity over vehicle-treated control. Activation of VDR-GFP
caused a 13-fold transactivation of the luciferase reporter gene,
whereas AF-2del-VDR-GFP and LBDdel-VDR-GFP were inactive. Analog
specificity was shown by lack of transactivation by
D3.
View larger version (75K):
[in a new window]
Fig. 3.
Hormone treatment induces VDR-GFP
translocation in different cell lines. VDR-GFP was used for
transfection of melanoma (A and B), 3134 (C and D), and ROS 17/2.8 (E and
F) cells. 24 h after transfection, living cells were
evaluated with confocal fluorescent microscopy as described under
"Experimental Procedures." Before hormone (A, C, and
E), VDR-GFP was both in the cytoplasm and in the nucleus.
After treatment with 100 nM calcitriol (B, D,
and F), VDR-GFP translocated to the nuclei (N) in
every cell line tested. Nucleoli (n) remained void of
receptors. Intranuclear foci (f) were detected after hormone
addition, here most apparent in F. Receptors were not
detected at the plasma membrane (P). Bars = 10 µm.
View larger version (79K):
[in a new window]
Fig. 4.
Real time recording shows calcitriol-induced
VDR-GFP translocation into the nucleus. COS-7 cells were grown on
chambered coverglasses and transfected with VDR-GFP as described under
"Experimental Procedures." 4 h after transfection, cells were
treated with 10 nM calcitriol at 37 °C. Translocation of
VDR-GFP was followed up by confocal microscopy. Images were taken
before hormone treatment (A) and 6 (B), 10 (C), 12 (D), 14 (E), 20 (F), 25 (G), and 30 (H) min after
hormone treatment. Arrowheads point to plasma membrane
(P), nucleus (N), intranuclear foci
(f), and nucleolus (n). Bar = 10 µm.
View larger version (56K):
[in a new window]
Fig. 5.
VDR-GFP translocation is time- and
temperature-dependent. A, morphometric analysis
demonstrates timedependent translocation of VDR-GFP. COS-7
cells were transfected with 0.2 µg/well VDR-GFP. Cells were incubated
with 100 nM calcitriol at 37 °C. After the specified
incubation times with hormone, samples were fixed and evaluated by
confocal microscopy as described under "Experimental Procedures."
Data are integrated nuclear fluorescence expressed as a percentage of
total cellular fluorescence. VDR-GFP translocation was completed within
30 min of hormone treatment. B, morphometric analysis shows
temperature dependence of VDR-GFP translocation. COS-7 cells were
transfected with 0.2 µg/well VDR-GFP. 24 h after transfection,
cells were incubated with 100 nM calcitriol at 4, 20, or
37 °C. After 15-min incubation times, samples were fixed and
evaluated by confocal microscopy as described under "Experimental
Procedures." Data are integrated nuclear fluorescence expressed as a
percentage of total cellular fluorescence. Translocation of VDR-GFP
occurred at 37 °C but was not detectable at 4 or 20 °C.
C, COS-7 cells were transfected with VDR-GFP and treated for
30 min with vehicle (a), 100 nM calcitriol at
20 °C (b), or 100 nM calcitriol at 37 °C
(c). Cells were fixed and evaluated as described under
"Experimental Procedures." Receptor distribution did not change in
response to hormone treatment at 20 °C (compare a and
b); however, a complete translocation was detected at
37 °C (c). Bars = 10 µm.
View larger version (32K):
[in a new window]
Fig. 6.
Dose-response curves for transactivation and
translocation are similar for both calcitriol and KH1060. COS-7
cells were transfected with VDR-GFP and treated with hormones for
48 h, and then cells were harvested and transactivation assays
were carried out as described under "Experimental Procedures."
Open squares represent transactivation values expressed as
fold induction on the left axes. Data show that KH1060 is
about 100-fold more potent than calcitriol in inducing reporter
activity by VDR-GFP. Translocation was evaluated by microscopy and
morphometric analysis from VDR-GFP-transfected cells before and after
30 min of incubation with graded doses of calcitriol or KH1060.
Filled triangles represent nuclear fluorescence as a
percentage of whole cell fluorescence on the right axes. The
shift of fluorescence from the cytoplasm into the nucleus was hormone
concentration-dependent. The more potent calcitriol analog
KH1060 caused translocation at lower concentration than calcitriol.
