Distinct retinoid X receptor activation function-2 residues mediate transactivation in homodimeric and vitamin D receptor heterodimeric contexts.

The vitamin D receptor (VDR) stimulates transcription as a 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-activated heterodimer with retinoid X receptor (RXR). RXR also forms homodimers to mediate 9-cis retinoic acid (9-cis RA)-induced gene expression. Both receptors possess a C-terminal hormone-dependent activation function-2 (AF-2), a highly conserved region that binds coactivators to transduce the transcriptional signal. By replacing single amino acids within the AF-2 of human RXR alpha (hRXR alpha) or mouse RXR beta (mRXR beta), the contribution of these residues to transactivation by the RXR-VDR heterodimer and the RXR-RXR homodimer was evaluated. In 9-cis RA-responsive homodimers, the second and fourth positions of the AF-2 (leucine and glutamate respectively) are essential. However, in the context of an RXR-VDR heterodimer activated by 1,25(OH)(2)D(3), alteration of these two RXR residues has little effect. Instead, AF-2 residues located towards the C-terminus, such as the penultimate position (L455 in hRXR alpha or L441 in mRXR beta), are crucial for RXR-VDR heterodimers. Indeed, L455A mutant RXR exerts a dominant negative effect on RXR-VDR transcriptional responsiveness to 1,25(OH)(2)D(3). Further experiments with a mutant hRXR alpha (F313A) which elicits 9-cis RA-independent transactivation as a homodimer demonstrate that, when heterodimerized with VDR, this RXR mutant is incapable of activating the RXR-VDR heterocomplex in the absence of the VDR ligand. Taken together, these results indicate that RXR is a subordinate, yet essential transcriptional partner in RXR-VDR-mediated activation of gene expression. Furthermore, a functional switch in RXR AF-2 signaling occurs between RXR residues in the homodimeric versus the heterodimeric states, likely reflecting different interactions between subregions of the AF-2 and coactivator(s).

Functionally relevant polymorphisms in the human nuclear vitamin D receptor gene.

The functional significance of two unlinked human vitamin D receptor (hVDR) gene polymorphisms was evaluated in twenty human fibroblast cell lines. Genotypes at both a Fok I restriction site (F/f) in exon II and a singlet (A) repeat in exon IX (L/S) were determined, and relative transcription activities of endogenous hVDR proteins were measured using a transfected, 1,25-dihydroxyvitamin D(3)-responsive reporter gene. Observed activities ranged from 2--100-fold induction by hormone, with higher activity being displayed by the F and the L biallelic forms. Only when genotypes at both sites were considered simultaneously did statistically significant differences emerge. Moreover, the correlation between hVDR activity and genotype segregated further into clearly defined high and low activity groups with similar genotypic distributions. These results not only demonstrate functional relevance for both the F/f and L/S common polymorphisms in hVDR, but also provide novel evidence for a third genetic variable impacting receptor potency.

Molecular modeling, affinity labeling, and site-directed mutagenesis define the key points of interaction between the ligand-binding domain of the vitamin D nuclear receptor and 1 alpha,25-dihydroxyvitamin D3.

We have combined molecular modeling and classical structure-function techniques to define the interactions between the ligand-binding domain (LBD) of the vitamin D nuclear receptor (VDR) and its natural ligand, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. The affinity analogue 1alpha,25-(OH)(2)D(3)-3-bromoacetate exclusively labeled Cys-288 in the VDR-LBD. Mutation of C288 to glycine abolished this affinity labeling, whereas the VDR-LBD mutants C337G and C369G (other conserved cysteines in the VDR-LBD) were labeled similarly to the wild-type protein. These results revealed that the A-ring 3-OH group docks next to C288 in the binding pocket. We further mutated M284 and W286 (separately creating M284A, M284S, W286A, and W286F) and caused severe loss of ligand binding, indicating the crucial role played by the contiguous segment between M284 and C288. Alignment of the VDR-LBD sequence with the sequences of nuclear receptor LBDs of known 3-D structure positioned M284 and W286 in the presumed beta-hairpin of the molecule, thereby identifying it as the region contacting the A-ring of 1alpha, 25-(OH)(2)D(3). From the multiple sequence alignment, we developed a homologous extension model of the VDR-LBD. The model has a canonical nuclear receptor fold with helices H1-H12 and a single beta hairpin but lacks the long insert (residues 161-221) between H2 and H3. We docked the alpha-conformation of the A-ring into the binding pocket first so as to incorporate the above-noted interacting residues. The model predicts hydrogen bonding contacts between ligand and protein at S237 and D299 as well as at the site of the natural mutation R274L. Mutation of S237 or D299 to alanine largely abolished ligand binding, whereas changing K302, a nonligand-contacting residue, to alanine left binding unaffected. In the "activation" helix 12, the model places V418 closest to the ligand, and, consistent with this prediction, the mutation V418S abolished ligand binding. The studies together have enabled us to identify 1alpha,25-(OH)(2)D(3)-binding motifs in the ligand-binding pocket of VDR.

