Vitamin D-dependent Suppression of Human Atrial Natriuretic
Peptide Gene Promoter Activity Requires Heterodimer Assembly*
Songcang
Chen,
Claudia H. R. M.
Costa,
Karl
Nakamura,
Ralff C. J.
Ribeiro
, and
David G.
Gardner§
From the Metabolic Research Unit and Department of Medicine,
University of California, San Francisco, California 94143-0540 and
the
Department of Pharmaceutical Sciences, University of
Brasilia, Brasilia, DF, Brazil 70910-900
 |
ABSTRACT |
Crystallographic structures of the ligand-binding
domains for the retinoid X (RXR) and estrogen receptors have identified conserved surface residues that participate in dimer formation. Homologous regions have been identified in the human vitamin D receptor
(hVDR). Mutating Lys-386 to Ala (K386A) in hVDR significantly reduced
binding to glutathione S-transferase-RXR
in solution, whereas binding of an I384R/Q385R VDR mutant was almost undetectable. The K386A mutant formed heterodimers with RXR
on DR-3 (a direct repeat of AGGTCA spaced by three nucleotides), whereas the I384R/Q385R mutant completely eliminated heterodimer formation. Wild type hVDR
effected a 3-fold induction of DR-3-dependent thymidine
kinase-luciferase activity in cultured neonatal rat atrial myocytes, an
effect that was increased to 8-9-fold by cotransfected hRXR
.
Induction by K386A, in the presence or absence of RXR
, was only
slightly lower than that seen with wild type VDR. On the other hand,
I384R/Q385R alone displayed no stimulatory activity and less than
2-fold induction in the presence of hRXR
. Qualitatively similar
findings were observed with the negative regulation of the human atrial
natriuretic peptide gene promoter by these mutants. Collectively, these
studies identify specific amino acids in hVDR that play a critical role in heterodimer formation and subsequent modulation of gene transcription.
 |
INTRODUCTION |
The nuclear hormone receptors are a family of ligand-regulated
transcription factors that associate with cognate recognition sequences
in close proximity to target gene promoters and through an, as yet,
incompletely understood process regulate their transcriptional activity
(1, 2). There are two major classes of nuclear hormone receptors. Class
I receptors, which encompass the steroid hormone receptors
(i.e. receptors for glucocorticoids, mineralocorticoids, progestins, androgens, and estrogens), typically bind as homodimers to
palindromic sequences encoding the core recognition sequence. Class II
receptors, which include the vitamin D receptor
(VDR),1 thyroid receptor
(TR), and retinoic acid receptor (RAR), bind to direct repeat (DR)
elements as heterodimeric complexes with unliganded retinoid X receptor
(RXR) (see below). In contrast to class I receptors that invariably
recognize a palindrome spaced by 3 base pairs, the class II receptors
bind to DRs spaced by a variable length of nucleotides. This spacing
contributes to specificity in the types of receptors that associate
with a given recognition sequence (3).
VDR has been shown to interact with canonical recognition elements
termed vitamin D response elements (VDREs) in a variety of target
genes. In some cases these recognition elements function in a
stimulatory mode (e.g. osteopontin (4), osteocalcin (5, 6),
calbindin (7), 24-hydroxylase (8), and
3 integrin (9)),
whereas in others (e.g. parathyroid hormone (PTH) (10, 11)
and parathyroid hormone-related protein (PTHrP) (12, 13)) it is clearly
inhibitory. Although there is considerable sequence variation among the
stimulatory VDREs, the general structure suggests conservation of two
direct repeats of a consensus (A/G)G(G/T)TCA separated by a
three-nucleotide spacer (DR-3). VDR typically associates with this
element as a heterodimeric complex with RXR prior to effecting changes
in transcriptional activity. VDR homodimers have been described (14,
15), most notably with the receptors in an unliganded form (15);
however, it is generally accepted that the ligand-dependent
assembly of VDR-RXR heterodimeric complexes on the VDRE is the dominant
pathway leading to vitamin D-dependent activation of gene
expression. Less information is available regarding the inhibitory
effects of liganded VDR on gene expression. In the case of PTH and
PTHrP, the responsible element contains only one of the two tandem
hexameric sites found in VDREs involved in positive gene regulation,
and RXR does not appear to be involved in mediating the inhibitory
effect (10-13).
As already noted, RXR serves as a heterodimeric partner for a variety
of different nuclear receptors (1, 2). In addition, RXR can, in the
presence of its cognate ligand 9-cis-retinoic acid
(9-cis-RA), assemble as homodimers on a recognition sequence containing two DRs separated by a single nucleotide spacer (DR-1) (3).
Thus, there are a number of pathways by which this receptor can
regulate downstream transcriptional activity.
We have recently demonstrated that formation of VDR-RXR heterodimers is
important for activation of a DR-3-dependent reporter in
cultured neonatal rat atrial myocytes (16). However, whereas VDR-dependent inhibition of ANP promoter activity is
amplified by cotransfection with RXR, the dependence of this inhibition on heterodimerization of these two receptors remains unclear. A VDR
mutant (L262G), which demonstrates impaired heterodimerization with RXR
(17), retains the ability to suppress human atrial natriuretic peptide
(hANP) promoter activity (16) in transfected myocytes. Thus, the
dependence of hANP promoter suppression on heterodimer formation
remains open to question.
The crystallographic structures of RXR
(18) and the estrogen
receptor (ER) (19) imply an important role for several surface residues
within helix (H) 10 (for RXR) and 11 (for ER) in homodimer formation.