Error bars, 1 S.E.
View larger version (29K):
[in a new window]
Fig. 7.
Translocation of VDR-GFP is hormone
analog-dependent and contingent upon intact ligand-binding
domain. COS-7 cells were transfected with VDR-GFP (open
columns) and treated with vehicle, 100 nM
D3, KH060, or calcitriol. After 30 min, cells were fixed
and evaluated as described under "Experimental Procedures."
D3 caused no translocation of VDR-GFP, whereas calcitriol
and KH1060 caused complete translocation of VDR-GFP. COS-7 cells were
also transfected with LBDdel-VDR-GFP (solid columns) and
treated with vehicle or 100 nM calcitriol at 37 °C. No
hormone-dependent translocation was observed. Error
bars, 1 S.E.
View larger version (56K):
[in a new window]
Fig. 8.
Intact AF-2 region is required for
complete translocation of VDR-GFP. COS-7 cells were transfected
with wild-type and AF-2del-VDR-GFP and treated with vehicle
(A), 100 nM calcitriol (B), or 100 nM KH1060 (C) at 37 °C. Cells were fixed and
pictures were taken with confocal microscopy after 30 min of incubation
with hormone (bottom). Bars = 10 µm.
Morphometric analysis of translocation was carried out as described
under "Experimental Procedures" (top left panel).
Transactivation was measured after 48 h treatment with hormones as
described under "Experimental Procedures" (top right
panel). Both microscopy and morphometric analysis show that
extreme high doses of calcitriol and KH060 induce a small but
significant translocation and transactivation of AF-2del-VDR-GFP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (16) and
mineralocorticoid receptor (13), whereas it is different from androgen
receptor (14) and GR (15, 17, 34), which reside exclusively in the
cytoplasm without hormone, and differs from estrogen receptor (35) and
progesterone receptor (36), which are exclusively nuclear. It is
possible to speculate that the presence of unliganded VDR in the
nucleus has functional significance. These receptors may bind target
DNA sequences and act as silencers (37). The intranuclear distribution of VDR, however, was similar to the intranuclear distribution of all
other steroid hormone receptors, as the nucleoli are void of receptors.
Rapid, so-called nongenomic actions of steroid hormones, including
calcitriol, were suggested to be mediated by plasma membrane receptors
(38, 39). In the majority of cultured cells, VDR-GFP was not localized
to the plasma membrane, but occasionally small amounts of receptors
were detectable in the plasma membrane of COS-7 cells (Fig. 4D,
at P). The nature of this subset of VDR-GFP and its
functional significance are not clear. Plasma membrane VDR-GFP could
represent alternate splicing products, degradation products, or
differentially processed proteins. The ability to directly observe VDR
in living cells has allowed us to confirm that VDR resides both in the
cytoplasm and in the nucleus without ligand binding.
. Two NLS sequences were identified in the VDR (11, 40). The
functional importance of the NLS signal C-terminal from the second zinc
finger (40) has not been explored in detail. However, a very important
recent study from Haussler's laboratory (11) showed that mutations
that disrupt the NLS between the two zinc fingers within the
DNA-binding domain of the VDR abolish hormone-independent translocation
but allow the hormone-dependent translocation to take
place. Furthermore, we found significant heterogeneity in the extent of
hormone-independent translocation within the same cell population,
suggesting additional level of controls. In a cultured cell population,
cells are in different stages of cell cycle. Differences in
nuclear/cytoplasmic ratios of VDR could be the result of regulatory
mechanisms related to the cell cycle, as was suggested by previous
studies with GR (41) and thyroid receptor (16). Taken together, these
results suggest that the hormone-independent translocation of VDR
requires receptor interactions with importins and is regulated by
additional factors.
