Identification of an autonomous transactivation domain in helix H3 of the vitamin D receptor

The vitamin D receptor (VDR) contains an α‐helical, ligand‐inducible activation function (AF‐2) at the COOH‐terminus of the ligand‐binding domain (LBD). In this study, a second distinct activation domain was identified in the VDR LBD. Using a yeast‐based system to screen a random mutant library of G...

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Bibliographic Details
Published in:	Journal of cellular biochemistry Vol. 75; no. 1; pp. 82 - 92
Main Authors:	Kraichely, Dennis M., Nakai, Yuichiro D., MacDonald, Paul N.
Format:	Journal Article
Language:	English
Published:	New York John Wiley & Sons, Inc 01-10-1999
Subjects:	1,25‐(OH)2D3 25-(OH)2D3 AF-2 domain Calcitriol - pharmacology DNA Mutational Analysis DNA-Binding Proteins Fungal Proteins - genetics helix H3 Humans Mutagenesis, Site-Directed Protein Structure, Secondary Receptors, Calcitriol - chemistry Receptors, Calcitriol - genetics Recombinant Fusion Proteins - genetics Saccharomyces cerevisiae Proteins Sequence Deletion transactivation transcription Transcription Factors - genetics Transcription, Genetic Transcriptional Activation - genetics Transfection VDR Yeasts
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Summary:	The vitamin D receptor (VDR) contains an α‐helical, ligand‐inducible activation function (AF‐2) at the COOH‐terminus of the ligand‐binding domain (LBD). In this study, a second distinct activation domain was identified in the VDR LBD. Using a yeast‐based system to screen a random mutant library of GAL4‐VDR (93–427), a mutant GAL4‐VDR fusion protein with constitutive transcriptional activity was isolated. Sequence analysis identified a C to T transition that introduced a stop codon at glutamine 239 eliminating a large portion of the LBD, including the AF‐2 domain. The GAL4‐VDR (93–238) mutant exhibited ligand‐independent transactivation activity both in yeast and in mammalian cells. Deletion analysis defined a minimal activation domain within helix H3 between D195 and I 238 in the VDR. An aspartic acid residue (D232) within helix H3 was essential for the autonomous transactivation activity since altering this residue to an alanine or an asparagine dramatically reduced its transactivation potential. Expression of the minimal helix H3 activation domain interfered with ligand‐activated transcription by full‐length VDR suggesting that helix H3 interacts with limiting cellular factors important for VDR‐activated transcription. Consequently, we have identified a novel activation domain in helix H3 of the VDR that apparently plays an important role in 1,25‐(OH)2D3‐activated transcription. J. Cell. Biochem. 75:82–92, 1999. © 1999 Wiley‐Liss, Inc.
Bibliography:	National Institutes of Health - No. R01DK50348; No. T32-GM08306 istex:74C0F928EF5D24993985597B4ADF9D9B5AA9FAE9 ark:/67375/WNG-7883XFQ1-5 ArticleID:JCB9
ISSN:	0730-2312 1097-4644
DOI:	10.1002/(SICI)1097-4644(19991001)75:1<82::AID-JCB9>3.0.CO;2-J