Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Role of PrP octapeptide repeats in ion metabolism

Using neuronal cells expressing PrP and various mutants we have continued investigating the mechanism by which PrP is internalised into cells in response to binding copper. PrP 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 PrP 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 PrP and copper caused PrP to move laterally out of detergent-insoluble lipid rafts into detergent-soluble regions of the plasma membrane. Using mutants of PrP 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 from lipid rafts, while the N-terminal polybasic region mediates its interaction with a transmembrane adaptor protein that engages the endocytic machinery of clathrin-coated pits.

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