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1.
Isolation of baculovirus-expressed human vitamin D receptor: DNA responsive element interactions and phosphorylation of the purified receptor 总被引:3,自引:0,他引:3
Jurutka PW MacDonald PN Nakajima S Hsieh JC Thompson PD Whitfield GK Galligan MA Haussler CA Haussler MR 《Journal of cellular biochemistry》2002,85(2):435-457
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Ochiai E Miura D Eguchi H Ohara S Takenouchi K Azuma Y Kamimura T Norman AW Ishizuka S 《Molecular endocrinology (Baltimore, Md.)》2005,19(5):1147-1157
We reported that (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) antagonizes vitamin D receptor (VDR)-mediated genomic actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] in human cells but is agonistic in rodent cells. Human and rat VDR ligand-binding domains are similar, but differences in the C-terminal region are important for ligand binding and transactivation and might determine the agonistic/antagonistic effects of TEI-9647. We tested TEI-9647 on 1alpha,25(OH)(2)D(3) transactivation using SaOS-2 cells (human osteosarcoma) or ROS 24/1 cells (rat osteosarcoma) cotransfected with human or rodent VDR and a reporter. In both cell lines, TEI-9647 was antagonistic with wild-type human (h)VDR, but agonistic with overexpressed wild-type rat (r)VDR. VDR chimeras substituting the hVDR C-terminal region (activation function 2 domain) with corresponding rVDR residues diminished antagonism and increased agonism of TEI-9647. However, substitution of 25 C-terminal rVDR residues with corresponding hVDR residues diminished agonism and increased antagonism of TEI-9647. hVDR mutants (C403S, C410N) demonstrated that Cys403 and/or 410 was necessary for TEI-9647 antagonism of 1alpha,25(OH)(2)D(3) transactivation. These results suggest that species specificity of VDR, especially in the C-terminal region, determines the agonistic/antagonistic effects of TEI-9647 that determine, in part, VDR interactions with coactivators and emphasize the critical interaction between TEI-9647 and the two C-terminal hVDR Cys residues to mediate the antagonistic effect of TEI-9647. 相似文献
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Jurutka PW Remus LS Whitfield GK Galligan MA Haussler CA Haussler MR 《Biochemical and biophysical research communications》2000,267(3):813-819
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. 相似文献
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B B Jones P W Jurutka C A Haussler M R Haussler G K Whitfield 《Molecular endocrinology (Baltimore, Md.)》1991,5(8):1137-1146
The human 1,25-dihydroxyvitamin D3 receptor (hVDR) has been recently shown to be phosphorylated in vitro by casein kinase-II. Most of the residues phosphorylated by this enzyme were shown to reside between Asn160 and Asp232, a region near the N-terminal boundary of the hormone-binding domain. We report here that the hVDR is also phosphorylated in vivo after transfection into ROS 17/2.8 cells. In addition to testing full-length hVDR, we analyzed several internally deleted hVDR mutants. The expression and phosphorylation of full-length and mutated hVDRs were monitored in transfected cells by metabolic labeling with either [35S]methionine or [32P]orthophosphate, followed by immunopurification using monoclonal anti-VDR antibody linked to agarose beads. Transfected hVDR is distinguishable from the endogenous rat VDR when the immunoprecipitated proteins are resolved on sodium dodecyl sulfate-polyacrylamide gels. Significant phosphorylation of transfected full-length hVDR was observed in ROS 17/2.8 cells, and it was less dependent on the presence of 1,25-dihydroxyvitamin D3 than that of the endogenous rat receptor. Most importantly, the region of in vivo phosphorylation, as defined by internal deletion mutants, resides between Met197 and Val234. Therefore, we have localized the major site of phosphorylation of hVDR to residues in the N-terminal region of the hormone-binding domain. The boundaries of this region fall within the amino acid segment defined for phosphorylation of hVDR by casein kinase-II in vitro, suggesting that VDR is an in vivo substrate for casein kinase-II or a related protein kinase. 相似文献
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Nicholas J. Koszewski Timothy A. Reinhardt Ronald L. Horst 《The Journal of steroid biochemistry and molecular biology》1996,59(5-6):377-388
The nature of the DNA binding interactions of the human vitamin D receptor (hVDR) with the murine osteopontin vitamin D response element (mOP VDRE) was examined. Both recombinant hVDR and human retinoid X receptor β (hRXRβ) proteins were obtained from baculovirus-infected Sf9 insect cells. Mixing extracts of the two recombinant proteins resulted in the strong, specific formation of a slower migrating complex in the electrophoretic mobility shift assay. Crude extracts of the expressed hVDR alone were also capable of binding with high affinity to the mOP sequence, and this binding was enhanced in the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Competition experiments confirmed the specificity of this interaction and revealed that the human osteocalcin VDRE was a poor competitor for this binding. Ethylation interference footprint analyses of hVDR/hRXRβ and hVDR complexes revealed only subtle differences in how these two different VDR-containing complexes interacted with the mOP VDRE. The footprints displayed contact points in both halves of the direct repeat format, confirming the dimeric and major groove interactions of both types of complexes. DNA affinity chromatography of labelled hVDR extracts revealed a peak eluting at ca. 290 mM KCl that was capable of rebinding to the mOP sequence in gel shift experiments. Ultraviolet (UV) light-crosslinking experiments of hVDR extracts alone to radiolabelled DNA were consistent with the existence of a homodimeric hVDR interaction. Additionally, these experiments confirmed the direct interaction of a hVDR/hRXRβ heterodimer when mixed extracts were utilized. From these results we infer that homodimers of the hVDR which respond with enhanced DNA binding to particular vitamin D response elements when exposed to 1,25-(OH)2D3 are possible. This may be of functional significance when RXR proteins are limiting or RXR ligand is present within a cell. 相似文献