This region resides within the 9th heptad repeat proposed for the TR
(20) and has been shown to be highly conserved in other receptors of
this class, suggesting conservation of the structural determinants that
govern dimerization in this family of regulatory proteins. In fact,
mutations have already been reported in this region of VDR (21), RXR
(22), and TR
(23) that appear to interfere with dimer formation.
However, based on the available structures (e.g. that for
TR
), a number of these mutations target residues placed internally
in the receptor molecule (24) where they might easily effect disruption
of receptor folding and structural integrity. We (25) have recently
shown that mutation of selected surface residues in the 9th heptad of
TR interferes with dimer formation yet preserves other receptor
functions such as ligand binding, DNA binding, and coactivator
interactions. Based on analogy to TR, we have placed homologous surface
mutations in human (h) RXR and hVDR in positions predicted to interfere selectively with dimer interactions but not with binding to DNA, ligand, or the relevant coactivators. We have examined the effects of
these mutations on dimer formation and functional activity in a
transfected atrial myocyte model.
 |
MATERIALS AND METHODS |
Plasmids--
Expression vectors for human VDR (26) and RXR
(27) have been described previously. Mutations K386A (VDRm1),
I384R/Q385R (VDRm2), and I384R (VDRm3) in hVDR and mutations R421A
(RXRm1) and L419R/L420R (RXRm2) in hRXR
were generated by polymerase chain reaction using a site-directed mutagenesis kit (Stratagene, La
Jolla, CA). GST-RXR
(full length) (28), GST-GRIP 1 (amino acids
730-1121) (29), VDR L262G (17), and
1150 hANP CAT (30) have been
reported previously. The reporter plasmid containing the synthetic VDR
response element has two copies of the DR, spaced by three nucleotides
(AGGTCAcagAGGTCA) (DR-3), cloned immediately upstream from a minimal
(
32/+45) thymidine kinase promoter linked to luciferase coding
sequence. GST-VDR was constructed by isolating an EcoRI
fragment from pSG5hVDR (26) followed by ligation into pGEX2T. The
structures of all new constructs were verified by DNA sequencing.
GST Pull-down Assay--
Wild type or mutant pSG5 hVDR vectors
and wild type or mutant pEThRXR
vectors were used to produce
radiolabeled full-length receptors in vitro using the
TNT-Coupled Reticulocyte Lysate System (Promega, Madison, WI) and
[35S]methionine. GST-hVDR, GST-hRXR
, and GST-GRIP 1 fusion proteins were prepared using conventional protocols (29).
Briefly, the plasmids were transformed into HB101, amplified in
culture, pelleted, resuspended in buffer IPAB-80 (20 mM
HEPES, 80 mM KCl, 6 mM MgCl2, 10%
glycerol, 1 mM dithiothreitol, 1 mM ATP, 0.2 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 µg/ml leupeptin) and sonicated (three times)
for 10 s. The debris was pelleted; the supernatant was incubated
for 2 h with 500 µl of glutathione-Sepharose 4B beads and
equilibrated with 5 volumes of IPAB-80. GST fusion protein beads were
washed with 5 volumes of phosphate-buffered saline containing 0.05%
Nonidet P-40 and resuspended in 0.5 ml of IPAB-80. All procedures above
were carried out at 4 °C. The concentrations of GST fusion proteins
were measured using the Coomassie protein reagent.
For the binding assay, the glutathione bead suspension containing 10 µg GST protein was incubated with 2 µl of 35S-labeled
protein in 150 µl of IPAB-80 buffer containing 2 µg/ml bovine serum
albumin, in the presence of 10 nM 1,25-dihydroxyvitamin D3 or vehicle. After incubation for 2 h at 4 °C,
the beads were washed (three times) using 1 ml of IPAB-80 buffer. The
beads were then heated to 100 °C for 3 min; associated proteins were
subjected to 10% SDS-PAGE and visualized by autoradiography. The
results were analyzed using NIH image.
Electrophoretic Mobility Shift Assay--
Gel shift assays using
35S-labeled proteins and nonradioactive DNA were performed
as described previously (31). In brief, 3 µl of
35S-labeled proteins were incubated with 10 ng of
oligonucleotide in DNA binding buffer (10 mM
NaHPO4, pH 7.6; 0.25 mM EDTA; 0.5 mM MgCl2; 5% glycerol) for 20 min at room
temperature in the presence or absence of 100 nM of the
appropriate ligand. The reaction mixtures were separated on 5%
nondenaturing polyacrylamide gels in TEA buffer (67 mM
Tris, pH 7.5; 10 mM EDTA; 33 mM sodium
acetate). The gel was run at 240 V for 3 h at 4 °C, washed
extensively with 30% methanol and 10% glacial acetic acid, and
amplified for 30 min (Amplifier; Amersham Pharmacia Biotech), dried,
and exposed for autoradiography.
Cell Culture and Transfection--
Atrial cells were obtained
from 1- to 2-day-old neonatal rat hearts by alternate cycles of trypsin
digestion and mechanical disruption as described previously (30). The
cells were transfected by electroporation (280 V and 250 µF) using
the plasmids indicated. All transfections were normalized for
equivalent DNA content with PUC18. After transfection, cells were
resuspended in Dulbecco's modified Eagle's medium H21 containing 10%
bovine calf serum (HyClone, Logan, UT) and cultured for 24 h. At
that time medium was changed to Dulbecco's modified Eagle's
medium/serum substitute (32), and the cultures were treated with 10 nM 1,25-dihydroxyvitamin D3 alone or in
combination with 10 nM 9- cis-RA for 48 h.
