Cellular prion protein associates with a multimolecular complex including LRP1 and glycosphingolipids within lipid rafts

Plasma membrane of different cell types contains microdomains, commonly referred to as lipid rafts (Simons and Ikonen, 1997). These domains are enriched in sphingolipids and cholesterol, moreover a number of proteins involved in signal transduction pathways co-purify with lipid rafts isolated on sucrose gradient (Langlet et al., 2000; Anderson et al., 2000). Like other GPI-anchored proteins, the evidence for the involvement of lipid rafts in the localization and trafficking of PrPc, as well as in PrPc-mediated cellular signaling, has been reported in neuronal and non neuronal cells (Mattei et al., 2002; Mattei et al., 2004; Lewis and Hooper, 2011). It has been suggested that Low density lipoprotein receptor-related protein (LRP1), which is a member of the LDL receptor gene family (Strickland et al., 2002) and controls both, the surface and biosynthetic trafficking of PrPc in neurons (Parkyn CJ et al. 2007), is the central organiser, but not the sole functional component, of a larger complex that traffics PrPc during biosynthesis and at the cell surface. LRP1 functions as an endocytic receptor for a broad range of structurally and functionally diverse ligands and also functions in cell signaling, directly, in response to ligand-binding, and indirectly, by regulating levels of other signaling receptors. We demonstrated that LRP1-initiated cell signaling is ligand-dependent. Proteins that activate cell signaling by binding to LRP1 assemble different co-receptor systems. Ligand-specific co-receptor recruitment provides a mechanism by which one receptor, LRP1, may trigger different signaling responses. (Mantuano et al., 2013). We hypothesized that the ability of PrP to induce cell signaling might involve a multi receptor complex, in which LRP1, in association with other receptors like TrkA receptor, plays a role in signal transduction within lipid rafts. In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of SK-N-BE2. PrPC was present in the Triton-insoluble fractions, corresponding to lipid rafts of cell plasma membrane. In addition scanning confocal microscopical analysis revealed a consistent colocalization between PrPC and GM1, and TLC immunostaining, showed that PrPC was associated with GM1 in PrPC immunoprecipitates. Moreover, signal transduction experiments on neuritogenic Erk pathway were performed using recombinant PrP (PrPrec). The results indicated that PrP signals through the lipid raft and that PrP-mediated Erk1/2 phosphorylation is regulated by LRP1, confirming that the multimolecular complex plays a role in signal trasduction within lipid rafts. Taken together, these findings demonstrate that LRP1 associates with a multimolecular complex, including PrPc and ganglioside GM1, which is dependent on the integrity of lipid raft and is involved in the neuritogenic signaling and trafficking.