The major goal of this project is to study the initial events leading to the formation of mineralized tissues. These studies will focus on the role of alkaline phosphatase (AP) in matrix vesicles, in chondrocytes and in osteoblasts to mediate hydroxyapatite formation. Hypotheses which posit AP as a nucleator of apatite and a booster of inorganic phosphate will be tested. The availability of two in vitro mineralization systems, one utilizing isolated matrix vesicles and the other using osteoblast cell lines which express differing levels of AP provides two complementary experimental approaches for investigating mineral formation. By comparing and contrasting MV mineralization with osteoblast mineralization we expect to isolate the AP dependent factors, and thus demonstrate the unique contribution of AP to these processes. With two good experimental systems for studying AP dependent mineralization in hand, and having access to AP gene clones, AP crystal structure coordinates and the tools of molecular biology and protein engineering, we hope to identify distinct domains in the AP molecule, and selectivity modify them. These mutant AP's can then be expressed in AP osteoblastoma cells (ROS 25/1) or incorporated into liposomes, and their mineralization potential tested. Finally,l we can use these same techniques in combination with conventional cell biology to test new hypotheses concerning AP function. These hypotheses are based on very recent studies of the cell surface in which it has been shown that AP and a number of other ectoproteins are attached to the cell membrane by a phosphatidylinositol (PIG) anchor. We propose to investigate the biological and biochemical consequences, for AP and cell function, of the fact that mature AP is anchored to the membrane by a PIG anchor domain. By modifying the anchor domain both genetically and chemically, we can test the fate of anchor components following release of AP, their effects on cell function, and the regulation of AP release by systemic hormones.
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