Functional domains of the human vitamin D3 receptor regulate osteocalcin gene expression.
The human vitamin D receptor (VDR) has been cloned recently. Two cDNAs comprising the full-length VDR were spliced, cloned into a mammalian expression vector, and transiently expressed in COS-1 cells. The protein product exhibited properties consistent with that observed for receptor in human cells. A series of 5'- and 3'-deletions of the full-length VDR cDNA was prepared and evaluated. Native DNA binding was localized to a peptide fragment (residues 1-114) whose most prominent feature is the cysteine rich region proven to represent the DNA binding domain in other steroid receptors. Steroid binding-competence required synthesis of a peptide that initiated C-terminal to the DNA-binding domain at residue 114 and which contained the remaining 313 residues. To determine the location of elements within the receptor necessary for transcription, an osteocalcin gene promoter-chloramphenicol acetyltransferase reporter gene was cotransfected together with wild type or mutant VDR cDNAs and the latter's effect on chloramphenicol acetyltransferase activity was assessed. Cotransfection of wild type receptor alone resulted in efficient transcription of the reporter plasmid. However, synthesis of a peptide containing the DNA binding domain as well as 76 residues carboxy terminal to this region exhibited some degree of activity, albeit constitutive. These results suggest that the functional domains of the VDR are similar to that of other steroid receptors and that these domains participate in the transcriptional regulation of the human osteocalcin gene.
McDonnell
D P
DP
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Scott
R A
RA
Kerner
S A
SA
O'Malley
B W
BW
Pike
J W
JW
D000818:Animals; D015153:Blotting, Western; D002117:Calcitriol / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002478:Cells, Cultured; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D009154:Mutation; D015675:Osteocalcin; D010957:Plasmids; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D002135:Calcium-Binding Proteins; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D015675:Osteocalcin; D004247:DNA; D002117:Calcitriol