ACKNOWLEDGEMENTS
FOOTNOTES
Current address: Diabetes Branch, NIDDK, National Institutes of
Health, 10 Center Dr., Bethesda, MD 20892.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Harmsen, A. S. Koster, J. H. Beijnen, J. H. M. Schellens, and I. Meijerman Comparison of Two Immortalized Human Cell Lines to Study Nuclear Receptor-Mediated CYP3A4 Induction Drug Metab. Dispos., June 1, 2008; 36(6): 1166 - 1171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Arnett-Mansfield, J. D. Graham, A. R. Hanson, P. A. Mote, A. Gompel, L. L. Scurr, N. Gava, A. de Fazio, and C. L. Clarke Focal Subnuclear Distribution of Progesterone Receptor Is Ligand Dependent and Associated with Transcriptional Activity Mol. Endocrinol., January 1, 2007; 21(1): 14 - 29. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Dusso, A. J. Brown, and E. Slatopolsky Vitamin D Am J Physiol Renal Physiol, July 1, 2005; 289(1): F8 - F28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. M. Schaaf, L. J. Lewis-Tuffin, and J. A. Cidlowski Ligand-Selective Targeting of the Glucocorticoid Receptor to Nuclear Subdomains Is Associated with Decreased Receptor Mobility Mol. Endocrinol., June 1, 2005; 19(6): 1501 - 1515. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. N. Feige, L. Gelman, C. Tudor, Y. Engelborghs, W. Wahli, and B. Desvergne Fluorescence Imaging Reveals the Nuclear Behavior of Peroxisome Proliferator-activated Receptor/Retinoid X Receptor Heterodimers in the Absence and Presence of Ligand J. Biol. Chem., May 6, 2005; 280(18): 17880 - 17890. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Schneider, C. El-Yazidi, A. Dace, M. Maraninchi, R. Planells, A. Margotat, and J. Torresani Expression of the 1,25-(OH)2 vitamin D3 receptor gene during the differentiation of mouse Ob17 preadipocytes and cross talk with the thyroid hormone receptor signalling pathway J. Mol. Endocrinol., February 1, 2005; 34(1): 221 - 235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Ruas, L. Poellinger, and T. Pereira Role of CBP in regulating HIF-1-mediated activation of transcription J. Cell Sci., January 15, 2005; 118(2): 301 - 311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. J. Squires, T. Sueyoshi, and M. Negishi Cytoplasmic Localization of Pregnane X Receptor and Ligand-dependent Nuclear Translocation in Mouse Liver J. Biol. Chem., November 19, 2004; 279(47): 49307 - 49314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Swales and M. Negishi CAR, Driving into the Future Mol. Endocrinol., July 1, 2004; 18(7): 1589 - 1598. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Arnett-Mansfield, A. deFazio, P. A. Mote, and C. L. Clarke Subnuclear Distribution of Progesterone Receptors A and B in Normal and Malignant Endometrium J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1429 - 1442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Koyano, K. Kurose, Y. Saito, S. Ozawa, R. Hasegawa, K. Komamura, K. Ueno, S. Kamakura, M. Kitakaze, T. Nakajima, et al. FUNCTIONAL CHARACTERIZATION OF FOUR NATURALLY OCCURRING VARIANTS OF HUMAN PREGNANE X RECEPTOR (PXR): ONE VARIANT CAUSES DRAMATIC LOSS OF BOTH DNA BINDING ACTIVITY AND THE TRANSACTIVATION OF THE CYP3A4 PROMOTER/ENHANCER REGION Drug Metab. Dispos., January 1, 2004; 32(1): 149 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Fan, T. Yanase, Y. Wu, H. Kawate, M. Saitoh, K. Oba, M. Nomura, T. Okabe, K. Goto, J. Yanagisawa, et al. Protein Kinase A Potentiates Adrenal 4 Binding Protein/Steroidogenic Factor 1 Transactivation by Reintegrating the Subcellular Dynamic Interactions of the Nuclear Receptor with Its Cofactors, General Control Nonderepressed-5/Transformation/ Transcription Domain-Associated Protein, and Suppressor, Dosage-Sensitive Sex Reversal-1: a Laser Confocal Imaging Study in Living KGN Cells Mol. Endocrinol., January 1, 2004; 18(1): 127 - 141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kawajiri, T. Ikuta, T. Suzuki, M. Kusaka, M. Muramatsu, K. Fujieda, M. Tachibana, and K.