Ternary complexes and cooperative interplay between NCoA-62/Ski-interacting protein and steroid receptor coactivators in vitamin D receptor-mediated transcription.

The vitamin D receptor (VDR) is a ligand-dependent transcriptional factor that binds to vitamin D-responsive elements as a heterodimer with retinoid X receptor (RXR) to regulate target gene transcription. The steroid receptor coactivator (SRC) proteins are coactivators that interact with the AF-2 domain of VDR to augment 1,25-dihydroxyvitamin D3-dependent transcription. In contrast, NCoA-62/Ski-interacting protein (SKIP) is a distinct, activation function-2-independent coactivator for VDR. The current study examined whether these two distinct classes of coactivators impact functionally on VDR-mediated transcription. Using a ternary complex binding assay, we observed a marked preference for the direct interaction of NCoA-62/SKIP with the VDR-RXR heterodimer as compared with the VDR-VDR homodimer or VDR monomer. The liganded VDR also formed a ternary complex with NCoA-62/SKIP and SRC proteins in vitro. Competition experiments using LXXLL peptides showed that NCoA-62/SKIP and SRC coactivators contact different domains of the VDR-RXR heterodimer. Synergistic interplays were observed between NCoA-62/SKIP and SRC coactivators in VDR-mediated transcriptional assays, and protein interference assays indicated a requirement for both NCoA-62/SKIP and SRCs in VDR- mediated transcription. These studies suggest that the ligand-dependent and simultaneous interaction of NCoA-62/SKIP and SRC coactivators with distinct interaction domains within the VDR-RXR heterodimer results in cooperative interplays between coactivators in VDR-mediated transcription.

Vitamin D receptor and nuclear receptor coactivators: crucial interactions in vitamin D-mediated transcription.

The nuclear actions of 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] are mediated by the vitamin D receptor (VDR). Binding of ligand induces conformational changes in the VDR which promote heterodimerization with retinoid X receptor (RXR) and recruitment of a number of nuclear receptor coactivator proteins including the steroid receptor coactivator (SRC) family members, select SMAD proteins, a novel coactivator complex referred to as DRIP, and a variety of other putative factors. We recently described a novel nuclear receptor coactivator termed NCoA-62 that interacts with the VDR to enhance 1alpha,25(OH)(2)D(3)-activated transcription. NCoA-62 is unrelated to the SRC family, the DRIP complex, as well as other nuclear receptor coactivators described thus far. The molecular mechanisms involved in NCoA-62 coactivator function are poorly understood, but protein-protein interactions are likely to play an important role. The purpose of this paper is to briefly review salient features of the coactivators involved in VDR-activated transcription and to focus on our current understanding of NCoA-62 and its interplay with other nuclear receptor coactivator proteins. It is clear from the studies described here that a concerted series of interactions with multiple coactivator proteins are essential for high order transactivation by 1alpha,25(OH)(2)D(3) and the VDR.

Selective inhibition of nuclear steroid receptor function by a protein from a human tumorigenic poxvirus.

The poxvirus molluscum contagiosum (MC) has a worldwide distribution and its prevalence is on the rise. Here we report that the MCV MC013L protein inhibits glucocorticoid and vitamin D, but not retinoid or estrogen, nuclear receptor transactivation. A direct interaction of MC013L with glucocorticoid and vitamin D receptor is supported by yeast two-hybrid, GST pull-down, and far Western blot analyses. Glucocorticoids act as potent inhibitors of keratinocyte proliferation, while vitamin D and retinoids promote and block terminal differentiation, respectively. Therefore, MC013L may promote efficient virus replication by blocking the differentiation of infected keratinocytes. MC013L may be the first member of a new class of poxvirus proteins that directly modulate nuclear receptor-mediated transcription.

Endocrine disrupting chemicals, phthalic acid and nonylphenol, activate Pregnane X receptor-mediated transcription.