Similar concentrations of ligand-free vehicle were used as controls.
Cells were washed with phosphate-buffered saline and lysed with lysis buffer (250 mM Tris-HCl, pH 7.5; 0.1% Triton X-100).
Soluble lysate protein concentration was determined using the Coomassie
protein reagent (Pierce). Luciferase activity was measured on equal
amounts of lysate protein using the Luciferase Assay System (Promega, Madison, WI). The CAT assay was performed as described previously (33).
Ligand Binding Assay--
Wild type or mutant hVDR proteins,
cloned in pSG5, were translated in vitro using the
TNT-Coupled Reticulocyte Lysate System (Promega; Madison, WI) and cold
methionine. Ten µl of the translation products was incubated with
increasing concentrations of
1,25-(OH)2-23,24[3H]vitamin D3
(98 Ci/mmol; Amersham Pharmacia Biotech) overnight at 4 °C in the
presence or absence of unlabeled 1,25-dihydroxyvitamin D3
(100-fold molar excess). Bound and free ligand were separated with
dextran-coated charcoal (Sigma) using the method of Dokoh et
al. (34). Scatchard analysis was carried out using the Graphpad Prism program.
 |
RESULTS |
Crystallographic structures of RXR and ER have identified surface
residues that participate in receptor dimerization. The majority of
these residues lie in helix 10 of RXR (18) and helix 11 of ER (19).
These, as well as homologous regions from the ligand-binding domains of
RAR, TR, and VDR are aligned in Fig. 1
for comparison. We have made mutations in several surface residues in
RXR
and homologous residues in VDR (identified by shaded
boxes) to evaluate their roles in generating dimeric complexes
in vitro and regulating transcription in
vivo.

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Fig. 1.
Alignment of amino acid residues from the
ligand binding domains of hRXR ,
hRAR , hTR ,
hER , and hVDR which, based on available
structural information, are thought to participate in dimer formation.
Shaded boxes identify conserved residues targeted for
mutation in hRXR and hVDR in the present study. Single
letter nomenclature for the individual amino acids is used.
Numbers at right identify the carboxyl-terminal
residue in each sequence.
|
|
By using a GST pull-down assay, a method that identifies
protein-protein interactions in solution, independent of the presence of a DNA recognition element, we examined the ability of wild type and
mutant VDRs to form complexes with RXR. As shown in Fig. 2A, GST-VDR showed little
propensity to self-associate with either of the VDR mutants. There was,
however, a small amount of homodimeric complex formed with wild type
VDR, and this association increased modestly with the addition of
1,25-dihydroxyvitamin D3. GST-RXR
strongly associated
with wild type VDR, and again, this interaction was
ligand-dependent. There was a reduced level of interaction with the K386A mutant of VDR (ligand-dependent) but
virtually no interaction with the I384R/Q385R mutant. In each instance
there was a modest increase in the presence of ligand. Of note, both the wild type and the mutant VDR proteins associated with GST-GRIP to
an equivalent degree and in a ligand-dependent fashion,
indicating that overall structure and function of these mutants were
preserved. Thus, both VDR mutants appear to selectively impair
heterodimerization with RXR in solution.

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Fig. 2.
Association of wild type or mutant VDR or
RXR (VDR mutants: Vm1, K386A;
Vm2, I384R/Q385R; RXR mutants: Rm1,
R421A; Rm2, L419R/L420R) with
GST-RXR , GST-VDR, and GST-GRIP1. A,
radiolabeled wild type or mutant VDR was synthesized in
vitro and incubated with GST-RXR , GST-VDR, or GST-GRIP1 in the
presence or absence of 10 nM 1,25-dihydroxyvitamin
D3. Columns were washed thoroughly; bound proteins were
separated from the beads by boiling for 3 min and subjected to 10%
SDS-PAGE. Bound proteins were identified by autoradiography.
B, wild type or mutant 35S-labeled RXR was
incubated with GST-VDR with or without 10 nM
1,25-dihydroxyvitamin D3. The remainder of the experiment
was performed as described above. Each experiment was repeated three
times. Representative experiments are shown.
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|
Similar findings were obtained when the RXR mutants were examined (Fig.
2B). Wild type RXR bound to GST-VDR in a
1,25-dihydroxyvitamin D3-dependent fashion. The
R421A and L419R/L420R mutants each displayed impaired capacity to
associate with VDR, and in both instances this limited interaction was
ligand-dependent. The L419R/L420R RXR mutant also
demonstrated poor heterodimerization with TR
and peroxisome
proliferator-activated receptor
(data not shown).
Since the mutants seemed to disrupt dimerization differentially and
because DNA may provide support for heterodimer interactions (35, 36),
we decided to test the ability of these mutations to disrupt homo- and
heterodimerization on DNA using conventional electrophoretic gel
mobility shift assays (EMSA). As shown in Fig.
3A, wild type RXR
effectively formed ligand-dependent homodimers and
hTR
1-dependent heterodimers on a conventional DR-4
element. Selective mutation at position 421 (R421A) in RXR
resulted
in a loss of homodimeric complexes while, if anything, it increased heterodimer formation. Mutation at positions 419 and 420 (L419R/L420R), which are exposed on the surface of RXR
at or near the dimeric interface (18), completely eliminated homodimer assembly and severely
reduced the formation of heterodimers. Of note, these latter reductions
were accompanied by the appearance of RXR monomers on the DR-4
template. Next, we studied RXR
dimer assembly on a DR-1 template, a
template that favors formation of homodimeric complexes of liganded RXR
(37). On the DR-1 template wild type RXR
assembled as a homodimer in
the presence or absence of ligand (Fig. 3B), ligand effected
a modest increase in the level of binding. Addition of TR
1 led only
to modest levels of heterodimerization with wild type RXR
(heterodimers do not bind effectively to the DR-1 template). Mutations
at positions 421 (R421A) or 419 and 420 (L419R/L420R) abolished
formation of wild type RXR
homodimers on DR-1, as well as the weak
TR
1-RXR
heterodimers noted above. Again, as in Fig.
