Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The action of 1 alpha, 25-dihydroxyvitamin D3 is mediated by its nuclear receptor (VDR), a ligand-dependent transcription regulator. We report the 1.8 A resolution crystal structure of the complex between a VDR ligand-binding domain (LBD) construct lacking the highly variable VDR-specific insertion domain and vitamin D. The construct exhibits the same binding affinity for vitamin D and transactivation ability as the wild-type protein, showing that the N-terminal part of the LBD is essential for its structural and functional integrity while the large insertion peptide is dispensable. The structure reveals the active conformation of the bound ligand and allows understanding of the different binding properties of some synthetic analogs.
Mol Cell 2000 Jan
PMID:The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. 1067 79

We examined the effects of cyclic adenosine 3',5'-monophosphate (cAMP) and protein kinase A (PKA) on the ligand-dependent transactivation mediated via the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptor (VDR). A human VDR expression plasmid was transfected into HeLa, Saos-2 and MG63 cells with a luciferase reporter gene construct containing the vitamin D responsive element. With the addition of 0.5 mM 8 bromo-cAMP, the response to 1,25(OH)2D3 was suppressed to 61 and 78% in the HeLa and Saos-2 cells, respectively. The suppressive effect of 8 bromo-cAMP was observed without the introduction of the VDR expression plasmid in the MG63 cells. In the HeLa cells the co-expression of PKA reduced the ligand-inducible transactivation to 61% and the fold induction by 1,25(OH)2D3 to 89% of that without PKA. The CREB binding protein (CBP) was recently reported to integrate the intracellular signals via the cAMP/PKA cascade and nuclear hormone receptors. However, the suppressive effect of cAMP was not influenced by the co-expression of CBP. Lastly, we introduced point mutations at possible PKA phosphorylation sites into the VDR expression vector at serine-172 and threonine-175, but both mutant receptors still exhibited reduced transactivation with the co-expression of PKA. These results indicate that the phosphorylation of proteins other than the VDR may also be involved in the inhibitory effect mediated by the cAMP/PKA cascade.
Mol Cell Endocrinol 2000 Jan 25
PMID:Effect of cyclic adenosine 3',5'-monophosphate and protein kinase A on ligand-dependent transactivation via the vitamin D receptor. 1068 51

The human vitamin D receptor (hVDR) is a ligand-regulated transcription factor that mediates the actions of the 1,25-dihydroxyvitamin D3 hormone to effect bone mineral homeostasis. Employing mutational analysis, we characterized Arg-18/Arg-22, hVDR residues immediately N-terminal of the first DNA binding zinc finger, as vital for contact with human basal transcription factor IIB (TFIIB). Alteration of either of these basic amino acids to alanine also compromised hVDR transcriptional activity. In contrast, an artificial hVDR truncation devoid of the first 12 residues displayed both enhanced interaction with TFIIB and transactivation. Similarly, a natural polymorphic variant of hVDR, termed F/M4 (missing a FokI restriction site), which lacks only the first three amino acids (including Glu-2), interacted more efficiently with TFIIB and also possessed elevated transcriptional activity compared with the full-length (f/M1) receptor. It is concluded that the functioning of positively charged Arg-18/Arg-22 as part of an hVDR docking site for TFIIB is influenced by the composition of the adjacent polymorphic N terminus. Increased transactivation by the F/M4 neomorphic hVDR is hypothesized to result from its demonstrated enhanced association with TFIIB. This proposal is supported by the observed conversion of f/M1 hVDR activity to that of F/M4 hVDR, either by overexpression of TFIIB or neutralization of the acidic Glu-2 by replacement with alanine in f/M1 hVDR. Because the f VDR genotype has been associated with lower bone mineral density in diverse populations, one factor contributing to a genetic predisposition to osteoporosis may be the F/f polymorphism that dictates VDR isoforms with differential TFIIB interaction.
Mol Endocrinol 2000 Mar
PMID:The polymorphic N terminus in human vitamin D receptor isoforms influences transcriptional activity by modulating interaction with transcription factor IIB. 1070 58