Recently, Pregnane X receptor (PXR), a new member of the nuclear receptor superfamily, was shown to mediate the effects of several steroid hormones, such as progesterone, glucocorticoid, pregnenolone, and xenobiotics on cytochrome P450 3A genes (CYP3A) through the specific DNA sequence for CYP3A, suggesting that PXR may play a role in steroid hormone metabolism. In this paper, we demonstrated that phthalic acid and nonylphenol, endocrine-disrupting chemicals (EDCs), stimulated PXR-mediated transcription at concentrations comparable to those at which they activate estrogen receptor-mediated transcription using a transient reporter gene expression assay in COS-7 cells. However, bisphenol A, another EDC, had no effect on PXR-mediated transcription, although this chemical significantly enhanced ER-mediated transcription. In the yeast two-hybrid protein interaction assay, PXR interacted with two nuclear receptor coactivator proteins, steroid hormone receptor coactivator-1 and receptor interacting protein 140, in the presence of phthalic acid or nonylphenol. Thus, EDC-occupied PXR may regulate its specific gene expression through the receptor-coactivator interaction. In contrast, these EDCs had no effect on the interaction between PXR and suppressor for gal 1, a component of proteasome. Finally, the expression of CYP3A1 mRNA in the liver of rats exposed to phthalic acid or nonylphenol markedly increased compared with that in rats treated with estradiol, bisphenol A, or ethanol as assessed by competitive RT-PCR. These data suggest that EDCs may affect endocrine functions by altering steroid hormone metabolism through PXR.

Identification of an autonomous transactivation domain in helix H3 of the vitamin D receptor.

The vitamin D receptor (VDR) contains an alpha-helical, ligand-inducible activation function (AF-2) at the COOH-terminus of the ligand-binding domain (LBD). In this study, a second distinct activation domain was identified in the VDR LBD. Using a yeast-based system to screen a random mutant library of GAL4-VDR (93-427), a mutant GAL4-VDR fusion protein with constitutive transcriptional activity was isolated. Sequence analysis identified a C to T transition that introduced a stop codon at glutamine 239 eliminating a large portion of the LBD, including the AF-2 domain. The GAL4-VDR (93-238) mutant exhibited ligand-independent transactivation activity both in yeast and in mammalian cells. Deletion analysis defined a minimal activation domain within helix H3 between D195 and I 238 in the VDR. An aspartic acid residue (D232) within helix H3 was essential for the autonomous transactivation activity since altering this residue to an alanine or an asparagine dramatically reduced its transactivation potential. Expression of the minimal helix H3 activation domain interfered with ligand-activated transcription by full-length VDR suggesting that helix H3 interacts with limiting cellular factors important for VDR-activated transcription. Consequently, we have identified a novel activation domain in helix H3 of the VDR that apparently plays an important role in 1,25-(OH)(2)D(3)-activated transcription.

The autonomous transactivation domain in helix H3 of the vitamin D receptor is required for transactivation and coactivator interaction.

A ligand-inducible transactivation function (AF-2) exists in the extreme carboxyl terminus of the vitamin D receptor (VDR) that is essential for 1alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-activated transcription and p160 coactivator interaction. Crystallographic data of related nuclear receptors suggest that binding of 1, 25-(OH)2D3 by VDR induces conformational changes in the ligand-binding domain (LBD), the most striking of which is a packing of the AF-2 helix onto the LBD adjacent to helices H3 and H4. In this study, a panel of VDR helix H3 mutants was generated, and residues in helix H3 that are important for ligand-activated transcription by the full-length VDR were identified. In particular, one mutant (VDR (Y236A)) exhibited normal ligand binding and heterodimerization with the retinoid X receptor (RXR) but was transcriptionally inactive. Yeast two-hybrid studies and in vitro protein interaction assays demonstrated that VDR (Y236A) was selectively impaired in interaction with AF-2-interacting coactivator proteins such as SRC-1 and GRIP-1. These data indicate an importance of helix H3 in the mechanism of VDR-mediated transcription, and they support the concept that helix H3 functions in concert with the AF-2 domain to form a transactivation surface for binding the p160 class of nuclear receptor coactivators.

Proteasome-mediated degradation of the vitamin D receptor (VDR) and a putative role for SUG1 interaction with the AF-2 domain of VDR.