Biological activity of CD-ring modified 1alpha,25-dihydroxyvitamin D analogues: C-ring and five-membered D-ring analogues.

Nonsteroidal analogues of 1alpha,25(OH)2D3, lacking either the full five-membered D ring (C-ring analogues) or the full six-membered C ring (D-ring analogues) are more potent inhibitors of cell proliferation or inducers of cell differentiation than is 1alpha,25(OH)2D3. Maximal superagonistic activity was seen for the C-ring analogue with a 24(R)-hydroxyl group in the side chain [30- to 60-fold the activity of 1alpha,25(OH)2D3]. The 19-nor-16-ene-26,27-bishomo C-ring analogue showed the best ratio of antiproliferative to calcemic effects (1275-fold better than 1alpha,25(OH)2D3 and severalfold better than all vitamin D analogues so far described). The analogues are able to stimulate specific vitamin D-dependent genes and are active in transfection assays using an osteocalcin promoter VDRE. Low binding affinity to the vitamin D binding protein, differences in metabolism, or affinity for the vitamin D receptor (VDR) are not the most important explanations for the enhanced intrinsic activity. However, the analogues are able to induce conformational changes in the VDR, which makes the VDR-ligand complex more resistant against protease digestion than is 1alpha,25(OH)2D3. In contrast to 20-epimer steroidal vitamin D analogues, 20-epimer C-ring analogues were less potent than analogues with a natural C-20 configuration. In conclusion, several nonsteroidal vitamin D analogues are superagonists of 1alpha,25(OH)2D3 despite lower receptor affinity and, for the C-ring analogues, higher flexibility of the side chain; moreover, they have a better selectivity profile than all analogues yet published.

The polymorphic N terminus in human vitamin D receptor isoforms influences transcriptional activity by modulating interaction with transcription factor IIB.

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.

Biochemical evidence for a 170-kilodalton, AF-2-dependent vitamin D receptor/retinoid X receptor coactivator that is highly expressed in osteoblasts.

Human vitamin D receptor (hVDR) fused to glutathione S-transferase was utilized to detect a VDR-interacting protein (VIP) of approximately 170 kDa. VIP(170) is expressed in osteoblast-like ROS 17/2.8 cells and, to a lesser extent, in COS-7 and HeLa cells. VIP(170) may be a coactivator because it interacts only with 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) ligand-bound hVDR and because a mutation (E420A) in the activation function-2 (AF-2) of hVDR abolishes both receptor-mediated transactivation and VIP(170) binding. Unlike L254G hVDR, a heterodimerization mutant with an intact AF-2, the E420A mutant is only partially attenuated in its association with the retinoid X receptor (RXR) DNA-binding partner. Finally, the ability of overexpressed hVDR to squelch glucocorticoid receptor-mediated transactivation is lost in both the L254G and E420A mutants. These results suggest that several protein-protein interactions, including VDR association with RXR and VIP(170), are required for stabilization of a multimeric complex that transduces the signal for 1,25(OH)(2)D(3)-elicited transactivation.

Characterization of unique DNA-binding and transcriptional-activation functions in the carboxyl-terminal extension of the zinc finger region in the human vitamin D receptor.

The vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D(3) and mediates its actions on gene transcription by heterodimerizing with retinoid X receptors (RXRs) on direct repeat (DR+3) vitamin D responsive elements (VDREs) located in target genes. The VDRE binding function of VDR has been primarily ascribed to the zinc finger region (residues 24-87). To define the minimal VDRE binding domain for human VDR (hVDR), a series of C-terminally truncated hVDR mutants (Delta134, Delta113, Delta102, Delta90, Delta84, Delta80, and Delta60) was generated and expressed in bacteria. Only the Delta134 and Delta113 mutants bound the VDRE (predominantly as monomers), suggesting that, in addition to the conserved zinc finger region of hVDR, as many as 25 amino acids in a C-terminal extension (CTE) participate in DNA binding. Site-directed mutagenesis of conserved charged residues in full-length hVDR was then performed to dissect the functional significance of the CTE (residues 88-112) in the context of the complete hVDR-RXR-VDRE interaction. Functional assays revealed that E98K/E99K, R102A/K103A/R104A, and K109A/R110A/K111A mutant hVDRs possessed dramatically reduced DNA binding and transcriptional activities, whereas distinct point mutants, such as K103A, bound to DNA normally but lacked transcriptional activity. Therefore, the boundary for the minimal DNA-binding domain in hVDR extends C-terminal of the zinc fingers to Lys-111, with clusters of highly conserved charged amino acids playing a crucial role in binding to the DR+3 element. Further, individual residues in this region (e.g., Lys-103) may lie on the opposing face of a DNA-binding alpha-helix, where they could contact transcriptional coactivators or basal transcription factors.

Vitamin D receptor displays DNA binding and transactivation as a heterodimer with the retinoid X receptor, but not with the thyroid hormone receptor.

The vitamin D receptor (VDR) is a transcription factor believed to function as a heterodimer with the retinoid X receptor (RXR). However, it was reported [Schräder et al., 1994] that, on putative vitamin D response elements (VDREs) within the rat 9k and mouse 28k calcium binding protein genes (rCaBP 9k and mCaBP 28k), VDR and thyroid hormone receptor (TR) form heterodimers that transactivate in response to both 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and triiodothyronine (T(3)). We, therefore, examined associations of these receptors on the putative rCaBP 9k and mCaBP 28k VDREs, as well as on established VDREs from the rat osteocalcin (rOC) and mouse osteopontin (mOP) genes, plus the thyroid hormone response element (TRE) from the rat myosin heavy chain (rMHC) gene. In gel mobility shift assays, we found no evidence for VDR-TR heterodimer interaction with any tested element. Further, employing these hormone response elements linked to reporter genes in transfected cells, VDR and TR mediated responses to their cognate ligands only from the rOC/mOP and rMHC elements, respectively, while the CaBP elements were unresponsive to any combination of ligand(s). Utilizing the rOC and mOP VDREs, two distinct repressive actions of TR on VDR-mediated signaling were demonstrated: a T(3)-independent action, presumably via direct TR-RXR competition for DNA binding, and a T(3)-dependent repression, likely by diversion of limiting RXR from VDR-RXR toward the formation of TR-RXR heterodimers. The relative importance of these two mechanisms differed in a response element-specific manner. These results may provide a partial explanation for the observed association between hyperthyroidism and bone demineralization/osteoporosis.

Steroid hormone receptors: evolution, ligands, and molecular basis of biologic function.

The characterization of the superfamily of nuclear receptors, in particular the steroid/retinoid/thyroid hormone receptors, has resulted in a more complete understanding of how a repertoire of hormonally and nutritionally derived lipophilic ligands controls cell functions to effect development and homeostasis. As transducers of hormonal signaling in the nucleus, this superfamily of DNA-binding proteins appears to represent a crucial link in the emergence of multicellular organisms. Because nuclear receptors bind and are conformationally activated by a chemically diverse array of ligands, yet are closely related in general structure, they present an intriguing example of paralogous evolution. It is hypothesized that an ancient prototype receptor evolved into an intricate set of dimerizing isoforms, capable of recognizing an ensemble of hormone-responsive element motifs in DNA, and exerting ligand-directed combinatorial control of gene expression. The effector domains of nuclear receptors mediate transcriptional activation by recruiting coregulatory multisubunit complexes that remodel chromatin, target the initiation site, and stabilize the RNA polymerase II machinery for repeated rounds of transcription of the regulated gene. Because some nuclear receptors also function in gene repression, while others are constitutive activators, this superfamily of proteins provides a number of avenues for investigating hormonal regulation of gene expression. This review surveys briefly the latest findings in the nuclear receptor field and identifies particular areas where future studies should be fruitful. J. Cell. Biochem. Suppls. 32/33:110-122, 1999.

