Characterization of transcriptional activation and DNA-binding functions in the hinge region of the vitamin D receptor.
The vitamin D receptor (VDR) is a ligand-responsive transcription factor that forms active, heterodimeric complexes with the 9-cis retinoic acid receptor (RXR) on vitamin D response elements (VDREs). Both proteins consist of an N-terminal DNA-binding domain, a C-terminal ligand-binding domain, and an intervening hinge region. The length requirements of the hinge for both transcriptional regulation and DNA binding have not been studied to date for any member of the steroid hormone superfamily. We have generated a series of internal deletion mutants of the VDR hinge and found that deletion of as few as five amino acids from the C-terminus of the hinge significantly reduces transcriptional activation in vivo. Replacing deleted residues in the C-terminus of the hinge with alanines restored activity, indicating that this section of the hinge acts as a sequence-independent spacer. The hinge region of VDR forms a long helix, and the geometric consequences of this structure may explain the requirement of the hinge region for transcriptional activity. Interestingly, all of the deletion mutants, even those that do not activate transcription, bind VDREs with equal and high affinity, indicating that the defect in these mutants is not their ability to bind VDREs. In contrast to VDR, constructs of RXR containing deletions of up to 14 amino acids in the hinge region exhibit near wild-type transcriptional activity. The ability to delete more of the RXR hinge may be related to the additional plasticity required by its role as the common heterodimer partner for nuclear receptors on differing DNA elements.
Shaffer
Paul L
PL
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
McDonnell
Donald P
DP
Gewirth
Daniel T
DT
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D018167:Receptors, Calcitriol / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D047490:Retinoid X Receptor alpha / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017384:Sequence Deletion / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D014162:Transfection
D004268:DNA-Binding Proteins; D018167:Receptors, Calcitriol; D047490:Retinoid X Receptor alpha; D015534:Trans-Activators