The AF-2 helix of nuclear receptors is essential for ligand-activated transcription, and it may function to couple the receptor to transcriptional coactivator proteins. This domain also contacts components of the proteasome machinery, suggesting that nuclear receptors may be targets for proteasome-mediated proteolysis. In the present study, we demonstrate that mSUG1 (P45), a component of the 26S proteasome, interacts in a 1,25-(OH)2D3-dependent manner with the AF-2 domain of the vitamin D receptor (VDR). Furthermore, treatment of ROS 17/2.8 osteosarcoma cells with the proteasome inhibitors MG132 or beta-lactone increased steady-state levels of the VDR protein. In the presence cycloheximide (10 microg/ml), the liganded VDR protein was degraded with a half-life of approximately 8 h, and this rate of degradation was completely blocked by 0.05 mM MG132. The role of SUG1 -VDR interaction in this process was investigated in transient expression studies. Overexpression of wild-type mSUG1 in ROS17/2.8 cells generated a novel proteolytic VDR fragment of approximately 50 kDa, and its production was blocked by proteasome inhibitors or by a nonhydrolyzable ATP analog. Parallel studies with SUG1 (K196H), a mutant that does not interact with the VDR, did not produce the 50 kDa VDR fragment. Functionally, expression of SUG1 in a VDR-responsive reporter gene assay resulted in a profound inhibition of 1,25-(OH)2D3-activated transcription, while expression of SUG1 (K196H) had no significant effect in this system. These data show that the AF-2 domain of VDR interacts with SUG1 in a 1,25-(OH)2D3-dependent fashion and that this interaction may target VDR to proteasome-mediated degradation as a means to downregulate the 1,25-(OH)2D3-activated transcriptional response.

Transcriptional activation through the vitamin D receptor in osteoblasts.

Osteoblasts are bone-forming cells that play an essential role in the development and maintenance of a mineralized bone extracellular matrix and they are target cells for vitamin D. Osteoblasts express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates the expression of osteoblastic-specific genes such as osteocalcin and osteopontin. VDR is a ligand-inducible transcription factor which heterodimerizes with retinoid X receptor (RXR) and binds as a heterodimer to vitamin D-responsive elements (VDREs) in the promoter region of vitamin-D responsive genes, ultimately leading to their increased transcription. Important structural aspects of the VDR and the role that each functional domain plays in mediating VDR action in the context of the osteoblast are discussed. A summary of the potential molecular mechanisms involved in VDR-activated transcription highlighting the importance of interactions between the VDR and general transcription factors (GTFs), TBP-associated factors (TAFIIs), and nuclear receptor coactivator and corepressor proteins are reviewed. These interactions have a role in linking the VDR-RXR heterodimer to the transcriptional pre-initiation complex (PIC) and in regulating the transcription of vitamin D-dependent genes. In addition, recent findings suggest that these interactions are important for regulating the accessibility to promoters by modifying the acetylation state of histones. The complex interplay that occurs between VDR and these various factors to determine the overall transcriptional activity of vitamin D-responsive genes will be summarized.

Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription.

The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR.RXR heterodimer is not well understood, but it may involve interactions of VDR.RXR with transcriptional coactivator or corepressor proteins. Here, a yeast two-hybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors. One cDNA clone designated NCoA-62, encoded a 62, 000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression. Yeast two-hybrid studies and in vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR. Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1, 25-dihydroxyvitamin D3-activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription. NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression. These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D- and other nuclear receptor-mediated transcriptional pathways.

Evidence for ligand-dependent intramolecular folding of the AF-2 domain in vitamin D receptor-activated transcription and coactivator interaction.

A ligand-dependent transcriptional activation domain (AF-2) exists in region E of the nuclear receptors. This highly conserved domain may contact several coactivators that are putatively involved in nuclear receptor-mediated transcription. In this study, a panel of vitamin D receptor (VDR) AF-2 mutants was created to examine the importance of several conserved residues in VDR-activated transcription. Two AF-2 mutants (L417S and E420Q) exhibited normal ligand binding, heterodimerization with retinoid X receptor, and vitamin D-responsive element interaction, but they were transcriptionally inactive in a VDR-responsive reporter gene assay. All AF-2 mutations that abolished VDR-mediated transactivation also eliminated interactions between VDR and several putative coactivator proteins including suppressor of gal1 (SUG1), steroid hormone receptor coactivator-1 (SRC-1), or receptor interacting protein (RIP140), suggesting that coactivator interaction is important for AF-2-mediated transcription. In support of this concept, the minimal AF-2 domain [VDR(408-427)] fused to the gal4 DNA binding domain was sufficient to mediate transactivation as well as interaction with putative coactivators. Introducing the L417S and E420Q mutations into the minimal AF-2 domain abolished this autonomous transactivation and coactivator interactions. Finally, we demonstrate that the minimal AF-2 domain interacted with an AF-2 deletion mutant of the VDR in a 1,25-(OH)2D3-dependent manner, suggesting a ligand-induced intramolecular folding of the VDR AF-2 domain. The L417S mutant of this domain disrupted the interaction with VDR ligand-binding domain, while the E420Q mutant did not affect this interaction. These studies suggest that the conserved AF-2 motif may mediate transactivation through ligand-dependent intermolecular interaction with coactivators and through ligand-induced intramolecular contacts with the VDR ligand-binding domain itself.