Normal vitamin D receptor concentration and responsiveness to 1, 25-dihydroxyvitamin D3 in skin fibroblasts from patients with absorptive hypercalciuria.

To evaluate whether there is an increase in vitamin D receptor (VDR) concentration which could raise intestinal calcium absorption in absorptive hypercalciuric (AH) patients and promote hypercalciuria, we measured VDR concentration and VDR mRNA levels in skin fibroblasts from 16 patients with AH and 17 age-matched normal subjects before and following a 16-hour incubation in the presence of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. There were no significant differences in VDR concentration between normal subjects and AH patients in the basal state (30 +/- 11 vs. 30 +/- 15 ng/mg protein, respectively) or following 1,25(OH)2D3-mediated upregulation (43 +/- 18 vs. 42 +/- 16 ng/mg protein) as measured by immunoblot methodology. Analysis of VDR mRNA/beta-actin mRNA ratios demonstrated no significant differences between normal subjects and AH patients prior to (2.1 +/- 1.7 vs. 1.8 +/- 2.4) or following (2.7 +/- 2.8 vs. 1.9 +/- 1.8) 1,25(OH)2D3 exposure. As a measure of VDR bioactivity, we quantitated 1,25(OH)2D3-mediated induction of 25-hydroxyvitamin D3-24-hydroxylase. Again, no significant differences were observed between normal subjects and all patients (2.1 +/- 1.6 vs. 1.9 +/- 1.6 pmol/mg/30 min, respectively). These findings indicate that there is neither an increase in VDR concentration in skin fibroblasts, a recognized vitamin D responsive cell, nor increased sensitivity to upregulation of VDR numbers by 1, 25(OH)2D3 in patients with AH. This suggests an alternative cause of intestinal hyperabsorption of calcium in AH other than alteration of the VDR number.

Novel nuclear localization signal between the two DNA-binding zinc fingers in the human vitamin D receptor.

The human vitamin D receptor (hVDR) possesses a unique array of five basic amino acids positioned between the two DNA-binding zinc fingers that is similar to well-characterized nuclear localization sequences in other proteins. When residues within this region are mutated to nonbasic amino acids, or when this domain is deleted, the receptor is still well expressed, but it no longer associates with the vitamin D-responsive element in DNA, in vitro, and hVDR-mediated transcriptional activation is abolished in transfected cells. Concomitantly, the mutated hVDRs exhibit a significant shift in hVDR cellular distribution favoring cytoplasmic over nuclear retention as assessed by subcellular fractionation and immunoblotting. Independent immunocytochemical studies employing a VDR-specific monoclonal antibody demonstrate that mutation or deletion of this basic domain dramatically attenuates hVDR nuclear localization in transfected COS-7 cells. Although wild-type hVDR is partitioned predominantly to the nucleus in the absence of the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone, treatment with ligand further enhances nuclear translocation, as it does to some degree in receptors with the basic region altered. The role of 1,25(OH)2D3 may be to facilitate hVDR heterodimerization with retinoid X receptors, stimulating subsequent DNA binding and ultimately enhancing nuclear retention. Taken together, these data reveal that the region of hVDR between Arg-49 and Lys-55 contains a novel constitutive nuclear localization signal, RRSMKRK.

Suppression of ANP gene transcription by liganded vitamin D receptor: involvement of specific receptor domains.

