Previous investigations have demonstrated that besides the intestine at least 20 tissues including kidney, pancreas, skin and brain all have receptors for 1,25dihydroxyvitamin D3 (1,25(OH)2D3) and/or a vitamin D induced calcium binding protein (CaBP), thus suggesting a wider role for vitamin D in calcium metabolism than merely intestinal calcium absorption. The object of this proposal is to obtain a better understanding of the multiple actions of the vitamin D endocrine system by studying the function and regulation of the mammalian 28,000 Mr vitamin D dependent CaBP. Two working hypotheses will be tested: (1) that CaBP may have an important fundamental role in mediating intracellular calcium dependent processes and (2) that factors other than or in addition to 1,25(OH)2D3 may modulate CaBP mRNA and the subsequent synthesis of the protein. In an effort to establish the functional significance of CaBP, provocative preliminary results concerning a dose-dependent inhibition by CaBP of Ca++ dependent protein kinase will be pursued. In order to examine the possibility that CaBP may participate in synaptic events, we will extend our preliminary findings concerning the presence of CaBP in synaptosomes and we will test the ability of CaBP to activate Ca-Mg ATPase. We will use the gel overlay technique in order to determine whether specific binding proteins for CaBP can be detected and identified. In addition, using the DNA probe complementary to CaBP mRNA and recombinant DNA techniques, we will study the regulation of mammalian CaBP both in vivo and in vitro. Changes in CaBP gene expression during development will be examined and studies concerning the regulation of human CaBP will be initiated. The nucleotide sequence of CaBP will be determined and studies concerning the regulation of CaBP gene expression using transfected cells will be initiated. It is likely that important advances to the understanding of vitamin D regulated calcium homeostasis can be made by examining molecular level changes. Implicit in this study is facilitation of a more detailed understanding of how 1,25(OH)2D3 is involved in the many disease processes involved with abnormalities in the calcium homeostatic process such as osteomalacia, osteoporosis and perturbations of parathyroid function. Additionally, this study may lead to an increased understanding of the role of the vitamin D endocrine system in normal brain function as well as in brain disorder involving calcium dependent functions.
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