• Whitfield, G Kerr (PI)

Project: Research project

Grant Details


The main objective of the proposed research project is to gain further
information and insight into the mode of action of the vitamin D receptor
(VDR). This is to be approached by developing a model system consisting
of a) the VDR cDNA cloned into a vector which will allow for the
expression of normal or mutagenized receptor in transfected cells, b) the
cloned 5' region of the calcium-binding protein (CaBP) gene, a gene known
to be induced at the level of transcription by the VDR, and c) recipient
cell lines which are VDR-deficient, such as the ROS 24/1 line. This
system, a large portion of which is already available, will permit
extensive testing of the functional domains of the VDR, including those
for hormone-binding, DNA binding, and transcriptional activation. The regions of the CaBP gene which interact with the VDR will be
delineated, first by testing partially deleted versions of the CaBP gene,
and then by more direct methods, such as DNase I footprinting. Next,
those regions of the VDR necessary for hormone binding and for VDR
binding to the site(s) on the CaBP gene, already known in broad outline
from earlier studies, will be precisely determined by partial deletion of
the VDR cDNA, and then by site-directed mutagenesis of specific amino
acid codons. Finally, this approach should permit the identification of
the region(s) of the VDR responsible for transcriptional activation of
CaBP. The functional domains of the VDR can ultimately be tested by
inserting them into heterologous proteins, such as the glucocorticoid
receptor, to see if they are necessary and sufficient for their function. The availability of fibroblasts from a patient with vitamin-D dependent
rickets type II will be exploited as a means of analyzing an in vivo VDR-
deficient mutant. It will be seen whether transfection of a human VDR
cDNA into these cells can restore their responsiveness to the vitamin D
hormone, as indicated by an induction of 24-hydroxylase. If restoration
is achieved, confirming that the VDR is the site of defect, then a
project will be initiated to determine the exact nature of the lesion in
the VDR from these cells. A characterization of the mode of action of the
VDR, a member of the family of steroid and thyroid hormone receptors, is
significant to basic biology and should enhance our understanding of
tissue-specific gene regulation.
Effective start/end date7/1/8912/31/94


  • National Institutes of Health


  • Medicine(all)


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