Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS

Signal transduction pathways mediated by PrP: Cis- and trans-interactions between PrP and NCAM are important for NCAM stabilisation in lipid rafts

PrP interacts directly with NCAM in lipid raft

We previously studied different signal transduction pathways involved in neurite outgrowth and neuronal survival elicited by PrP in cell culture of primary neurons. These pathways include the nonreceptor Src-related family member p59(Fyn), PI3 kinase/Akt, cAMP-dependent protein kinase A, and MAP kinase (Chen et al 2003). p59fyn has been further described as to be involved in PrP mediated signalling (Mouillet-Richard et al., 2000). Accumulation of NCAM in lipid rafts is necessary for NCAM-mediated neurite outgrowth and implies activation of the fyn kinase pathway (Niethammer et al., 2002). We asked therefore whether PrP could play a role in the same paradigm. First we analysed the association between PrP and NCAM in cultured hippocampal neurons. PrP partially co-localised with NCAM along neurites and in growth cones. As a GPI-anchored protein, PrP mostly localises to lipid rafts (Walmsley et al., 2003; Gorodinsky and Harris, 1995).

We therefore analysed whether NCAM co-localises with PrP in lipid rafts by extracting neurons with cold 1% Triton X-100, a procedure used to isolate cytoskeleton-bound and raft-associated proteins (Niethammer et al., 2002; Ledesma, et al., 1998; Leshchyns'ka et al., 2003). In extracted neurons, PrP and NCAM showed a similar pathcy distribution suggesting that both proteins form a complex in lipid rafts. We thus cross-linked NCAM at the neuronal surface with NCAM antibodies applied to live neurons. NCAM clustering induced partial redistribution of PrP to NCAM containing clusters. To further investigate this phenomenon, we analysed by an ELISA binding assay whether PrP and NCAM directly interact using recombinant PrP-Fc, which contains the extracellular domain of mouse PrP fused to the Fc portion of human IgG (Chen et al., 2003), and NCAM purified from mouse brain. NCAM bound to PrP-Fc in a concentration-dependent manner, but not to BSA. NCAM is the carrier of polysialic acid (PSA) which may influence its binding to PrP. To verify whether PSA influences binding to PrP, we analysed by ELISA the interaction between PrP and non-polysialylated NCAM-Fc produced in CHO cells using recombinant PrP-AP, which contains the extracellular domain of mouse PrP fused to alkaline phosphatase (Chen et al., 2003).

We found that non-polysialylated NCAM-Fc also bound to PrP-AP in a concentration-dependent manner, but not to BSA. To investigate whether NCAM and PrP interact in brain tissue, we immunoprecipitated NCAM from brain homogenates and analysed immunoprecipitates with antibodies against PrP. PrP co-immunoprecipitated with NCAM, indicating that the two proteins are associated in brain. Next we examined whether NCAM and PrP exist in a complex in the same plasma membrane microenvironment by inducing covalent binding between primary amino groups of adjacent proteins in the lipid raft fraction from total brain homogenates using the homobifunctional BS3 chemical cross-linker with a spacer arm of 11.4Å. The overall observations show that NCAM interacts with PrP and that both molecules form a complex in lipid rafts.

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