Crosstalk during Ca2+-, cAMP-, and glucocorticoid-induced gene expression in lymphocytes.

In the WEHI7.2 thymoma cell line, cAMP, glucocorticoids, or increases in cytosolic Ca2+ concentration lead to cell death by apoptosis. In the present study, we examined the effects of these compounds on cAMP response element (CRE)-mediated gene expression. Thapsigargin and A23187 were employed to increase cytosolic Ca2+ levels and induce apoptosis. Both compounds enhanced transcription from a CRE preceding apoptotic death. Moreover, the transcriptional response to the combination of forskolin and either thapsigargin or A23187 was synergistic mirroring the effect on cell death. Importantly, dexamethasone treatment, which causes an efflux of Ca2+ from the ER, induced transcription from a CRE alone or in synergy with forskolin. The increase in CRE-controlled gene expression correlated with a decrease in cell viability. Following treatment with forskolin, thapsigargin, or dexamethasone, the CRE binding protein (CREB) was phosphorylated at levels correlating with the level of induced gene expression. These data suggest that transcriptional crosstalk between independent signaling pathways occurs in lymphocytes, and CREB may play a central role in the mediation of CRE-dependent transcription by these diverse set of apoptotic agents.

The N-terminal domain of transcription factor IIB is required for direct interaction with the vitamin D receptor and participates in vitamin D-mediated transcription.

The interaction of the vitamin D receptor (VDR) with transcription factor IIB (TFIIB) represents a potential physical link between the VDR-DNA complex and the transcription preinitiation complex. However, the functional relevance of the VDR-TFIIB interaction in vitamin D-mediated transcription is not well understood. In the present study, we used site-directed mutagenesis to demonstrate that the structural integrity of the amino-terminal zinc finger of TFIIB is essential for VDR-TFIIB complex formation. Altering the putative zinc-coordinating residues (C15, C34, C37, or H18) to serines abolished TFIIB interaction with the VDR as assessed in a yeast two-hybrid system and by in vitro protein interaction assays. This N-terminal, VDR-interactive domain functioned as a selective, dominant-negative inhibitor of vitamin D-mediated transcription. Expressing amino acids 1-124 of human TFIIB (N-TFIIB) in COS-7 cells or in osteoblastic ROS17/2.8 cells effectively suppressed 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-induced transcription, but had no effect on basal or glucocorticoid-activated transcription. A mutant N-terminal domain [N-TFIIB(C34S:C37S)] that does not interact with VDR had no impact on 1,25-(OH)2D3-induced transcription. Interestingly, both in vitro and in vivo protein interaction assays showed that the VDR-TFIIB protein complex was disrupted by the 1,25-(OH)2D3 ligand. Mechanistically, these data establish a functional role for the N terminus of TFIIB in VDR-mediated transcription, and they allude to a role for unliganded VDR in targeting TFIIB to the promoter regions of vitamin D-responsive target genes.

The Nuclear Receptor Resource Project.

We have expanded the original Glucocorticoid Receptor Resource (GRR) database to include several individual resources as part of a larger project called the Nuclear Receptor Resource (NRR). In addition to the GRR, the NRR currently features the Thyroid Hormone Receptor Resource, the Androgen Receptor Resource, the Mineralocorticoid Receptor Resource, the Vitamin D Receptor Resource, and the Steroid Receptor Associated Proteins Resource. The goal of the NRR project is to provide a comprehensive resource for information on the nuclear receptor superfamily, and to provide a forum for the dissemination and discussion of both published and unpublished material on these proteins. Although the individual resources are managed from different servers, all the files are integrated and can be accessed through the project's Home Page, housed at http://nrr. georgetown.edu/nrr.html. In the near future, we hope to expand the project to contain information on other nuclear receptors and to better our electronic publication system. To accomplish this, we encourage the involvement of nuclear receptor investigators in the NRR.

