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. 1996 Nov 15;24(22):4513–4518. doi: 10.1093/nar/24.22.4513

The high affinity ligand binding conformation of the nuclear 1,25-dihydroxyvitamin D3 receptor is functionally linked to the transactivation domain 2 (AF-2).

S Nayeri 1, J P Kahlen 1, C Carlberg 1
PMCID: PMC146265  PMID: 8948643

Abstract

The nuclear receptor for 1,25-dihydroxyvitamin D3 (VD), VDR, is a transcription factor that mediates all genomic actions of the hormone. The activation of VDR by ligand induces a conformational change within its ligand binding domain (LBD). Due to the lack of a crystal structure analysis, biochemical methods have to be applied in order to investigate the details of this receptor-ligand interaction. The limited protease digestion assay can be used as a tool for the determination of a functional dissociation constant (K(df)) of VDR with any potential ligand. This method provided with the natural hormone VD two protease-resistant fragments of the VDR LBD and with the 20-epi conformation of VD, known as MC1288, even an additional fragment of intermediate size. These fragments were interpreted as different receptor conformations and their decreasing size was found to be associated with decreasing ligand binding affinity. A critical amino acid for VDR's high ligand binding conformation has been identified by C-terminal receptor truncations and point mutations as phenylalanine 422. This amino acid appears to directly contact the ligand and belongs to the ligand-inducible activation function-2 (AF-2) domain. Moreover, functional assays supported the observation that high affinity ligand binding is directly linked to transactivation function.