Vitamin D is enzymatically modified to more than 35 metabolites. While many of these are thought to represent degradation products, some have been shown to exhibit biological activity. We tested whether 3-epi-1alpha,25-dihydroxyvitamin D(3) (3-epi-1alpha, 25(OH)(2)D(3)), 1alpha,25-dihydroxy-24-oxo-vitamin D(3) (1alpha, 25(OH)(2)-24-oxo-D(3)), and 1alpha,25(OH)(2)D(3)-26,23-lactone can stimulate transcription of vitamin D responsive genes. MC3T3-E1 cells transfected with a 25-hydroxyvitamin D 24-hydroxylase (CYP24) promoter construct displayed a 6 fold response when treated with either 1alpha,25(OH)(2)D(3) or 3-epi-1alpha,25(OH)(2)D(3). Caco-2 cells were transfected with the wild type CYP24 promoter construct, or a Vitamin D Response Element (VDRE)-mutated form. Cells acquiring the wild type reporter responded to 1alpha,25(OH)(2)D(3) and 3-epi-1alpha,25(OH)(2)D(3) but not cells which acquired the mutated reporter. Additionally, VDR-negative COS-7 cells transfected with the wild type promoter responded (approximately 13 fold) to 1alpha, 25(OH)(2)D(3) and 3-epi-1alpha,25(OH)(2)D(3), only when co-transfected with the VDR. These results were confirmed using shorter incubation times and serum-free conditions. This strongly suggested that 3-epi-1alpha,25(OH)(2)D(3) mediates its effects through the VDR and its cognate binding site. Similar results were obtained with 1alpha,25(OH)(2)-24-oxo-D(3) using VDR-negative P19 cells. We could never detect activity from 1alpha,25(OH)(2)D(3)-26, 23-lactone on vitamin D-responsive target promoters. Our results firmly conclude that both 3-epi-1alpha,25(OH)(2)D(3) and the 1alpha, 25(OH)(2)-24-oxo-D(3) elicit their biological effects by acting through the VDR/VDRE.
J Steroid Biochem Mol Biol
PMID:The 3-epi- and 24-oxo-derivatives of 1alpha,25 dihydroxyvitamin D(3) stimulate transcription through the vitamin D receptor. 1073 35

Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D(3) receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.
Mol Cell Biol 2000 Apr
PMID:The DRIP complex and SRC-1/p160 coactivators share similar nuclear receptor binding determinants but constitute functionally distinct complexes. 1073 74

The TRAP coactivator complex is a large, multisubunit complex of nuclear proteins which associates with nuclear hormone receptors (NRs) in the presence of cognate ligand and stimulates NR-mediated transcription. A single subunit, TRAP220, is thought to target the entire complex to a liganded receptor through a domain containing two of the signature LXXLL motifs shown previously in other types of coactivator proteins to be essential for mediating NR binding. In this work, we demonstrate that each of the two LXXLL-containing regions, termed receptor binding domains 1 and 2 (RBD-1 and RBD-2), is differentially preferred by specific NRs. The retinoid X receptor (RXR) displays a weak yet specific activation function 2 (AF2)-dependent preference for RBD-1, while the thyroid hormone receptor (TR), vitamin D(3) receptor (VDR), and peroxisome proliferator-activated receptor all exhibit a strong AF2-dependent preference for RBD-2. Using site-directed mutagenesis, we show that preference for RBD-2 is due to the presence of basic-polar residues on the amino-terminal end of the core LXXLL motif. Furthermore, we show that the presence and proper spacing of both RBD-1 and RBD-2 are required for an optimal association of TRAP220 with RXR-TR or RXR-VDR heterodimers bound to DNA and for TRAP220 coactivator function. On the basis of these results, we suggest that a single molecule of TRAP220 can interact with both subunits of a DNA-bound NR heterodimer.
Mol Cell Biol 2000 Aug
PMID:Specific structural motifs determine TRAP220 interactions with nuclear hormone receptors. 1089 84

