title: Assigning functions to distinct regions of the N-terminus of the prion protein that are involved in its copper-stimulated, clathrin-dependent endocytosis.
abstract: The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. Although PrP(C) 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 PrP(C) 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 PrP(C). Copper caused PrP(C) to move laterally out of detergent-insoluble lipid rafts into detergent-soluble regions of the plasma membrane. Using mutants of PrP(C) 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 PrP(C) 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.
authors.affiliation: Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: David R
authors.initials: DR
authors.lastname: Taylor
authors.forename: Nicole T
authors.initials: NT
authors.lastname: Watt
authors.forename: W Sumudhu S
authors.initials: WS
authors.lastname: Perera
authors.forename: Nigel M
authors.initials: NM
authors.lastname: Hooper
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D002966:Clathrin / Q000502:physiology*; D003300:Copper / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D003902:Detergents; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism; D051379:Mice; D034041:Monomeric Clathrin Assembly Proteins / Q000235:genetics; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D018621:PrPC Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid; D012995:Solubility; D014162:Transfection; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D002966:Clathrin; D003902:Detergents; D034041:Monomeric Clathrin Assembly Proteins; D010446:Peptide Fragments; D018621:PrPC Proteins; D020032:Tyrphostins; C439023:clathrin assembly protein AP180; D003300:Copper; C060641:tyrphostin A23