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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
- Source :
- Biochemistry. 38(49)
- Publication Year :
- 1999
-
Abstract
- 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.
- Subjects :
- Molecular Sequence Data
Biochemistry
Calcitriol receptor
chemistry.chemical_compound
Humans
Point Mutation
Amino Acid Sequence
Conserved Sequence
Sequence Deletion
Zinc finger
General transcription factor
Chemistry
Zinc Fingers
Zinc finger nuclease
Peptide Fragments
Cell biology
VDRE
Protein Structure, Tertiary
RING finger domain
DNA-Binding Proteins
Amino Acid Substitution
Mutagenesis, Site-Directed
Trans-Activators
Receptors, Calcitriol
Dimerization
DNA
Binding domain
Subjects
Details
- ISSN :
- 00062960
- Volume :
- 38
- Issue :
- 49
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....3ad10c16a927e287255733209c35db06