Response Element and Coactivator-Mediated Conformational Change of the Vitamin D3 Receptor Permits Sensitive Interaction with Agonists
- Michaela Herdick1,
- Yvonne Bury1,
- Marcus Quack1,
- Milan R. Uskokovic2,
- Patsie Polly1 and
- Carsten Carlberg1
- 1Institut für Physiologische Chemie I and Biomedizinisches Forschungszentrum, Heinrich-Heine-Universität, Düsseldorf, Germany (M.H., Y.B., M.Q., P.P., C.C.); and 2Hoffmann-La Roche Inc., Nutley, New Jersey (M.R.U.)
Abstract
The vitamin D receptor (VDR) is the nuclear receptor for 1,25-dihydroxyvitamin D3[1α,25(OH)2D3] that acts as a ligand-dependent transcription factor via combined contact with coactivator proteins (steroid receptor coactivator-1, transcriptional intermediary factor 2, and receptor associated coactivator 3) and specific DNA binding sites [vitamin D response elements (VDREs)]. Ligand-mediated conformational changes of the VDR contribute to the key mechanisms in this nuclear hormone signaling process. 1α,25(OH)2D3, MC1288 [20-epi-1α,25(OH)2D3], ZK161422 [20-methyl-1α,25(OH)2D3], and Ro27–2310 (also called Gemini, having two side chains at carbon 20) were used as model VDR agonists. The analysis of agonist-induced VDR conformations and coactivator interactions were found to be insufficient for extrapolating in vivo activities. In DNA-independent assays, such as classical limited protease digestions and glutathioneS-transferase pull downs, Gemini seemed to be up to 10,000-fold and the other VDR agonists 10- to 100-fold weaker than in functional in vivo assays. A more accurate description of the gene regulatory potential of VDR agonists was obtained with all tested VDR agonists by analyzing VDR conformations in the context of VDRE-bound VDR-retinoid X receptor heterodimers, in such assays as gel supershift, gel shift clipping, and limited protease digestion in the presence of DNA and cofactor. Coactivators were found to shift the ligand sensitivity (by a factor of 4 for Gemini) and the ratio of VDR conformations in the presence of DNA toward the high-affinity ligand binding conformation (c1LPD). In conclusion, the induction of response element- and coactivator-modulated VDR conformations appears to be a key step for the gene regulatory function of a VDR agonist. The quantification of these effects would be of central importance for the evaluation of the cell-specific efficacy of systemically applied 1α,25(OH)2D3 analogs.
Footnotes
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Send reprint requests to: Dr. Carsten Carlberg, Institut für Physiologische Chemie I, Heinrich-Heine-Universität, Düsseldorf, Postfach 10 10 07, D-40001 Düsseldorf, Germany. E-mail: carlberg{at}uni-duesseldorf.de
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This work was supported by the Sonderforschungsbereich 503, project A6, the Medical Faculty of the Heinrich-Heine University, Düsseldorf, and the Fonds der Chemischen Industrie (all to C.C.). P.P. is the recipient of a fellowship from the Alexander von Humboldt Foundation. M.H. and Y.B. contributed equally to this study.
- Abbreviations:
- 1α,25(OH)2D3
- 1α,25-dihydroxyvitamin D3
- VDR
- 1α,25-dihydroxyvitamin D3 receptor
- VDRE
- 1α,25-dihydroxyvitamin D3response element
- DR3
- direct repeat spaced by 3 nucleotides
- IP9
- inverted palindrome spaced by nine nucleotides
- DBD
- DNA binding domain
- aa
- amino acids
- LBD
- ligand binding domain
- SRC-1
- steroid receptor coactivator-1
- TIF2
- transcriptional intermediary factor 2
- RAC3
- receptor associated coactivator 3
- RXR
- retinoid X receptor
- PCR
- polymerase chain reaction
- GST
- glutathioneS-transferase
- ANF
- atrial natriuretic factor
- DMEM
- Dulbecco's modified Eagle's medium
- LPD
- limited protease digestion
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- Received November 29, 1999.
- Accepted February 8, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