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Selected References

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  1. Allan G. F., Leng X., Tsai S. Y., Weigel N. L., Edwards D. P., Tsai M. J., O'Malley B. W. Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation. J Biol Chem. 1992 Sep 25;267(27):19513–19520. [PubMed] [Google Scholar]
  2. Bourguet W., Ruff M., Chambon P., Gronemeyer H., Moras D. Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-alpha. Nature. 1995 Jun 1;375(6530):377–382. doi: 10.1038/375377a0. [DOI] [PubMed] [Google Scholar]
  3. Breen E. C., van Wijnen A. J., Lian J. B., Stein G. S., Stein J. L. In vivo occupancy of the vitamin D responsive element in the osteocalcin gene supports vitamin D-dependent transcriptional upregulation in intact cells. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12902–12906. doi: 10.1073/pnas.91.26.12902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carlberg C., Bendik I., Wyss A., Meier E., Sturzenbecker L. J., Grippo J. F., Hunziker W. Two nuclear signalling pathways for vitamin D. Nature. 1993 Feb 18;361(6413):657–660. doi: 10.1038/361657a0. [DOI] [PubMed] [Google Scholar]
  5. Carlberg C. Mechanisms of nuclear signalling by vitamin D3. Interplay with retinoid and thyroid hormone signalling. Eur J Biochem. 1995 Aug 1;231(3):517–527. [PubMed] [Google Scholar]
  6. Cavaillès V., Dauvois S., L'Horset F., Lopez G., Hoare S., Kushner P. J., Parker M. G. Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor. EMBO J. 1995 Aug 1;14(15):3741–3751. doi: 10.1002/j.1460-2075.1995.tb00044.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Danielian P. S., White R., Lees J. A., Parker M. G. Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J. 1992 Mar;11(3):1025–1033. doi: 10.1002/j.1460-2075.1992.tb05141.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Durand B., Saunders M., Gaudon C., Roy B., Losson R., Chambon P. Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity. EMBO J. 1994 Nov 15;13(22):5370–5382. doi: 10.1002/j.1460-2075.1994.tb06872.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kahlen J. P., Carlberg C. Functional characterization of a 1,25-dihydroxyvitamin D3 receptor binding site found in the rat atrial natriuretic factor promoter. Biochem Biophys Res Commun. 1996 Jan 26;218(3):882–886. doi: 10.1006/bbrc.1996.0157. [DOI] [PubMed] [Google Scholar]
  10. Keidel S., LeMotte P., Apfel C. Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping. Mol Cell Biol. 1994 Jan;14(1):287–298. doi: 10.1128/mcb.14.1.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Le Douarin B., Zechel C., Garnier J. M., Lutz Y., Tora L., Pierrat P., Heery D., Gronemeyer H., Chambon P., Losson R. The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18. EMBO J. 1995 May 1;14(9):2020–2033. doi: 10.1002/j.1460-2075.1995.tb07194.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Leid M. Ligand-induced alteration of the protease sensitivity of retinoid X receptor alpha. J Biol Chem. 1994 May 13;269(19):14175–14181. [PubMed] [Google Scholar]
  13. Leng X., Tsai S. Y., O'Malley B. W., Tsai M. J. Ligand-dependent conformational changes in thyroid hormone and retinoic acid receptors are potentially enhanced by heterodimerization with retinoic X receptor. J Steroid Biochem Mol Biol. 1993 Dec;46(6):643–661. doi: 10.1016/0960-0760(93)90306-h. [DOI] [PubMed] [Google Scholar]
  14. Luckow B., Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. doi: 10.1093/nar/15.13.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mangelsdorf D. J., Thummel C., Beato M., Herrlich P., Schütz G., Umesono K., Blumberg B., Kastner P., Mark M., Chambon P. The nuclear receptor superfamily: the second decade. Cell. 1995 Dec 15;83(6):835–839. doi: 10.1016/0092-8674(95)90199-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nayeri S., Danielsson C., Kahlen J. P., Schräder M., Mathiasen I. S., Binderup L., Carlberg C. The anti-proliferative effect of vitamin D3 analogues is not mediated by inhibition of the AP-1 pathway, but may be related to promoter selectivity. Oncogene. 1995 Nov 2;11(9):1853–1858. [PubMed] [Google Scholar]
  17. Nayeri S., Mathiasen I. S., Binderup L., Carlberg C. High-affinity nuclear receptor binding of 20-epi analogues of 1,25-dihydroxyvitamin D3 correlates well with gene activation. J Cell Biochem. 1996 Sep 1;62(3):325–333. doi: 10.1002/(SICI)1097-4644(199609)62:3%3C325::AID-JCB3%3E3.0.CO;2-T. [DOI] [PubMed] [Google Scholar]
  18. Peleg S., Sastry M., Collins E. D., Bishop J. E., Norman A. W. Distinct conformational changes induced by 20-epi analogues of 1 alpha,25-dihydroxyvitamin D3 are associated with enhanced activation of the vitamin D receptor. J Biol Chem. 1995 May 5;270(18):10551–10558. doi: 10.1074/jbc.270.18.10551. [DOI] [PubMed] [Google Scholar]
  19. Pothier F., Ouellet M., Julien J. P., Guérin S. L. An improved CAT assay for promoter analysis in either transgenic mice or tissue culture cells. DNA Cell Biol. 1992 Jan-Feb;11(1):83–90. doi: 10.1089/dna.1992.11.83. [DOI] [PubMed] [Google Scholar]
  20. Renaud J. P., Rochel N., Ruff M., Vivat V., Chambon P., Gronemeyer H., Moras D. Crystal structure of the RAR-gamma ligand-binding domain bound to all-trans retinoic acid. Nature. 1995 Dec 14;378(6558):681–689. doi: 10.1038/378681a0. [DOI] [PubMed] [Google Scholar]
  21. Schräder M., Müller K. M., Carlberg C. Specificity and flexibility of vitamin D signaling. Modulation of the activation of natural vitamin D response elements by thyroid hormone. J Biol Chem. 1994 Feb 25;269(8):5501–5504. [PubMed] [Google Scholar]
  22. Tate B. F., Allenby G., Janocha R., Kazmer S., Speck J., Sturzenbecker L. J., Abarzúa P., Levin A. A., Grippo J. F. Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha. Mol Cell Biol. 1994 Apr;14(4):2323–2330. doi: 10.1128/mcb.14.4.2323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tate B. F., Grippo J. F. Mutagenesis of the ligand binding domain of the human retinoic acid receptor alpha identifies critical residues for 9-cis-retinoic acid binding. J Biol Chem. 1995 Sep 1;270(35):20258–20263. doi: 10.1074/jbc.270.35.20258. [DOI] [PubMed] [Google Scholar]
  24. Wagner R. L., Apriletti J. W., McGrath M. E., West B. L., Baxter J. D., Fletterick R. J. A structural role for hormone in the thyroid hormone receptor. Nature. 1995 Dec 14;378(6558):690–697. doi: 10.1038/378690a0. [DOI] [PubMed] [Google Scholar]
  25. Walters M. R. Newly identified actions of the vitamin D endocrine system. Endocr Rev. 1992 Nov;13(4):719–764. doi: 10.1210/edrv-13-4-719. [DOI] [PubMed] [Google Scholar]
  26. Yu V. C., När A. M., Rosenfeld M. G. Transcriptional regulation by the nuclear receptor superfamily. Curr Opin Biotechnol. 1992 Dec;3(6):597–602. doi: 10.1016/0958-1669(92)90002-z. [DOI] [PubMed] [Google Scholar]
  27. vom Baur E., Zechel C., Heery D., Heine M. J., Garnier J. M., Vivat V., Le Douarin B., Gronemeyer H., Chambon P., Losson R. Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1. EMBO J. 1996 Jan 2;15(1):110–124. [PMC free article] [PubMed] [Google Scholar]

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