2A, most of the L419R/L420R mutant assembled as monomers on
the DR-1 template.

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Fig. 3.
DNA binding activity of wild type and mutant
RXR in presence or absence of
TR 1. A, radiolabeled RXR or the
relevant RXR mutant, with or without unlabeled TR 1, was incubated
with a DR-4 oligonucleotide in the presence or absence of 100 nM 9-cis-RA. Reaction mixtures were subjected to
5% nondenaturing PAGE. The gel was amplified, dried, and exposed for
autoradiography. B, gel mobility shift assay was performed
in the same fashion as in A except that DR-1 was used
instead of DR-4. Similar results were obtained from two additional
experiments.
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|
As expected, wild type VDR formed a stable complex on a DR-3 template
with wild type RXR
(Fig. 4). The R421A
mutant of RXR
also formed stable heterodimeric complexes with VDR on
this template. On the other hand, the L419R/L420R RXR
mutant formed
only very low levels of heterodimeric complex with wild type VDR.
Mutation of VDR at position 386 (K386A), analogous to the R421A
mutation in RXR
, still bound to wild type RXR
and the R421A
mutant of that protein (albeit in a somewhat reduced fashion relative
to that seen with wild type VDR), and like wild type VDR, it failed to
complex with the L419R/L420R mutant of RXR
. No homodimeric complexes
were seen with either wild type VDR or its K386A mutant. Mutation of
VDR at positions 384 and 385 (I384R/Q385R), analogous to the
L419R/L420R mutation of RXR
, displayed modest heterodimer formation
only with wild type RXR
. Neither of the RXR mutants showed
appreciable interaction with I384R/Q385R. These complexes were replaced
by monomers of the VDR mutant on the DR-3 template.

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Fig. 4.
Gel mobility shift assay of wild type and
mutant VDR and RXR. Radiolabeled VDR, VDR mutant, RXR , or RXR
mutant was incubated with the DR-3 oligonucleotide in presence of 100 nM 1,25-dihydroxyvitamin D3. Mixtures were then
separated on nondenaturing PAGE and subjected to autoradiography. This
autoradiograph is representative of three independent
experiments.
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Each of these mutants was next tested for the capacity to activate a
reporter plasmid linking the DR-3 sequence upstream from a thymidine
kinase promoter-driven luciferase reporter (DR-3-TKLuc). As shown in
Fig. 5, liganded VDR alone effected an
~3-fold increment in luciferase activity compared with the untreated
control. K386A was somewhat less effective, and I384R/Q385R was devoid
of activity. Transfection with wild type RXR
alone led to DR-3-TKLuc
activities that were not different from control (+VD3).
Activity declined still further with the R421A and L419R/L420R mutants.
Wild type RXR
significantly amplified the VDR effect, as did RXR
R421A, whereas RXR L419R/L420R effected only a 2-fold increment in
reporter activity over that seen with VDR alone. A similar activity
profile was seen with the K386A mutant when it was substituted for wild type VDR. The I384R/Q385R VDR mutant, on the other hand, proved incapable of interacting functionally with either the wild type RXR
or the RXR
mutants.

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Fig. 5.
VDR activates DR-3-TK-luciferase reporter in
ligand- and heterodimer-dependent fashion. VDR or the
relevant VDR mutant (5 µg) was cotransfected with DR-3-TK-Luc (10 µg), in presence or absence of wild type or mutant RXR (5 µg),
into primary cultures of neonatal rat atrial myocytes. Twenty-four
hours later, cells were treated with vehicle or 10 nM
1,25-dihydroxyvitamin D3. Cells were collected after
48 h for measurement of luciferase activity. C
represents control samples. Data obtained from 5 to 7 independent
experiments are presented as means ± S.D.
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The ANP gene promoter has been shown previously to be a target for the
liganded VDR (16, 27, 38, 39). 1,25-Dihydroxyvitamin D3, as
well as a number of non-hypercalcemic analogues of vitamin D, effects a
VDR-dependent reduction in hANP promoter activity. With
this in mind, we examined the ability of the various VDR and RXR
mutants to impact on this inhibitory activity. As shown in Fig.
6, liganded VDR effected ~50%
inhibition in hANP-CAT reporter activity, whereas liganded RXR
produced only a 20% reduction, levels that are in agreement with those
previously reported (27). The VDR K386A mutant was slightly less
effective than wild type VDR in promoting the inhibition, whereas the
I384R/Q385R mutant was virtually devoid of activity. Neither of the
RXR
mutants proved capable of inhibiting hANP promoter activity.

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Fig. 6.
Effect of VDR, RXR ,
or their mutants on 1150 hANP-CAT activity. Five µg VDR,
RXR , or the relevant mutants was cotransfected with 20 µg of
hANP-CAT into neonatal rat atrial myocytes. After 24 h, cells were
treated with vehicle, 10 nM 1,25-dihydroxyvitamin
D3, or 10 nM 9-cis-RA for 48 h.
Cells were harvested, and CAT activity was measured. Pooled data,
derived from four independent experiments, are presented as means ± S.D.