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The polymorphic N terminus in human vitamin D receptor isoforms influences transcriptional activity by modulating interaction with transcription factor IIB 总被引:10,自引:0,他引:10
Jurutka PW Remus LS Whitfield GK Thompson PD Hsieh JC Zitzer H Tavakkoli P Galligan MA Dang HT Haussler CA Haussler MR 《Molecular endocrinology (Baltimore, Md.)》2000,14(3):401-420
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Hsieh JC Dang HT Galligan MA Whitfield GK Haussler CA Jurutka PW Haussler MR 《Biochemical and biophysical research communications》2004,324(2):801-809
The human vitamin D receptor (hVDR), which is a substrate for several protein kinases, mediates the actions of its 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ligand to regulate gene expression. To determine the site, and functional impact, of cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of hVDR, we generated a series of C-terminally truncated and point mutant receptors. Incubation of mutant hVDRs with PKA and [gamma-32P]ATP, in vitro, or overexpressing them in COS-7 kidney cells labeled with [32P]orthophosphate, revealed that serine-182 is the predominant residue in hVDR phosphorylated by PKA. An aspartate substituted mutant (S182D), incorporating a negative charge to mimic phosphorylation, displayed only 50% of the transactivation capacity in response to 1,25(OH)2D3 of either wild-type or an S182A-altered hVDR. When the catalytic subunit of PKA was overexpressed, a similar reduction in wild-type but not S182D hVDR transactivity was observed. In a mammalian two-hybrid system, S182D bound less avidly than wild-type or S182A hVDR to the retinoid X receptor (RXR) heterodimeric partner that co-mediates vitamin D responsive element recognition and transactivation. These data suggest that hVDR serine-182 is a primary site for PKA phosphorylation, an event that leads to an attenuation of both RXR heterodimerization and resultant transactivation of 1,25(OH)2D3 target genes. 相似文献
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F Alimirah X Peng L Yuan RR Mehta A von Knethen D Choubey RG Mehta 《Experimental cell research》2012,318(19):2490-2497
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Hsieh JC Whitfield GK Oza AK Dang HT Price JN Galligan MA Jurutka PW Thompson PD Haussler CA Haussler MR 《Biochemistry》1999,38(49):16347-16358
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Qi X Pramanik R Wang J Schultz RM Maitra RK Han J DeLuca HF Chen G 《The Journal of biological chemistry》2002,277(29):25884-25892
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Comparative modeling of the vitamin D receptor three-dimensional structure and computational docking of 1alpha,25-dihydroxyvitamin D(3) into the putative binding pocket of the two deletion mutant receptors: (207-423) and (120-422, Delta [164-207]) are reported and evaluated in the context of extensive mutagenic analysis and crystal structure of holo hVDR deletion protein published recently. The obtained molecular model agrees well with the experimentally determined structure. Six different conformers of 1alpha,25-dihydroxyvitamin D(3) were used to study flexible docking to the receptor. On the basis of values of conformational energy of various complexes and their consistency with functional activity, it appears that 1alpha,25-dihydroxyvitamin D(3) binds the receptor in its 6-s-trans form. The two lowest energy complexes obtained from docking the hormone into the deletion protein (207-423) differ in conformation of ring A and orientation of the ligand molecule in the VDR pocket. 1alpha,25-Dihydroxyvitamin D(3) possessing the A-ring conformation with axially oriented 1alpha-hydroxy group binds receptor with its 25-hydroxy substituent oriented toward the center of the receptor cavity, whereas ligand possessing equatorial conformation of 1alpha-hydroxy enters the pocket with A ring directed inward. The latter conformation and orientation of the ligand is consistent with the crystal structure of hVDR deletion mutant (118-425, Delta [165-215]). The lattice model of rVDR (120-422, Delta [164-207]) shows excellent agreement with the crystal structure of the hVDR mutant. The complex obtained from docking the hormone into the receptor has lower energy than complexes for which homology modeling was used. Thus, a simple model of vitamin D receptor with the first two helices deleted can be potentially useful for designing a general structure of ligand, whereas the advanced lattice model is suitable for examining binding sites in the pocket. 相似文献