We showed previously that liganded vitamin D receptor (VDR) effects a suppression of human atrial natriuretic peptide (hANP) gene-promoter activity in cultured neonatal rat atrial myocytes. In the present study, we have attempted to identify the structural domains of the VDR that are involved in mediating this suppression. We examined the effects of a series of VDR mutants on a cotransfected hANP promoter-driven chloramphenicol acetyltransferase (CAT) reporter. Neither the native VDR nor any of the mutants tested displayed inhibitory activity in the absence of the 1,25-dihydroxyvitamin D3 (VD3) ligand. Delta134, a deletant harboring solely the DNA binding region of the VDR, and L254G, a mutant shown to be defective in retinoid X receptor (RXR) heterodimer formation in other systems, were as effective as the native VDR in reducing promoter activity. HBD, a deletant containing only the hormone-binding domain of the VDR, and K246G, a point mutant that is defective in the activation function of the receptor, did not attenuate reporter activity. A similar activity profile was displayed when a positively regulated promoter containing a direct-repeat vitamin D responsive element (DR3-CAT) was examined in these cells. Liganded VDR, the delta134 mutant, and liganded L254G effected increases in DR3-CAT activity of 2.5-, 2-, and 4-fold, respectively. Two nonhypercalcemic analogues of VD3 (RO 23-7553 and RO 25-6760) displayed the same inhibitory activity as VD3. These studies suggest that the inhibition of hANP promoter activity requires both the DNA binding and activation functions of the receptor but does not appear to require formation of a classic RXR alpha-VDR heterodimer.

Heterodimeric DNA binding by the vitamin D receptor and retinoid X receptors is enhanced by 1,25-dihydroxyvitamin D3 and inhibited by 9-cis-retinoic acid. Evidence for allosteric receptor interactions.

Gel mobility shift analysis was utilized to investigate the molecular function of 1alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and 9-cis-retinoic acid (9-cis-RA) ligands in the binding of the vitamin D receptor (VDR) and retinoid X receptor (RXR) to mouse osteopontin and rat osteocalcin vitamin D-response elements (VDREs). At physiological ionic strength and reduced concentrations of expressed proteins, efficient binding to either VDRE occurs as a VDR. RXR heterodimer, not as a VDR homodimer. 1,25-(OH)2D3 dramatically enhances heterodimer-VDRE interaction, whereas somewhat higher concentrations of 9-cis-RA inhibit this association, perhaps related to the role of this retinoid in facilitating RXR homodimer formation. Interestingly, if VDR is occupied by 1,25-(OH)2D3 prior to complexing with RXR, the resulting heterodimer is relatively resistant to dissociation and diversion to other pathways by 9-cis-RA. Therefore, a proposed molecular action of 1,25-(OH)2D3 is to generate an allosteric switch in VDR to a form that not only binds to the VDRE with high affinity and specificity as a heterodimer with RXR, but also interacts with the RXR partner to conformationally restrict the action of its cognate ligand.

The vitamin D hormone and its nuclear receptor: molecular actions and disease states.