Association of 1 alpha,25-dihydroxyvitamin D3-occupied vitamin D receptors with cellular membrane acceptance sites.

We previously reported nongenomic activation of ROS 17/2.8 cells by vitamin D metabolites (1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], 25-hydroxyvitamin D3, 22-oxa-calcitriol, etc.). The hormone 1 alpha,25-(OH)2D3, or calcitriol, mediated rapid transient changes in intracellular free calcium levels and concomitant stimulation of inositol polyphosphate and diacylglycerol production. These effects resemble the mechanism of cell activation induced by ligands with plasma membrane (PM) receptors. As preliminary studies indicated that PM isolated from ROS 17/2.8 cells lacked specific binding sites for calcitriol alone, we studied the association between calcitriol-occupied vitamin D receptors (VDR) and ROS 17/2.8 cellular membranes. Saturable binding to the PM and the endoplasmic reticulum (ER) of calcitriol-occupied VDR was demonstrated. Binding of the VDR-[3H]calcitriol complex was displaceable by nonradioactive VDR/calcitriol, but not by the unoccupied VDR or by calcitriol alone. ER binding, but not PM binding, was competitively inhibited by a peptide from the VDR sequence recognized by an ER protein, calreticulin, and by an anticalreticulin antibody. The monoclonal antibody (9A7) against the VDR inhibited PM and ER binding of the hormone-occupied VDR. These results were substantiated by studies using baculovirus-expressed human VDR for binding studies with the PM and ER and for immunoblot analysis. We conclude that specific PM and ER sites of association for calcitriol-occupied VDR exist and suggest that these associations could participate in the nongenomic rapid actions of 1 alpha,25-(OH)2D3.

Phosphorus restriction prevents parathyroid gland growth. High phosphorus directly stimulates PTH secretion in vitro.

Dietary phosphorus (P) restriction is known to ameliorate secondary hyperparathyroidism in renal failure patients. In early renal failure, this effect may be mediated by an increase in 1,25-(OH)2D3, whereas in advanced renal failure, P restriction can act independent of changes in 1,25-(OH)2D3 and serum ionized calcium (ICa). In this study, we examined the effects of dietary P on serum PTH, PTH mRNA, and parathyroid gland (PTG) hyperplasia in uremic rats. Normal and uremic rats were maintained on a low (0.2%) or high (0.8%) P diet for 2 mo. PTG weight and serum PTH were similar in both groups of normal rats and in uremic rats fed the 0.2% P diet. In contrast, there were significant increases in serum PTH (130 +/- 25 vs. 35 +/- 3.5 pg/ml, P < 0.01), PTG weight (1.80 +/- 0.13 vs. 0.88 +/- 0.06 microg/gram of body weight, P < 0.01), and PTG DNA (1.63 +/- 0.24 vs. 0.94 +/- 0.07 microg DNA/gland, P < 0.01) in the uremic rats fed the 0.8% P diet as compared with uremic rats fed the 0.2% P diet. Serum ICa and 1,25-(OH)2D3 were not altered over this range of dietary P, suggesting a direct effect of P on PTG function. We tested this possibility in organ cultures of rat PTGs. While PTH secretion was acutely (30 min) regulated by medium calcium, the effects of medium P were not evident until 3 h. During a 6-h incubation, PTH accumulation was significantly greater in the 2.8 mM P medium than in the 0.2 mM P medium (1,706 +/- 215 vs. 1,033 +/- 209 pg/microg DNA, P < 0.02); the medium ICa was 1.25 mM in both conditions. Medium P did not alter PTH mRNA in this system, but cycloheximide (10 microg/ml) abolished the effect of P on PTH secretion. Thus, the effect of P is posttranscriptional, affecting PTH at a translational or posttranslational step. Collectively, these in vivo and in vitro results demonstrate a direct action of P on PTG function that is independent of ICa and 1,25-(OH)2D3.

Affinity labeling of the 1 alpha,25-dihydroxyvitamin D3 receptor.

