title: Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells.
abstract: GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insoluble complexes, enriched in cholesterol, glycolipids, and GPI-linked proteins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cellular origin and molecular protein components of this complex remain unknown, we have begun to characterize these low-density insoluble complexes isolated from MDCK cells. We find that these complexes, which represent 0.4-0.8% of the plasma membrane, ultrastructurally resemble caveolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many other plasma membrane associated molecules and organelle-specific marker enzymes, suggesting that they represent microdomains of the plasma membrane. In addition to caveolin, these insoluble complexes contain a subset of hydrophobic plasma membrane proteins and cytoplasmically-oriented signaling molecules, including: (a) GTP-binding proteins--both small and heterotrimeric; (b) annex II--an apical calcium-regulated phospholipid binding protein with a demonstrated role in exocytic fusion events; (c) c-Yes--an apically localized member of the Src family of non-receptor type protein-tyrosine kinases; and (d) an unidentified serine-kinase activity. As we demonstrate that caveolin is both a transmembrane molecule and a major phospho-acceptor component of these complexes, 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-mediated signal transduction events. In addition, our results have implications for understanding v-Src transformation and the actions of cholera and pertussis toxins on hetero-trimeric G proteins.
authors.affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479.
authors.forename: M
authors.initials: M
authors.lastname: Sargiacomo
authors.forename: M
authors.initials: M
authors.lastname: Sudol
authors.forename: Z
authors.initials: Z
authors.lastname: Tang
authors.forename: M P
authors.initials: MP
authors.lastname: Lisanti
mesh_terms: D000818:Animals; D051242:Caveolin 1; D022461:Caveolins; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D016764:Cell Polarity; D003902:Detergents; D004285:Dogs; D005347:Fibroblasts / Q000737:chemistry / Q000648:ultrastructure; D019204:GTP-Binding Proteins / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000502:physiology*; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051575:Proto-Oncogene Proteins c-yes; D051381:Rats; D015398:Signal Transduction; D012995:Solubility; D013961:Thyroid Gland / Q000378:metabolism; D019061:src-Family Kinases
chemical_list: C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D051575:Proto-Oncogene Proteins c-yes; D019061:src-Family Kinases; D019204:GTP-Binding Proteins