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When used in combination, wild type VDR and RXR
effected a >90%
inhibition of
1150 hANP CAT activity (Fig.
7). RXR
R421A was less active than
wild type in amplifying VDR activity, whereas inhibition in the
presence of I384R/Q385R was reduced to ~50%, the level seen with
wild type VDR alone (see above). In agreement with the observations
made with DR-3 TKCAT (see above), the combination of VDR and the RXR
mutants resulted in a stepwise loss of ANP promoter inhibition that
varied as a function of the "severity" of the mutation. The most
significant loss of inhibitory activity was seen with the combination
of RXR
L419R/L420R and VDR I384R/Q385R. This combination had
virtually no effect on the
1150 hANP CAT reporter. These findings
support the hypothesis that residues critical for heterodimerization in
these two nuclear receptors are also critical for maintenance of
transcriptional regulatory activity (in this case, either stimulatory
or inhibitory in nature).

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Fig. 7.
VDR-dependent suppression of
1150 hANP-CAT activity requires an intact heterodimerization
function. Five µg of VDR or the relevant mutant was
cotransfected with 20 µg of hANP-CAT, with or without wild type or
mutant RXR (5 µg), into neonatal rat atrial myocytes. After
24 h of culture, cells were exposed to vehicle, 10 nM
1,25-dihydroxyvitamin D3, or vitamin D plus 10 nM 9-cis-RA for 48 h. Cells were lysed, and
CAT activity was measured. Pooled data from 4 to 7 experiments are
presented as means ± S.D.
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A trivial explanation of these findings arises from the possibility
that the mutations, which we assume are selectively targeted to the
dimer interface, actually lead to global changes in receptor structure.
Since these mutations are positioned in the ligand-binding domain of
the receptor, alterations in ligand binding could account for both the
loss of heterodimerization and the impairment in functional activity.
To address this question, we examined the ligand binding properties of
both the wild type and mutant VDRs in a cell-free system. As shown in
Fig. 8, affinity of the receptors for
[3H]dihydroxyvitamin D3 was almost identical
for each of the three receptors while, if anything, total binding
capacity was modestly increased with the mutants. This, together with
the observation that each of the VDRs bound equivalently to GRIP-1
(Fig. 2A), argues against major structural changes as
accounting for the loss of functional activity in the mutants and
implies that the latter results from selective impairment in the
ability of these mutants to form heterodimers.

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Fig. 8.
Ligand binding assays of wild type and mutant
VDRs. Ten µl of programmed reticulocyte lysate for VDR or a VDR
mutant were incubated with increasing concentrations of
1,25-(OH)2-23,24[3H]vitamin D3 at
4 °C for 14-16 h, as described under "Materials and Methods."
Bound and free ligand were separated and quantified. Scatchard analyses
of the data are presented in A-C. The same experiment was
repeated twice with similar results.
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We employed a double mutation to probe the VDR heterodimerization
function to maximize the probability of interfering with the dimer
interface. One of the amino acids mutated here (Gln-385) has previously
been shown to reduce VDR interaction with an auxiliary factor
(presumably RXR) present in COS-7 cells (21). To address the selective
role of Ile-384 in the dimerization process, we introduced a
site-directed mutation at this position, and we examined the effects of
this perturbation on the RXR binding and functional properties of VDR.
I384R, like the double mutant (I384R/Q385R), was ineffective in
activating DR-3-TK-Luc (Fig.
9A) or inhibiting
1150 hANP
CAT (Fig. 9B) in atrial myocytes. In addition, this mutant
displayed a markedly reduced affinity for RXR
in the GST pull-down
assay (Fig. 9C). Placed in the context of the earlier results of Nakajima et al. (21), it would appear that both
Ile-384 and Gln-385 play equivalently important roles in heterodimer
assembly.

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Fig. 9.
Impact of Ile to Arg mutation at position 384 (VDR I384R: VDRm3) on the functional and RXR binding
properties of VDR. Five µg of wild type VDR or VDRm3, alone or
together with RXR (or one of the RXR mutants), was cotransfected
with DR-3-TK-luciferase (10 µg) or hANP-CAT (20 µg) into neonatal
rat atrial myocytes. Extracts were analyzed for luciferase or CAT
activity 48 h later. Pooled data from 3 to 4 independent
experiments are presented in A and B.
C, 35S-labeled VDR or VDRm3 was incubated with
GST-RXR in the presence or absence of 10 nM
1,25-dihydroxyvitamin D3 for 2 h at 4 °C. Bound
protein was then analyzed by SDS-PAGE, as described in Fig. 2. The
experiment was repeated twice, with comparable results.
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|
 |
DISCUSSION |
Recent crystallographic analyses of individual nuclear receptors
suggest conservation of structural features involved in dimer assembly.
Specifically amino acid residues in helix 10 of hRXR
(18) (these
residues are located in helix 11 in hTR and hER) and helix 11 of hTR
(24) and hER (19) appear to play a key role in establishing contacts
required for dimer formation. The present study demonstrates that
mutation of two residues (Leu-419 and Leu-420), which structural
studies place on the surface of RXR
at or near the dimer interface
(18), participate in functional heterodimer assembly in the atrial
myocyte, as do two homologous residues in VDR (Ile-384 and Gln-385).
Contiguously positioned residues in RXR
(Arg-421) or VDR (Lys-386)
do not inhibit heterodimer formation on DR-3 and consequently have very
little impact on receptor-dependent transcriptional regulation.
A number of mutations in helices 10 and 11 have been shown to alter
receptor dimerization for different nuclear receptors (21-23).
