LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neurobla
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RESEARCH ARTICLE
LRP6 mediated signal transduction pathway triggered by tissue plasminogen activator acts through lipid rafts in neuroblastoma cells Gloria Riitano 1 & Valeria Manganelli 1 & Antonella Capozzi 1 & Vincenzo Mattei 2 & Serena Recalchi 1 & Stefano Martellucci 2 & Agostina Longo 1 & Roberta Misasi 1 & Tina Garofalo 1 & Maurizio Sorice 1 Received: 10 September 2019 / Accepted: 10 February 2020 # The International CCN Society 2020
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-βcyclodextrin. 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. Keywords LRP6 . Lipid rafts . tPA . β-Catenin . Neuroblastoma cells
Abbreviations LRPs LDL receptor–related proteins tPA Tissue plasminogen activator MβCD Methyl-β-cyclodextrin mAb Monoclonal antibody SDS-PAGE Sodium-dodecyl sulfate polyacrylamide gel electrophoresis PVDF Polyvinylidene difluoride HRP Horseradish peroxidase Gloria Riitano, Valeria Manganelli, Tina Garofalo and Maurizio Sorice contributed equally to this work. * Maurizio Sorice [email protected] 1
Department of Experimental Medicine, University of Rome “La Sapienza”, viale Regina Elena 324, 00161 Rome, Italy
2
Laboratory of Experimental Medicine and Environmental Pathology, Rieti, Italy
Introduction LRPs (LDL receptor–related proteins) are transmembrane proteins, belonging to the LDL receptor gene f
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