Genomic actions of the calciotropic hormone 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) involves a multistep process that is triggered by the highly specific binding of 1,25(OH)2D3 to 1 alpha, 25-dihydroxyvitamin D3 receptor, VDR. In order to study this key step in the cascade, we synthesized 1 alpha,25-dihydroxy[26(27)-3H]vitamin D3-3-deoxy-3 beta-bromoacetate (1,25(OH)2[3H]D3-BE) and 1 alpha,25-dihydroxyvitaminD3-3 beta-[1-14C]bromoacetate(1,25(OH)2D3-[14C]BE) binding-site directed analogs of 1,25(OH)2D3, and affinity-labeled baculovirus-expressed recombinant human VDR (with 1,25(OH)2[3H]D3-BE), and naturally occurring VDRs in cytosols from calf thymus homogenate and rat osteosarcoma (ROS 17/2.8) cells (with 1,25(OH)2D3-[14C]BE). In each case, specificity of labeling was demonstrated by the drastic reduction in labeling when the incubation was carried out in the presence of an excess of nonradioactive 1 alpha,25(OH)2D3. These results strongly suggested that 1,25(OH)2[3H]D3-BE and 1,25(OH)2D3-[14C]BE covalently modified the 1,25(OH)2D3-binding sites in baculovirus-expressed recombinant human VDR and naturally occurring calf thymus VDR and rat osteosarcoma VDR, respectively.

A highly conserved region in the hormone-binding domain of the human vitamin D receptor contains residues vital for heterodimerization with retinoid X receptor and for transcriptional activation.

Residues located between amino acids 244 and 263 in the human vitamin D receptor (hVDR) show extensive homology with other members of the steroid/thyroid/retinoid hormone receptor superfamily. The corresponding region of the glucocorticoid receptor has been shown to interact with the 90-kilodalton heat shock protein (hsp90), yet hVDR does not appear to bind to hsp90. Herein we report a study of hVDR in which the functional role of five conserved residues was tested by replacing Phe-244, Lys-246, Leu-254, Gln-259, and Leu-262 with glycines by site-directed mutagenesis. Initial screening of these mutants indicated that all were significantly impaired in their ability to activate transcription from a vitamin D-responsive reporter construct when expressed in transfected VDR-deficient COS-7 cells. Further characterization revealed two classes of mutants: the predominant class binds the 1,25-dihydroxyvitamin D3 ligand normally but is defective in its ability to form a heterodimeric complex with the retinoid X receptor (RXR) on a vitamin D responsive element (VDRE). A second unique class, represented by a single mutant at Lys-246, is normal both with respect to ligand binding and complex formation but still very impaired in transactivation ability. The distinction between these two classes was confirmed by the demonstration that a member of the first class, with a mutation at Gln-259, could be restored to near wild type transactivation ability by supplying excess RXR, while the Lys-246 mutant could not be so rescued. We therefore conclude that the primary function of this conserved domain in hVDR is the mediation of heterodimerization with RXR, leading to VDRE binding and transactivation. The possibility also exists that the Lys-246 mutant may be impaired in a step of transactivation that is distal to complex formation with RXR on the VDRE, perhaps in interactions with the transcriptional machinery itself.

Genomic actions of 1,25-dihydroxyvitamin D3.

Recent studies have identified a heterodimer of the vitamin D receptor (VDR) and the retinoid X receptor (RXR) as the active complex for mediating positive transcriptional effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active hormonal form of vitamin D. The VDR-RXR heterodimer has been shown to bind to direct repeat vitamin D-responsive elements (VDREs) upstream of positively controlled genes in the target tissues for vitamin D, including bone (osteocalcin, osteopontin, and beta 3 integrin), kidney (24-hydroxylase) and intestine (calbindin). Residues that participate in heterodimer formation have been identified in the C-terminal hormone-binding domain by analysis of VDR mutants. The role of the 1,25(OH)2D3 ligand in transcriptional activation by the VDR-RXR heterodimer is not entirely clear, but studies of two natural VDR mutants suggest that the binding of both hormone and RXR are required to induce a receptor conformation that is competent to activate transcription. A final level of complexity is added by recent observations that VDR is modified by phosphorylation. Thus, the VDR-mediated action of 1,25(OH)2D3 is now known to involve multiple factors that may provide a conceptual basis for future understanding of the tissue-specific genomic effects of 1,25(OH)2D3.