However, interpretation of these findings is complicated by the fact
that, at least in some instances, they have targeted nonsurface
residues of the receptor, a manipulation that is likely to disrupt
protein folding and/or alter ligand binding. For example, the leucines
that border the heptad repeats of TR
were previously thought to be
involved in dimerization (20), but structural studies have shown that
these leucines are engaged in intramolecular interactions and do not
participate directly in dimer formation (24). For this reason we
confined our mutations to those residues that are known or, based on
sequence homology, would be predicted to lie on the surface of RXR
and VDR, respectively.
On the DR-4 template, RXR
R421A demonstrated impaired formation of
RXR
homodimers, whereas heterodimer formation with TR
1 was
relatively normal. If anything, the latter was modestly increased, perhaps reflecting diversion of the mutant RXR
, incapable of assembling with itself, into complexes with a heterodimeric partner. The double mutation (L419R/L420R) further upstream completely eliminated both homo- and heterodimer assembly with RXR
. Noteworthy, the double mutation still permitted monomer binding to the DR-4 template, implying that the DNA binding function, per se, is
not perturbed in this mutant.
We noted no VDR homodimer formation on the DR-3 template in the EMSA
and only minimal interaction in the GST pull-down assay. VDR homodimers
have been identified in in vitro binding studies by others
(14, 15), and in the unliganded form they may function as suppressors
of gene transcription (15). However, most studies suggest that the
functionally relevant complex in transducing the positive vitamin D
signal in the target cell is the liganded VDR-RXR heterodimer (39). VDR
I384R/Q385R completely disrupted heterodimeric pairings with either
wild type RXR
or the RXR mutants. Mutations in this region of the
hVDR molecule have been reported previously to interfere with
heterodimer formation (21). Specifically, VDR mutations K382E, M383G,
Q385K, and L390G reduced assembly of a VDR/accessory factor complex on
VDRE. Studies with the Q385K mutant (21) support our findings with
I384R/Q385R. Additional studies focusing specifically on Ile-384
(mutation I384R) indicate that this residue, as well, plays an
important role in heterodimer formation. Thus, it would appear that
both Ile-384 and Gln-385 participate directly in dimer assembly.
Lys-386, on the other hand, despite its contiguous location on the
receptor surface, does not appear to play a critical role in this process.
The EMSA analyses indicate that both VDR K386A and RXR R421A retain the
capacity to interact with heterodimeric partners (RXR
in the case of
VDR and TR
1 in the case of RXR
) at near wild type levels. The
conclusion, at least as it applies to the RXR mutant, R421A, stands in
contrast to those of Lee et al. (22) who identified this
residue as critical for heterodimerization. This difference remains
unexplained since both EMSA and the functional analyses indicated that
this particular mutant displays close to wild type activity in our
system. It should be noted, however, that the GST pull-down assays
showed impaired heterodimeric interactions of these two mutants
(i.e. VDR K386A and RXR R421A) (see Fig. 3). This
discrepancy (GST pull-down versus EMSA) likely reflects differences in the end points being addressed in these two assays. The
GST pull-down assay assesses the ability of proteins to associate in
solution. Such associations, by definition, have to be of sufficient affinity to preclude disruption during the washing procedure used to
reduce "nonspecific" protein-protein interactions. The EMSA is
carried out in the presence of DNA template. Positioning of nuclear
receptors next to each other on DNA may promote dimer contacts between
the DNA binding domains of the subunits (35, 36), and receptor-DNA
contacts may further stabilize the dimer complex. Thus, the GST
pull-down assay is probably a more sensitive method to detect subtle
impairment of protein-protein interactions that might otherwise be
obscured when the same proteins are bound to DNA. With reference to the
current study, although VDR K386A and RXR
R421A displayed obvious
impairment in their capacity to establish protein-protein interactions
in solution, the impairment was not seen when they were permitted to
assemble on DNA, and the latter, rather than the former, is probably
most reflective of their functional activity in the intact cell (see
Figs. 4 and 7).
Collectively, our data suggest that the surfaces involved in homo-
versus heterodimerization overlap (i.e.
impairment of both homo- and heterodimerization is seen with the double
mutants). They also reveal a critical role for selected residues in
dimer assembly. Disruption of the hydrophobic residues in both VDR
(Ile-384 and Gln-385) and RXR (Leu-419 and Leu-420) abolishes
heterodimerization on DNA, whereas disruption of the charged residue
flanking this hydrophobic patch (Lys-386 or Arg-421, respectively) has
no effect on this process. Homodimerization appears to be equally
affected by any of these mutations suggesting that homodimer assembly
is less stable than that of heterodimers, a finding that is in
agreement with the fact that heterodimers form preferentially (over
homodimers) in solution or on DNA (see Figs. 2-4) (2).
We have shown previously that the liganded VDR exerts anti-hypertrophic
activity and suppresses ANP gene transcription in cultured neonatal rat
atrial (16, 38, 39) and ventricular (27) myocytes. This effect was
clearly amplified by cotransfection with RXR
(16, 27); however, a
VDR mutant (L262G) with impaired capacity for heterodimer formation
(17) was found to retain the ability to suppress the hANP gene promoter
(16). This places into question the inferred requirement for VDR
heterodimerization in generating the inhibitory effect. Our studies
with the I384R/Q385R mutant clearly demonstrate that capacity for
heterodimerization closely parallels the ability of the receptor to
suppress hANP gene promoter activity. By inference, this would suggest
that the L262G mutant described above retains the capacity to interact with a heterodimeric partner, albeit not RXR
(17), as a prelude to
initiating its biological activity.