-i. Morohashi Role of the LXXLL-Motif and Activation Function 2 Domain in Subcellular Localization of Dax-1 (Dosage-Sensitive Sex Reversal-Adrenal Hypoplasia Congenita Critical Region on the X Chromosome, Gene 1) Mol. Endocrinol., June 1, 2003; 17(6): 994 - 1004. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. M. Schaaf and J. A. Cidlowski Molecular Determinants of Glucocorticoid Receptor Mobility in Living Cells: the Importance of Ligand Affinity Mol. Cell. Biol., March 15, 2003; 23(6): 1922 - 1934. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kawana, T. Ikuta, Y. Kobayashi, O. Gotoh, K. Takeda, and K. Kawajiri Molecular Mechanism of Nuclear Translocation of an Orphan Nuclear Receptor, SXR Mol. Pharmacol., March 1, 2003; 63(3): 524 - 531. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhao, K. Goto, M. Saitoh, T. Yanase, M. Nomura, T. Okabe, R. Takayanagi, and H. Nawata Activation Function-1 Domain of Androgen Receptor Contributes to the Interaction between Subnuclear Splicing Factor Compartment and Nuclear Receptor Compartment. IDENTIFICATION OF THE p102 U5 SMALL NUCLEAR RIBONUCLEOPROTEIN PARTICLE-BINDING PROTEIN AS A COACTIVATOR FOR THE RECEPTOR J. Biol. Chem., August 9, 2002; 277(33): 30031 - 30039. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Prufer and J. Barsony Retinoid X Receptor Dominates the Nuclear Import and Export of the Unliganded Vitamin D Receptor Mol. Endocrinol., August 1, 2002; 16(8): 1738 - 1751. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. W. Braun, M.-N. Vo, and K. H. Kim Positive Regulation of Retinoic Acid Receptor Alpha by Protein Kinase C and Mitogen-Activated Protein Kinase in Sertoli Cells Biol Reprod, July 1, 2002; 67(1): 29 - 37. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Prufer, C. Schroder, K. Hegyi, and J. Barsony Degradation of RXRs Influences Sensitivity of Rat Osteosarcoma Cells to the Antiproliferative Effects of Calcitriol Mol. Endocrinol., May 1, 2002; 16(5): 961 - 976. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Saitoh, R. Takayanagi, K. Goto, A. Fukamizu, A. Tomura, T. Yanase, and H. Nawata The Presence of Both the Amino- and Carboxyl-Terminal Domains in the AR Is Essential for the Completion of a Transcriptionally Active Form with Coactivators and Intranuclear Compartmentalization Common to the Steroid Hormone Receptors: A Three-Dimensional Imaging Study Mol. Endocrinol., April 1, 2002; 16(4): 694 - 706. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Schwartz, V. L. Sylvia, D. Larsson, I. Nemere, D. Casasola, D. D. Dean, and B. D. Boyan 1alpha ,25(OH)2D3 Regulates Chondrocyte Matrix Vesicle Protein Kinase C (PKC) Directly via G-protein-dependent Mechanisms and Indirectly via Incorporation of PKC during Matrix Vesicle Biogenesis J. Biol. Chem., March 29, 2002; 277(14): 11828 - 11837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Sunn, T.-A. Cock, L. A. Crofts, J. A. Eisman, and E. M. Gardiner Novel N-Terminal Variant of Human VDR Mol. Endocrinol., September 1, 2001; 15(9): 1599 - 1609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Zelko, T. Sueyoshi, T. Kawamoto, R. Moore, and M. Negishi The Peptide Near the C Terminus Regulates Receptor CAR Nuclear Translocation Induced by Xenochemicals in Mouse Liver Mol. Cell. Biol., April 15, 2001; 21(8): 2838 - 2846. [Abstract] [Full Text]
|
|
|
|
|
|
D. L. Stenoien, M. G. Mancini, K. Patel, E. A. Allegretto*, C. L. Smith, and M. A. Mancini Subnuclear Trafficking of Estrogen Receptor-{alpha} and Steroid Receptor Coactivator-1 Mol. Endocrinol., April 1, 2000; 14(4): 518 - 534. [Abstract] [Full Text]
|
|
|
|
|
|
K. Prufer, A. Racz, G. C. Lin, and J. Barsony Dimerization with Retinoid X Receptors Promotes Nuclear Localization and Subnuclear Targeting of Vitamin D Receptors J. Biol. Chem., December 22, 2000; 275(52): 41114 - 41123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Tomura, K. Goto, H. Morinaga, M. Nomura, T. Okabe, T. Yanase, R. Takayanagi, and H. Nawata The Subnuclear Three-dimensional Image Analysis of Androgen Receptor Fused to Green Fluorescence Protein J. Biol. Chem., July 20, 2001; 276(30): 28395 - 28401. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |