There are important links between the biochemical regulators, calcium (Ca++) and calcium binding proteins (CaBP's), and the vitamin D endocrine system. However, the precise functions of many of the CaBP's are unknown, as are details of the mechanism(s) by which 1,25(OH)2D participates in their regulation. The overall goal of this proposal is to understand two aspects of these complex and interrelated problems: (a) the functions and characteristics of 1,25(OH)2D-induced calmodulin (CaM)-acceptor proteins (185 KD and 115 KD) which we recently identified by the 125-I-CaM gel overlay procedure in the rat kidney cytosol, and (b) whether the functions of the vitamin D-related calbindin-D28K (CaBP-D28K) also involve specific acceptor proteins. Whether 1,25(OH)2D induction of the CaM-acceptor sites is a 1,25(OH)2D3 receptor-mediated event will be investigated by comparing the time course of induction of the CaM-acceptors to those of known biochemical sequelae of the 1,25(OH)2D receptors. In addition, we will determine the steroid specificity of their induction, whether the degree of their induction correlates with receptor levels, and whether protein and RNA synthesis are required for their induction. Both in vivo (vitamin D deficient rats vs. 1,25(OH)2D-stimulated rats) and in vitro (kidney cell lines known to contain 1,25(OH)2D receptors) models will be tested. Functional characteristics of the 1,25(OH)2D-induced CaM-acceptors will be investigated by establishing their location along the nephron and in other subcellular fractions, by testing for re-distribution within the cell after 1,25(OH)2D stimulation, and by determining whether they bind CaBP's other than CaM. The 1,25(OH)2D-induced CaM acceptors will be purified for antibody production and for determining a partial amino acid sequence to compare to other known sequences. With the antibodies, 1,25(OH)2D- induction of the CaM-acceptor proteins will be compared to 1,25(OH)2D enhancement of their CaM-binding capability. Finally, whether CaBP-D28K also interacts with specific acceptor proteins will be tested by modifying the gel overlay procedure and testing for binding under a variety of different biochemical and physiological conditions. In particular, these studies will utilize CaBP-D28K purified without exposure to Ca-chelating agents, which seem to complex to the protein. Further, possible weak (and thus non-functional) interactions of the CaBP-D28K with CaM-acceptors will also be tested. If putative CaBP-D28K acceptors are identified, they will be characterized by techniques similar to those proposed for the CaM- acceptors. These studies will provide a solid basis for understanding the functions of CaBP-D28K and CaM-acceptor sites and their roles in mediating the effects of 1,25(OH)2D in its principal target tissues.
