NR ATHI
AU Taylor,D.R.; Watt,N.T.; Perera,W.S.S.; Hooper,N.M.
TI Assigning functions to distinct regions of the N-terminus of the prion protein that are involved in its copper-stimulated, clathrin-dependent endocytosis
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Cell Biology of PrPc and PrPsc CELL-14
PT Konferenz-Poster
AB The cellular prion protein (PrPc) is essential for the pathogenesis and transmission of prion diseases. PrPc is located in detergent-insoluble lipid rafts at the surface of neuronal cells, however, the mechanism of its internalisation is unclear with both raft/caveolae-based and clathrin-mediated processes being proposed. We have investigated the mechanism of copper-induced internalisation of PrPc in neuronal cells by immunofluorescence microscopy, surface biotinylation assays and buoyant sucrose density gradient centrifugation in the presence of Triton X-100. Clathrin-mediated endocytosis was selectively blocked with tyrphostin A23, that disrupts the interaction between tyrosine motifs in the cytosolic domains of integral membrane proteins and the adaptor complex AP2, and a dominant-negative mutant of the adaptor protein AP180. Both these agents inhibited the copper-induced endocytosis of PrPc. Copper caused PrPc to move laterally out of detergent-insoluble lipid rafts into detergent-soluble regions of the plasma membrane. Using mutants of PrPc that lack either the octapeptide repeats or the N-terminal polybasic region, and a construct with a transmembrane anchor, we show that copper binding to the octapeptide repeats promotes dissociation of PrPc from lipid rafts, while the N-terminal polybasic region mediates its interaction with a transmembrane adaptor protein that engages the clathrin endocytic machinery. Our results provide an experimental basis for reconciling the apparently contradictory observations that although being localised in lipid rafts, the prion protein undergoes clathrin-dependent endocytosis and, in addition, we have been able to assign distinct functions in the endocytic process to separate regions of the protein.
AD David R. Taylor, Nicole T. Watt, W. Sumudhu S. Perera, Nigel M. Hooper, Proteolysis Research Group, School of Biochemistry and Molecular Biology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds, LS2 9JT, U.K
SP englisch
PO Deutschland