Numerous cell surface proteins are anchored to the cell membrane by covalent linkage to a complex lipid moiety, glycosylphosphatidylinositol (GPI). The proteins which utilize the GPI anchor are functionally and evolutionarily diverse, and have been identified on enzymes, adhesion molecules, receptors, etc. in all eukaryotic cell types examined so far. It has been proposed that the presence of a GPI anchor on a particular protein facilitates specific interactions with other molecules at the cell surface. It also provides a means for rapid release from the membrane as a result of degradation by specific phospholipases. However, the reason why such a complex membrane protein anchoring mechanism should be so widely distributed in nature remains obscure. In mammalian cells, GPI anchors can become acylated on the inositol ring which blocks their degradation by phospholipase C, but not by phospholipase D. Although inositol acylation has a profound effect on the biochemical properties of the GPI-anchored proteins, little is known about the biosynthesis of this modification or what effect it has on the function of GPI-anchored proteins. In this proposed research, the mechanism of biosynthesis and functional significance of inositol acylation will be investigated in human tumor cell lines. The subcellular location of the inositol acylation biosynthetic process will be determined by pulse-chase studies. In vitro enzyme assays and mutant cell lines will be developed to facilitate studies of the inositol-acylation process at the molecular level. The function of inositol acylation will be determined by studying its effect on several important properties of GPI-anchored proteins: (I) specific interactions with cell surface lipids and non-receptor tyrosine kinases, (ii) accessibility in the cell surface to degradation by plasma phospholipase D, and (iii) participation in T lymphocyte activation. The results of these studies will provide valuable insights into the biochemistry and cell biology of an increasingly important mechanism of membrane anchoring. GPI-anchored proteins have many important functions on the surface of human cells and inositol acylation is likely to have a major influence on their ability to carry-out those functions correctly. The results of these studies will therefore have significant implications for understanding the molecular basis of human disease.
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