Vitamin D plays a major role in bone mineral homeostasis by promoting the transport of calcium and phosphate to ensure that the blood levels of these ions are sufficient for the normal mineralization of type I collagen matrix in the skeleton. In contrast to classic vitamin D-deficiency rickets, a number of vitamin D-resistant rachitic syndromes are caused by acquired and hereditary defects in the metabolic activation of the vitamin to its hormonal form, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or in the subsequent functions of the hormone in target cells. The actions of 1,25(OH)2D3 are mediated by the nuclear vitamin D receptor (VDR), a phosphoprotein which binds the hormone with-high affinity and regulates the expression of genes via zinc finger-mediated DNA binding and protein-protein interactions. In hereditary hypocalcemic vitamin D-resistant rickets (HVDRR), natural mutations in human VDR that confer patients with tissue insensitivity to 1,25(OH)2D3 are particularly instructive in revealing VDR structure function relationships. These mutations fall into three categories: (i) DNA binding/nuclear localization, (ii) hormone binding and (iii) heterodimerization with retinoid X receptors (RXRs). That all three classes of VDR mutations generate the HVDRR phenotype is consistent with a basic model of the active receptor as a DNA-bound, 1,25(OH)2D3-liganded heterodimer of VDR and RXR. Vitamin D responsive elements (VDREs) consisting of direct hexanucleotide repeats with a spacer of three nucleotides have been identified in the promoter regions of positively controlled genes expressed in bone, such as osteocalcin, osteopontin, beta 3-integrin and vitamin D 24-OHase. The 1,25(OH)2D3 ligand promotes VDR-RXR heterodimerization and specific, high affinity VDRE binding, whereas the ligand for RXR, 9-cis retinoic acid (9-cis RA), is capable of suppressing 1,25(OH)2D3-stimulated transcription by diverting RXR to form homodimers. However, initial 1,25(OH)2D3 liganding of a VDR monomer renders it competent not only to recruit RXR into a heterodimer but also to conformationally silence the ability of its RXR partner to bind 9-cis RA and dissociate the heterodimer. Additional probing of protein-protein interactions has revealed that VDR also binds to basal transcription factor IIB (TFIIB) and, in the presence of 1,25(OH)2D3, an RXR-VDR-TFIIB ternary complex can be created in solution. Moreover, for transcriptional activation by 1,25(OH)2D3, both VDR and RXR require an intact short amphipathic alpha-helix, known as AF-2, positioned at their extreme C-termini. Because the AF-2 domains participate neither in VDR-RXR heterodimerization nor in TFIIB association, it is hypothesized that they contact, in a ligand-dependent fashion, transcriptional coactivators such as those of the steroid receptor coactivator family, constituting yet a third protein-protein interaction for VDR. Therefore, in VDR-mediated transcriptional activation, 1,25(OH)2D3 binding to VDR alters the conformation of the ligand binding domain such that it: (i) engages in strong heterodimerization with RXR to facilitate VDRE binding, (ii) influences the RXR ligand binding domain such that it is resistant to the binding of 9-cis RA but active in recruiting coactivator to its AF-2 and (iii) presents the AF-2 region in VDR for coactivator association. The above events, including bridging by coactivators to the TATA binding protein and associated factors, may position VDR such that it is able to attract TFIIB and the balance of the RNA polymerase II transcription machinery, culminating in repeated transcriptional initiation of VDRE-containing, vitamin D target genes. Such a model would explain the action of 1,25(OH)2D3 to elicit bone remodeling by stimulating osteoblast and osteoclast precursor gene expression, while concomitantly triggering the termination of its hormonal signal by inducing the 24-OHase catabolizing enzyme.

Mutations in the 1,25-dihydroxyvitamin D3 receptor identifying C-terminal amino acids required for transcriptional activation that are functionally dissociated from hormone binding, heterodimeric DNA binding, and interaction with basal transcription factor IIB, in vitro.

To investigate a potential ligand-dependent transcriptional activation domain (AF-2) in the C-terminal region of the human vitamin D receptor (hVDR), two conserved residues, Leu-417 and Glu-420, were replaced with alanines by site-directed mutagenesis (L417A and E420A). Transcriptional activation in response to 1, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) was virtually eliminated when either point mutant was transfected into several mammalian cell lines. Furthermore, both mutants exhibited a dominant negative phenotype when expressed in COS-7 cells. Scatchard analysis at 4 degrees C and a ligand-dependent DNA binding assay at 25 degrees C revealed essentially normal 1,25-(OH)2D3 binding for the mutant hVDRs, which were also equivalent to native receptor in associating with the rat osteocalcin vitamin D responsive element as a presumed heterodimer with retinoid X receptor. Glutathione S-transferase-human transcription factor IIB (TFIIB) fusion protein linked to Sepharose equally coprecipitated the wild-type hVDR and the AF-2 mutants. These data implicate amino acids Leu-417 and Glu-420, residing in a putative alpha-helical region at the extreme C terminus of hVDR, as critical in the mechanism of 1, 25-(OH)2D3-stimulated transcription, likely mediating an interaction with a coactivator(s) or a component of the basal transcriptional machinery distinct from TFIIB.

