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. RibeiroDagger , and David G. Gardner§

From the Metabolic Research Unit and Department of Medicine, University of California, San Francisco, California 94143-0540 and the Dagger  Department of Pharmaceutical Sciences, University of Brasilia, Brasilia, DF, Brazil 70910-900

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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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-RXRalpha in solution, whereas binding of an I384R/Q385R VDR mutant was almost undetectable. The K386A mutant formed heterodimers with RXRalpha 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 hRXRalpha . Induction by K386A, in the presence or absence of RXRalpha , 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 hRXRalpha . 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.

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INTRODUCTION
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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 beta 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 RXRalpha (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 TRbeta (23) that appear to interfere with dimer formation. However, based on the available structures (e.g. that for TRalpha ), 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.

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Plasmids-- Expression vectors for human VDR (26) and RXRalpha (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 hRXRalpha were generated by polymerase chain reaction using a site-directed mutagenesis kit (Stratagene, La Jolla, CA). GST-RXRalpha (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 pEThRXRalpha 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-hRXRalpha , 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.

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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 RXRalpha 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 hRXRalpha , hRARgamma , hTRbeta , hERalpha , and hVDR which, based on available structural information, are thought to participate in dimer formation. Shaded boxes identify conserved residues targeted for mutation in hRXRalpha 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-RXRalpha 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 RXRalpha (VDR mutants: Vm1, K386A; Vm2, I384R/Q385R; RXR mutants: Rm1, R421A; Rm2, L419R/L420R) with GST-RXRalpha , GST-VDR, and GST-GRIP1. A, radiolabeled wild type or mutant VDR was synthesized in vitro and incubated with GST-RXRalpha , 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 RXRalpha 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.

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 TRbeta and peroxisome proliferator-activated receptor gamma  (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 RXRalpha effectively formed ligand-dependent homodimers and hTRbeta 1-dependent heterodimers on a conventional DR-4 element. Selective mutation at position 421 (R421A) in RXRalpha 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 RXRalpha 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 RXRalpha 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 RXRalpha 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 TRbeta 1 led only to modest levels of heterodimerization with wild type RXRalpha (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 RXRalpha homodimers on DR-1, as well as the weak TRbeta 1-RXRalpha 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 RXRalpha in presence or absence of TRbeta 1. A, radiolabeled RXRalpha or the relevant RXR mutant, with or without unlabeled TRbeta 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.

As expected, wild type VDR formed a stable complex on a DR-3 template with wild type RXRalpha (Fig. 4). The R421A mutant of RXRalpha also formed stable heterodimeric complexes with VDR on this template. On the other hand, the L419R/L420R RXRalpha 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 RXRalpha , still bound to wild type RXRalpha 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 RXRalpha . 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 RXRalpha , displayed modest heterodimer formation only with wild type RXRalpha . 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, RXRalpha , 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.

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 RXRalpha 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 RXRalpha 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 RXRalpha or the RXRalpha 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 RXRalpha (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.

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 RXRalpha mutants to impact on this inhibitory activity. As shown in Fig. 6, liganded VDR effected ~50% inhibition in hANP-CAT reporter activity, whereas liganded RXRalpha 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 RXRalpha mutants proved capable of inhibiting hANP promoter activity.


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Fig. 6.   Effect of VDR, RXRalpha , or their mutants on -1150 hANP-CAT activity. Five µg VDR, RXRalpha , 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.

When used in combination, wild type VDR and RXRalpha effected a >90% inhibition of -1150 hANP CAT activity (Fig. 7). RXRalpha 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 RXRalpha 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 RXRalpha (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.

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.

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 RXRalpha 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 RXRalpha (or one of the RXRalpha 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-RXRalpha 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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ABSTRACT
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Recent crystallographic analyses of individual nuclear receptors suggest conservation of structural features involved in dimer assembly. Specifically amino acid residues in helix 10 of hRXRalpha (18) (these residues are located in helix 11 in hTR and hER) and helix 11 of hTRbeta (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 RXRalpha 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 RXRalpha (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 TRalpha 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 RXRalpha and VDR, respectively.

On the DR-4 template, RXRalpha R421A demonstrated impaired formation of RXRalpha homodimers, whereas heterodimer formation with TRbeta 1 was relatively normal. If anything, the latter was modestly increased, perhaps reflecting diversion of the mutant RXRalpha , 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 RXRalpha . 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 RXRalpha 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 (RXRalpha in the case of VDR and TRbeta 1 in the case of RXRalpha ) 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 RXRalpha 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 RXRalpha (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 RXRalpha (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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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