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The interaction of the vitamin D receptor with a vitamin D-responsive element (VDRE) derived from the human osteocalcin promoter in vitro has been shown to require a nuclear accessory factor (NAF) derived from monkey kidney cells. In this report we show that this factor is widely distributed in cells and tissues, including those that do not express the vitamin D receptor (VDR). NAF is required for VDR binding to a variety of known VDREs. VDR and NAF independently bind the VDRE weakly, as assessed by elution profiles generated during VDRE affinity chromatography. Together, however, both proteins coelute from this column with a profile that indicates a tighter strength of interaction. Analogous chromatography of the VDR derived from ROS 17/2.8 cells treated with 1,25-dihydroxyvitamin D3 in culture also reveals a dual profile of weak and strong binding, suggesting that in vivo modifications are unlikely to alter receptor DNA binding. NAF is a protein of 55 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and cross-linking experiments suggest that the VDR and NAF together form a heterodimer on a single VDRE with a mol wt of 103 kDa. These data demonstrate that NAF is required for VDR binding to specific DNA in vitro and suggest the possibility that NAF may be required for the transactivation capability of the VDR in vivo. 相似文献
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Adachi R Honma Y Masuno H Kawana K Shimomura I Yamada S Makishima M 《Journal of lipid research》2005,46(1):46-57
The vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the biological actions of the active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3). It regulates calcium homeostasis, immunity, cellular differentiation, and other physiological processes. Recently, VDR was found to respond to bile acids as well as other nuclear receptors, farnesoid X receptor (FXR) and pregnane X receptor (PXR). The toxic bile acid lithocholic acid (LCA) induces its metabolism through VDR interaction. To elucidate the structure-function relationship between VDR and bile acids, we examined the effect of several LCA derivatives on VDR activation and identified compounds with more potent activity than LCA. LCA acetate is the most potent of these VDR agonists. It binds directly to VDR and activates the receptor with 30 times the potency of LCA and has no or minimal activity on FXR and PXR. LCA acetate effectively induced the expression of VDR target genes in intestinal cells. Unlike LCA, LCA acetate inhibited the proliferation of human monoblastic leukemia cells and induced their monocytic differentiation. We propose a docking model for LCA acetate binding to VDR. The development of VDR agonists derived from bile acids should be useful to elucidate ligand-selective VDR functions. 相似文献
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The vitamin D receptor (VDR) is a member of the steroid/retinoid receptor superfamily of nuclear receptors that has potential tumor-suppressive functions. We show here that VDR interacts with and is regulated by BAG1L, a nuclear protein that binds heat shock 70-kDa (Hsp70) family molecular chaperones. Endogenous BAG1L can be co-immunoprecipitated with VDR from prostate cancer cells (ALVA31; LNCaP) in a ligand-dependent manner. BAG1L, but not shorter non-nuclear isoforms of this protein (BAG1; BAG1M/Rap46), markedly enhanced, in a ligand-dependent manner, the ability of VDR to trans-activate reporter gene plasmids containing a vitamin D response element in transient transfection assays. Mutant BAG1L lacking the C-terminal Hsc70-binding domain suppressed (in a concentration-dependent fashion) VDR-mediated trans-activation of vitamin D response element-containing reporter gene plasmids, without altering levels of VDR or endogenous BAG1L protein, suggesting that it operates as a trans-dominant inhibitor of BAG1L. Gene transfer-mediated elevations in BAG1L protein levels in a prostate cancer cell line (PC3), which is moderately responsive to VDR ligands, increased the ability of natural (1alpha,25(OH)(2) vitamin D(3)) and synthetic (1alpha, 25-dihydroxy-19-nor-22(E)-vitamin D(3)) VDR ligands to induce expression of the VDR target gene, p21(Waf1), and suppress DNA synthesis. Thus, BAG1L is a direct regulator of VDR, which enhances its trans-activation function and improves tumor cell responses to growth-suppressive VDR ligands. 相似文献
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Welsh J 《Archives of biochemistry and biophysics》2012,523(1):107-114
Epidemiologic data suggest that the incidence and severity of many types of cancer inversely correlates with indices of vitamin D status. The vitamin D receptor (VDR) is highly expressed in epithelial cells at risk for carcinogenesis including those resident in skin, breast, prostate and colon, providing a direct molecular link by which vitamin D status impacts on carcinogenesis. Consistent with this concept, activation of VDR by its ligand 1,25-dihydroxyvitamin D (1,25D) triggers comprehensive genomic changes in epithelial cells that contribute to maintenance of the differentiated phenotype, resistance to cellular stresses and protection of the genome. Many epithelial cells also express the vitamin D metabolizing enzyme CYP27B1 which enables autocrine generation of 1,25D from the circulating vitamin D metabolite 25-hydroxyvitamin D (25D), critically linking overall vitamin D status with cellular anti-tumor actions. Furthermore, pre-clinical studies in animal models has demonstrated that dietary supplementation with vitamin D or chronic treatment with VDR agonists decreases tumor development in skin, colon, prostate and breast. Conversely, deletion of the VDR gene in mice alters the balance between proliferation and apoptosis, increases oxidative DNA damage, and enhances susceptibility to carcinogenesis in these tissues. Because VDR expression is retained in many human tumors, vitamin D status may be an important modulator of cancer progression in persons living with cancer. Collectively, these observations have reinforced the need to further define the molecular actions of the VDR and the human requirement for vitamin D in relation to cancer development and progression. 相似文献