A large number of cell surface proteins are known to be anchored in the 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. However, the reason why such a complex membrane protein anchoring mechanism should be so widely distributed in nature is obscure. One attractive possibility is that degradation by a GPI-specific phospholipase would release GPI-anchored proteins from the membrane and thereby provide the cell with a versatile mechanism for regulating protein expression at the cell surface. Previous research has identified and characterized such an enzyme: the GPI-specific phospholipase D (GPI-PLD) which removes the phosphatidic acid (PA) moiety of the anchor. GPI-PLD is abundant in mammalian plasma but it is also found in a variety of different cell types (e.g. myeloid cells, keratinocytes and neurons). Studies of GPI-PLD have implications for human health since abnormal regulation of this enzyme would lead to the uncontrolled release of important cell surface molecules with potentially serious consequences. In the proposed research the function of GPI-PLD will be studied with particular emphasis on its role in the release of GPI-anchored proteins from the membrane and how this process might be regulated. The mechanism of GPl-anchored protein release will be examined by incubation of cells with GPI-PLD antisense oligonucleotides or GPI-PLD inhibitors. The effect of reducing the cholesterol/sphingolipid content of the membrane on protein release will also be studied. The subcellular location where GPI anchor degradation occurs will be identified. The mechanisms of GPI-PLD regulation will be investigated using purified GPI- PLD and GPI-anchored proteins. Structural features on GPI-PLD which might be important for regulation of its activity or localization within the cell (e.g. the integrin E-F hand Ca2+-binding sites- will be characterized by site-directed mutagenesis. These studies should establish the contribution of GPI-PLD to release of GPI-anchored proteins and provide valuable insights into the biochemistry and cell biology of an increasingly important mechanism of membrane protein anchoring.