A large number of proteins are modified by covalent attachment of a lipid moiety, in most cases myristate, palmitate, an isoprenoid group, or a glycosyl-phosphatidylinositol (GPI) anchor. The first three lipid modifications occur on intracellular proteins, often serving to anchor these proteins to the inner cytoplasmic face of the plasma membrane, whereas the GPI anchor attaches integral membrane proteins on the outside of the cell. The lipid modifications can be critical for the functions of the protein; for instance, myristoylation of the oncogene p60src or prenylation of p21ras is necessary for their membrane association and for their ability to transform cells. Studies of a cell signaling pathway initiated through the GPI anchor demonstrated an association of the GPI- anchored proteins and the protein tyrosine kinases p56lck and p59fyn, and this required palmitoylation of the kinase. Both GPI anchor attachment and palmitoylation resulted in localization of the lipid-modified proteins into caveolae, flask-shaped microinvaginations of the plasma membrane (non-clathrin-coated pits). This project has the long-range goals of understanding the biochemical structures and cell biological roles of lipid modifications of proteins, and of probing the functions of caveolae in cellular processes.
Specific aims are to delineate the consensus signal for GPI anchoring and palmitoylation, to define the signals for targeting of proteins to caveolae, and to investigate the role of palmitoylation and of caveolae in protein function. The consensus signals for GPI anchoring and palmitoylation will be analyzed by mutagenesis analysis and by the construction of synthetic polymeric sequences that can act as signals for lipid modifications. Signals for caveolar localization of proteins, including certain forms of lipid modification, will be investigated by mutagenesis of cloned caveolar proteins and by construction of chimeric proteins containing putative caveolar targeting signals. Functional studies of proteins that are palmitoylated and localized to caveolae will address three critical physiological functions: T cell activation through p56lck, uptake of oxidized low density lipoprotein through CD36, and apical sorting of proteins involving caveolin. Overall, the importance of this work is based on the functional consequences of lipid modifications and recent observations that have implicated caveolae in several critical cellular processes, including sorting of proteins along biosynthetic and endocytic pathways and participation in cell signaling pathways.
