STRUCTURE/FUNCTION STUDIES OF THE VITAMIN D RECEPTOR

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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.