Vitamin D receptors from patients with resistance to 1,25-dihydroxyvitamin D3: point mutations confer reduced transactivation in response to ligand and impaired interaction with the retinoid X receptor heterodimeric partner.

Hereditary hypocalcemic vitamin D-resistant rickets is attributable to defects in the nuclear receptor for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Two novel point mutations (I314S and R391C) identified in the hormone-binding domain of the human vitamin D receptor (VDR) from patients with hereditary hypocalcemic vitamin D-resistant rickets confer the receptor with sharply reduced 1,25-(OH)2D3-dependent transactivation. These natural mutations, especially R391C, also lead to a second specific consequence, namely impaired heterodimeric interaction with retinoid X receptor (RXR). While the transactivation ability of the I314S mutant can be largely restored by providing excess 1,25-(OH)2D3, R391C activity is more effectively restored with exogenous RXR. These observations are reflected also in the clinical course of each patient: the patient bearing the I314S mutation showed a nearly complete cure with pharmacological doses of a vitamin D derivative, whereas the patient bearing R391C responded only partially to such therapy. Further tests with patient fibroblasts and transfected cells show that the activity of the I314S VDR mutant is augmented somewhat by added RXR, while transactivation by the R391C mutant is best corrected by RXR in the presence of excess hormone. Thus, the effects of hormone vs. RXR in bolstering these mutant VDRs, such that they mediate efficient transactivation, are not entirely separable. The unique properties of these genetically altered receptors establish a new subclass of natural human VDR mutants that illustrate, in vivo, the importance of both 1,25-(OH)2D3 binding and heterodimerization with RXR in VDR action.

Human vitamin D receptor phosphorylation by casein kinase II at Ser-208 potentiates transcriptional activation.

The potential functional significance of human 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor (hVDR) phosphorylation at Ser-208 was evaluated by cotransfecting COS-7 kidney cells with hVDR constructs and the catalytic subunit of human casein kinase 11 (CK-11). Under these conditions, hVDR is intensely phosphorylated in a reaction that depends on both CK-II and the presence of Ser-208. The resulting hyperphosphorylated receptor is unaltered in its kinetics for binding the 1,25(OH)2D3 ligand, its partitioning into the nucleus, and its ability to associate with a vitamin D responsive element. Replacement of Ser-208 with glycine or alanine indicates that phosphorylation of hVDR at Ser-208 is not obligatory for 1,25(OH)2D3 action, but coexpression of wild-type hVDR and CK-11 elicits a dose-dependent enhancement of 1,25(OH)2D3-stimulated transcription of a vitamin D responsive element reporter construct. This enhancement by CK-II is abolished by mutating Ser-208 to glycine or alanine and does not occur with glucocorticoid receptor-mediated transcription. Therefore, phosphorylation of hVDR by CK-11 at Ser-208 specifically modulates its transcriptional capacity, suggesting that this covalent modification alters the conformation of VDR to potentiate its interaction with the machinery for DNA transcription.

Examination of the potential functional role of conserved cysteine residues in the hormone binding domain of the human 1,25-dihydroxyvitamin D3 receptor.

The significance of conserved cysteines at positions 288, 337, and 369 in the hormone binding domain of the human vitamin D receptor was evaluated by individual site-directed mutagenesis to glycine. Neither nuclear localization nor heterodimerization with retinoid X receptors in binding to the vitamin D-responsive element was appreciably affected by altering these cysteines, but vitamin D hormone (1,25-(OH)2D3) activated transcription was compromised significantly in the C288G and C337G mutants. Only the C288G mutant displayed depressed (3-fold) 1,25-(OH)2D3 ligand binding affinity at 4 degrees C, in vitro, although at elevated temperatures (23-37 degrees C), ligand binding was attenuated severely in C288G, moderately in C337G and very mildly in C369G. The degree of impairment of ligand binding at physiologic temperatures correlated with the requirement for increased concentrations of 1,25-(OH)2D3 ligand to maximally stimulate transcriptional activity in co-transfected COS-7 cells. Thus cysteine 288 and, to a lesser extent, cysteine 337 are important for high affinity hormone binding to the vitamin D receptor, which ultimately leads to ligand-dependent transcriptional activation.