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First published online October 27, 2005
doi: 10.1242/10.1242/jcs.02627
Research Article |
Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds LS2 9JT, UK
* Author for correspondence (e-mail: n.m.hooper{at}leeds.ac.uk)
Accepted 9 August 2005
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.
Key words: Prion protein, Copper, Lipid rafts, Clathrin, Endocytosis
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