NR ATUY
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 Journal of Cell Science 2005 Nov 1; 118(21): 5141-53
PT journal article
AB The cellular prion protein (PrPc) is essential for the pathogenesis and transmission of prion diseases. Although PrPc is known to be located in detergent-insoluble lipid rafts at the surface of neuronal cells, 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, which 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, whereas 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 the prion protein undergoes clathrin-dependent endocytosis despite being localised in lipid rafts. In addition, we have been able to assign distinct functions in the endocytic process to separate regions of the protein.
MH Animals; Binding Sites/genetics; Cell Line, Tumor; Clathrin/*physiology; Copper/antagonists & inhibitors/metabolism/*physiology; Detergents; Endocytosis/drug effects/genetics/*physiology; Humans; Membrane Microdomains/genetics/metabolism; Mice; Monomeric Clathrin Assembly Proteins/genetics; Peptide Fragments/antagonists & inhibitors/genetics/*physiology; PrPc Proteins/antagonists & inhibitors/genetics/*physiology; Protein Binding/genetics; Protein Transport/genetics; Repetitive Sequences, Amino Acid; Research Support, Non-U.S. Gov't; Solubility; Transfection; Tyrphostins/pharmacology
AD Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, UK
SP englisch
PO England