A 25-carboxylic ester analog of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], ZK159222 (compound 1), was recently described as a novel type of antagonist of 1alpha,25(OH)(2)D(3) signaling. In this study five derivatives of compound 1 (compounds 2-6) were selected because of their sensitivity in facilitating complex formation between the 1alpha,25(OH)(2)D(3) receptor (VDR) and the retinoid X receptor on a 1alpha,25(OH)(2)D(3) response element that was comparable to that of the natural hormone (0.2-0.9 nM). Most derivatives of compound 1 reacted as typical agonists, because they were able to promote ligand-dependent interaction of the VDR with the coactivator TIF2, stabilized the VDR preferentially in its agonistic conformation c1(LPD), and stimulated VDR-dependent gene activity with a potency similar to 1alpha,25(OH)(2)D(3). In contrast, only compound 2 showed the antagonistic profile of compound 1, which includes the incompetence to induce a VDR-TIF2 contact, the stabilization of the antagonistic conformation c2(LPD), and only a very weak and insensitive functional activity. Accordingly, only compounds 1 and 2, but not compounds 3 to 6, showed prominent antagonistic effects in cellular systems. The comparison of the structures of the compounds indicates that the essential requirements for an antagonistic function are a cyclopropyl ring at carbon 25, a hydroxy group at carbon 24, and at least a butylester. Interestingly, compound 2 was an approximately 3 times more sensitive antagonist than compound 1 and even displayed a lower residual agonistic activity. In conclusion, only a very limited number of structural variations of compound 1 are possible to keep its antagonistic profile, but the tools presented here for their in vitro evaluation allow an accurate prediction of the effects and are suited to screening for even more potent 1alpha, 25(OH)(2)D(3) antagonists.
Mol Pharmacol 2000 Nov
PMID:Structure activity relationship of carboxylic ester antagonists of the vitamin D(3) receptor. 1104 55

Twenty-epi analogs of 1alpha,25-dihydroxyvitamin D3 (1,25D3) are 100-1000 times more potent transcriptionally than the natural hormone. To determine whether this enhanced activity is mediated through modulation of the dimerization process or through interaction with coactivators, we performed quantitative protein-protein interaction assays with in vitro translated vitamin D receptor (ivtVDR) and fusion proteins containing glutathione-S-transferase (GST) and either the ligand-binding domain of retinoid X receptor (RXRalpha), or the nuclear receptor-interacting domain of the steroid receptor coactivator 1 (SRC-1), or the glucocorticoid receptor-interacting protein 1 (GRIP-1). We found that heterodimerization of the ligand-binding domains of RXRalpha and VDR was primarily deltanoid dependent as was the interaction of VDR with the SRC-1 or with GRIP-1. The ED50 for induction of heterodimerization was 2 nM for 1,25D3 and 0.05 nM for 20-epi-1,25D3. However, the ED50 for induction of VDR interaction with SRC-1 was similar for both 1,25D3 and the 20-epi analog (ED50 = 0.7-1.0 nM) as was the ED50 for ligand-mediated interaction of VDR with GRIP-1 (ED50 = 0.1-0.3 nM). Mutations in heptad 9 diminished both 1,25D3 and the 20-epi analog-mediated dimerization, without changing binding of these ligands to VDR. Mutations in VDR's activation function 2 (AF-2) domain/helix 12 residues diminished the ability of 1,25D3 to induce heterodimerization and interaction with SRC-1. These mutations did not change the ability of 20-epi-1,25D3 to induce dimerization but did diminish its ability to induce interaction with SRC-1. We hypothesize that both the hormone and the analog stabilize receptor conformations that expose VDR's functional interfaces. The mechanisms by which the two ligands expose these functional interfaces differ with respect to participation of the AF-2 domain.
Mol Endocrinol 2000 Nov
PMID:Regulation of ligand-induced heterodimerization and coactivator interaction by the activation function-2 domain of the vitamin D receptor. 1107 11