In summary, VDR and RXR mutations which, based on RXR and ER structural
studies, would be predicted to disrupt protein-protein interactions
involved in receptor dimerization do, in fact, demonstrate impairment
in dimerization in two independent in vitro assays. This is
accompanied by a commensurate reduction in functional activity,
assessed through activation of a DR-3-dependent promoter or
suppression of an hANP-dependent reporter, in transiently
transfected rat atrial myocytes. The studies provide support for the
suggested conservation of structure-function relationships across
different members of the nuclear receptor family and highlight the role of heterodimer formation in vivo as a prerequisite for
functional activity of VDR in activating or repressing target gene expression.
 |
ACKNOWLEDGEMENTS |
We are grateful to Mark Haussler and G. Kerr
Whitfield for advice on the ligand binding assays; to Jim Aprilletti
for assistance with processing the ligand binding data; and to Brian
West and Weijun Feng for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant HL-35753 and Knoll Pharmaceuticals Grant SYN-0297-08.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: Metabolic Research
Unit, University of California, San Francisco, CA 94143-0540. Tel.:
415-476-2729; Fax: 415-476-1660; E-mail: gardner{at}itsa.ucsf.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
VDR, vitamin D
receptor;
h, human;
RXR, retinoid X receptor;
ER, estrogen receptor;
PTH, parathyroid hormone;
PTHrP, parathyroid hormone-related protein;
TR, thyroid receptor;
RAR, retinoic acid receptor;
GST, glutathione
S-transferase;
DR-3, two copies of vitamin D response
element spaced by three nucleotides;
hANP, human atrial natriuretic
peptide;
PAGE, polyacrylamide gel electrophoresis;
EMSA, electrophoretic mobility shift assay;
GRIP1, glucocorticoid receptor
interacting protein 1;
CAT, chloramphenicol acetyltransferase coding
sequence;
TKLuc, thymidine kinase promoter linked to luciferase coding
sequence;
VDRE, vitamin D response elements;
9-cis-RA, 9-cis-retinoic acid.
 |
REFERENCES |
-
Mangelsdorf, D. J.,
Thummel, C.,
Beato, M.,
Herrlich, P.,
Schutz, G.,
Umesono, K.,
Blumberg, B.,
Kastner, P.,
Mark, M.,
Chambon, P.,
and Evans, R. M.
(1995)
Cell
83,
835-839[Medline]
[Order article via Infotrieve]
-
Ribeiro, R. C.,
Kushner, P. J.,
and Baxter, J. D.
(1995)
Annu. Rev. Med.
46,
443-453[CrossRef][Medline]
[Order article via Infotrieve]
-
Umesono, K.,
Murakami, K. K.,
Thompson, C. C.,
and Evans, R. M.
(1991)
Cell
65,
1255-1266[Medline]
[Order article via Infotrieve]
-
Noda, M.,
Vogel, R. L.,
Craig, A. M.,
Prahl, J.,
DeLuca, H. F.,
and Denhardt, D. T.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
9995-9999[Abstract]
-
Morrison, N. A.,
Shine, J.,
Fragonas, J.-C.,
Verkest, V.,
McMenemy, M. L.,
and Eisman, J. A.
(1989)
Science
246,
1158-1161[Medline]
[Order article via Infotrieve]
-
Ozono, K.,
Liao, J.,
Kerner, S. A.,
Scott, R. A.,
and Pike, J. W.
(1990)
J. Biol. Chem.
265,
21881-21888[Abstract/Free Full Text]
-
Schrader, M.,
Nayeri, S.,
Kahlen, J.-P.,
Muller, K. M.,
and Carlsberg, C.
(1995)
Mol. Cell. Biol.
15,
1154-1161[Abstract]
-
Ohyama, Y.,
Ozono, K.,
Uchida, M.,
Shinki, T.,
Kato, S.,
Suda, T.,
Yamamoto, O.,
Noshiro, M.,
and Kato, Y.
(1994)
J. Biol. Chem.
269,
10545-10550[Abstract/Free Full Text]
-
Cao, X.,
Ross, F. P.,
Zhang, L.,
MacDonald, P. N.,
Chappel, J.,
and Teitelbaum, S. L.
(1993)
J. Biol. Chem.
268,
27371-27380[Abstract/Free Full Text]
-
Demay, M. B.,
Kierman, M. S.,
DeLuca, H. F.,
and Kronenberg, H. M.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
8097-8101[Abstract]
-
Liu, S. M.,
Koszewski, N.,
Lupez, M.,
Malluche, H. H.,
Olivera, A.,
and Russell, J.
(1996)
Mol. Endocrinol
10,
206-215[Abstract]
-
Falzon, M.
(1996)
Mol. Endocrinol.
10,
672-681[Abstract]
-
Kremer, R.,
Sebag, M.,
Champigny, C.,
Meerovitch, K.,
Hendy, G. N.,
White, J.,
and Goltzman, D.
(1996)
J. Biol. Chem.
271,
16310-16316[Abstract/Free Full Text]
-
Carlsberg, C.,
Bendik, I.,
Wyss, A.,
Meier, E.,
Sturzenbecker, K. J.,
Grippo, J. F.,
and Hunziker, W.
(1993)
Nature
361,
657-660[CrossRef][Medline]
[Order article via Infotrieve]
-
Cheskis, B.,
and Freedman, L. P.
(1994)
Mol. Cell. Biol.
14,
3329-3338[Abstract]
-
Chen, S.,
Wu, J.,
Hsieh, J.-C.,
Whitfield, G. K.,
Jurutka, P. W.,
Haussler, M. R.,
and Gardner, D. G.
