SIGNAL-TRANSDUCING MOLECULES AND GLYCOSYL-PHOSPHATIDYLINOSITOL-LINKEDPROTEINS FORM A CAVEOLIN-RICH INSOLUBLE COMPLEX IN MDCK CELLS

GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insolubl e complexes, enriched in cholesterol, glycolipids, and GPI-linked prot eins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cel lular origin and molecular protein components of this complex remain u nknown, we have begun to characterize these low-density insoluble comp lexes isolated from MDCK cells. We find that these complexes, which re present 0.4-0.8% of the plasma membrane, ultrastructurally resemble ca veolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many ot her plasma membrane associated molecules and organelle-specific marker enzymes, suggesting that they represent microdomains of the plasma me mbrane. In addition to caveolin, these insoluble complexes contain a s ubset of hydrophobic plasma membrane proteins and cytoplasmically-orie nted signaling molecules, including: (a) GTP-binding proteins-both sma ll and heterotrimeric; (b) annexin II-an apical calcium-regulated phos pholipid binding protein with a demonstrated role in exocytic fusion e vents; (c) c-Yes-an apically localized member of the Src family of non -receptor type protein-tyrosine kinases; and (d) an unidentified serin e-kinase activity. As we demonstrate that caveolin is both a transmemb rane molecule and a major phospho-acceptor component of these complexe s, we propose that caveolin could function as a transmembrane adaptor molecule that couples luminal GPI-linked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPI-m ediated signal transduction events. In addition, our results have impl ications for understanding v-Src transformation and the actions of cho lera and pertussis toxins on hetero-trimeric G proteins.