(23S)-25-dehydro-1alpha-Dihydroxyvitamin D3-26,23-lactone (TEI-9647; MK) has been reported to antagonize the 1alpha,25-dihydroxyvitamin D3 nuclear receptor (VDR)- mediated increase in transcriptional activity. Using a transient transfection system incorporating the osteocalcin VDRE (vitamin D response element) in Cos-1 cells, we found that 20 nM MK antagonizes VDR-mediated transcription by 50% when driven by 1 nM 1alpha,25(OH)2D3. Four analogs of 1alpha,25(OH)2D3, also at 1 nM, were antagonized 25 to 39% by 20 nM MK. However, analogs with 16-ene/23-yne or 20-epi modifications, which have a significantly lower agonist ED50 for the VDR than 1alpha,25(OH)2D3, were antagonized by 20 nM MK only at 100 pM or 10 pM, respectively. One possible mechanism for antagonism is that the 25-dehydro alkene of MK might covalently bind the ligand-binding site of the VDR rendering it inactive. Utilization of a ligand exchange assay, however, demonstrated that MK bound to VDR is freely exchanged with 1alpha,25(OH)2D3 in vitro. These data support the apparent correlation between VDR transcriptional activation by agonists and the effective range of MK antagonism by competition. Furthermore, protease sensitivity analysis of MK bound to VDR indicates the presence of a unique conformational change in the VDR ligand-binding domain, showing a novel doublet of VDR fragments centered at 34 kDa, whereas 1alpha,25(OH)2D3 as a ligand produces only a single 34-kDa fragment. In comparison, the natural metabolite 1alpha,25-dihydroxyvitamin D3-26,23-lactone yields only the 30-kDa fragment that is produced by all ligands to varying degrees. Collectively, these results support that MK is a potent partial antagonist of the VDR for 1alpha,25(OH)2D3 and its analogs when in appropriate excess of the agonist.
Mol Endocrinol 2000 Nov
PMID:25-Dehydro-1alpha-hydroxyvitamin D3-26,23S-lactone antagonizes the nuclear vitamin D receptor by mediating a unique noncovalent conformational change. 1107 12

The nuclear receptor for the hormone 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), VDR, regulates gene expression via a ternary complex with the retinoid X receptor (RXR) and a 1alpha, 25(OH)(2)D(3) response element (VDRE). This complex mediates transcriptional repression through interaction with co-repressor proteins, such as NCoR, and transactivation through agonist-triggered contacts with co-activator proteins, such as SRC-1. This study demonstrates that the interaction of the VDR with NCoR results in a preferential stabilization of the VDR in a non-agonistic conformation (silent state), whereas within a complex with SRC-1 VDR is in its agonistic conformation (activated state). Helix 12 of the ligand-binding domain of the VDR was found to be a critical sensor for the differential stabilization of the activated and silent state of the receptor. VDR agonists that showed similar sensitivity in inducing VDR-RXR-VDRE complex formation were found to mediate a different dose-dependent release of NCoR from these complexes, which correlates with their ability to stabilize the silent state of the VDR in the presence of NCoR. Interestingly, up to 50 % of all VDR-NCoR complexes were found to be stable even in the presence of saturating agonist concentrations. This was confirmed by a quenching effect of overexpressed NCoR on agonist-induced gene activity mediated by VDR-RXR heterodimers. Taken together, co-activator and co-repressor proteins antagonize each other in stabilizing the activated and silent state of the receptor and modulate in this way the sensitivity and potency of the transcriptional activation by the ligand-responsive transcription factor VDR.
J Mol Biol 2000 Dec 15
PMID:Agonist-triggered modulation of the activated and silent state of the vitamin D(3) receptor by interaction with co-repressors and co-activators. 1112 27


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