(1998)
Hypertension
31,
1338-1342[Abstract/Free Full Text]
-
Whitfield, G. K.,
Hsieh, J.-C.,
Nakajima, S.,
MacDonald, P. N.,
Thompson, P. D.,
Jurutka, P. W.,
Haussler, C. A.,
and Haussler, M. R.
(1995)
Mol. Endocrinol.
9,
1166-1179[Abstract]
-
Bourguet, W.,
Ruff, M.,
Chambon, P.,
Gronemeyer, H.,
and Moras, D.
(1995)
Nature
375,
377-382[CrossRef][Medline]
[Order article via Infotrieve]
-
Brzozowski, A. M.,
Pike, A. C. W.,
Dauter, Z.,
Hubbard, R. E.,
Bonn, T.,
Engstrom, O.,
Ohman, L.,
Greene, G. L.,
Gustafsson, J.-A.,
and Carlquist, M.
(1997)
Nature
389,
753-758[CrossRef][Medline]
[Order article via Infotrieve]
-
Forman, B. M.,
and Samuels, H. H.
(1990)
Mol. Endocrinol
4,
1293-1301[Abstract]
-
Nakajima, S.,
Hsieh, J.-C.,
MacDonald, P. N.,
Galligan, M. A.,
Haussler, C. A.,
Whitfield, G. K.,
and Haussler, M. R.
(1994)
Mol. Endocrinol.
8,
159-172[Abstract]
-
Lee, S.-K.,
Na, S.-Y.,
Kim, H.-J.,
Soh, J.,
Choi, H.-S.,
and Lee, J. W.
(1998)
Mol. Endocrinol.
12,
325-332[Abstract/Free Full Text]
-
Nagaya, T.,
and Jameson, J. L.
(1993)
J. Biol. Chem.
268,
24278-24282[Abstract/Free Full Text]
-
Wagner, R. L.,
Apriletti, J. W.,
McGrath, M. E.,
West, B. L.,
Baxter, J. D.,
and Fletterick, R. J.
(1995)
Nature
378,
690-697[CrossRef][Medline]
[Order article via Infotrieve]
-
Ribeiro, R. C. J.,
Feng, W.,
Wagner, R. L.,
Costa, C. H. R. M.,
Pereira, A. C.,
Apriletti, J. W.,
Fletterick, R. J.,
Baxter, J. D.,
and West, B. L.
(1998)
Keystone Symposium: Nuclear Receptor Gene Family, March 28-April 3, 1998, p. 64, Abstract 150, Incline Village, NV
-
Baker, A. R.,
McDonnell, D. P.,
Hughes, M.,
Crisp, T. M.,
Mangelsdorf, D. J.,
Haussler, M. R.,
Pike, J. W.,
Shine, J.,
and O'Malley, B. W.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
3294-3298[Abstract]
-
Wu, J.,
Garami, M.,
Cheng, T.,
and Gardner, D. G.
(1996)
J. Clin. Invest.
97,
1577-1588[Abstract/Free Full Text]
-
Feng, W.,
Ribeiro, R. C. J.,
Wagner, R. L.,
Nguyen, H.,
Apriletti, J. W.,
Fletterick, R. J.,
Baxter, J. D.,
Kushner, P. J.,
and West, B. L.
(1998)
Science
280,
1747-1749[Abstract/Free Full Text]
-
Ding, X. F.,
Anderson, C. M.,
Ma, H.,
Hong, H.,
Uht, R. M.,
Kushner, P. J.,
and Stallcup, M. R.
(1998)
Mol. Endocrinol.
12,
302-313[Abstract/Free Full Text]
-
Wu, J. P.,
LaPointe, M. C.,
West, B. L.,
and Gardner, D. G.
(1989)
J. Biol. Chem.
264,
6472-6479[Abstract/Free Full Text]
-
Ribeiro, R. C. J.,
Apriletti, J. W.,
Yen, P. M.,
Chin, W. W.,
and Baxter, J. D.
(1994)
Endocrinology
135,
2076-2085[Abstract]
-
Bauer, R. F.,
Arthur, L. O.,
and Fine, D. L.
(1976)
In Vitro
12,
558-563[Medline]
[Order article via Infotrieve]
-
Neumann, J. R.,
Morency, C. A.,
and Russian, K. O.
(1987)
BioTechniques
5,
444-447
-
Dokoh, S.,
Pike, J. W.,
Chandler, J. S.,
Mancini, J. M.,
and Haussler, M. R.
(1981)
Anal. Biochem.
116,
211-222[Medline]
[Order article via Infotrieve]
-
Luisi, B. F.,
Xu, W. X.,
Otwinowski, Z.,
Freedman, L. P.,
Yamamoto, K. R.,
and Sigler, P. B.
(1991)
Nature
352,
497-505[CrossRef][Medline]
[Order article via Infotrieve]
-
Rastinejad, F.,
Perlmann, T.,
Evans, R. M.,
and Sigler, P. B.
(1995)
Nature
375,
203-211[CrossRef][Medline]
[Order article via Infotrieve]
-
Glass, C. K.
(1994)
Endocr. Rev.
15,
391-405[Medline]
[Order article via Infotrieve]
-
Li, Q.,
and Gardner, D. G.
(1994)
J. Biol. Chem.
269,
4934-4939[Abstract/Free Full Text]
-
Wu, J.,
Garami, M.,
Cao, L.,
Li, Q.,
and Gardner, D. G.
(1995)
Am. J. Physiol.
268,
E1108-E1113[Abstract/Free Full Text]
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