LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells.

Abstract

LDL receptor-related proteins 6 (LRP6) is a type I transmembrane receptor (C-terminus in cytosol), which appears to be essential in numerous biological processes, since it is an essential co-receptor of Wnt ligands for canonical β-catenin dependent signal transduction. It was shown that tissue plasminogen activator (tPA), physically interacting with LRP6, induces protein phosphorylation, which may have large implication in the regulation of neural processes. In this investigation we analyzed whether LRP6 is associated with lipid rafts following tPA triggering in neuroblastoma cells and the role of raft integrity in LRP6 cell signaling. Sucrose gradient separation revealed that phosphorylated LRP6 was mainly, but not exclusively present in lipid rafts; this enrichment became more evident after triggering with tPA. In these microdomains LRP6 is strictly associated with ganglioside GM1, a paradigmatic component of these plasma membrane compartments, as revealed by coimmunoprecipitation experiments. As expected, tPA triggering induced LRP6 phosphorylation, which was independent of LRP1, as revealed by knockdown experiments by siRNA, but strictly dependent on raft integrity. Moreover, tPA induced β-catenin phosphorylation was also significantly prevented by previous pretreatment with methyl-β-cyclodextrin. Our results demonstrate that LRP6 mediated signal transduction pathway triggered by tPA acts through lipid rafts in neuroblastoma cells. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents. Indeed, these data, pointing to the key role of lipid rafts in tPA triggered signaling involving β-catenin, may have pharmacological implications, suggesting that cyclodextrins, and potentially other drugs, such as statins, may represent an useful tool.