The Role of Neu1 Sialidase in Epidermal Growth Factor Receptor Activation
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The epidermal growth factor receptor (EGFR) exists as a single, highly glycosylated subunit receptor on the plasma membrane of a cell. Upon ligand binding to its extracellular domain, the EGFR dimerizes with an adjacent receptor. This results in activation of the EGFR’s intracellular tyrosine kinase domain, and consequently, autophosphorylation of specific tyrosine residues on the receptor’s cytoplasmic tail. Adaptor proteins bind to these phosphorylated tyrosine residues and transduce the message internally, initiating a multitude of signalling cascades which stimulate cell growth, division, and movement. Despite all that has been elucidated regarding the activation and signalling pathways of the EGFR, the parameters controlling dimerization and activation remain unknown. Recently, Neu1 sialidase, an enzyme which cleaves α-2,3-linked sialic acids from glycosylated substrates, has been implicated as a critical mediator of TrkA receptor activation. Upon activation, the sialidase desialylates the external receptor glycosylation, removing a physical barrier which was formerly hindering receptor dimerization, and thus, receptor activation. Based on the known sialylation of EGFR glycosylation, as well as the demonstrated importance of receptor glycosylation in EGFR activation, we hypothesized that the EGFR may be activated by a similar mechanism. Here, we report an identical membrane signalling paradigm initiated by epidermal growth factor (EGF) binding to EGFR to rapidly induce Neu1 sialidase activity in live NIH3T3-EGFR cells but not in live Neu1-deficient human fibroblast cells. Furthermore, we report that Neu1 sialidase activity is required for EGFR activation, supported by the finding that tyrosine phosphorylation is inhibited in EGF-stimulated NIH3T3-hEGFR cells which have been pretreated with both broad-range (oseltamivir phosphate) and specific (anti-Neu1 neutralizing antibody) sialidase inhibitors. MMP-9 plays a role in the initiation of Neu1 sialidase post-ligand binding, as pre-treatment of NIH3T3-hEGFR cells with specific MMP-9 inhibitor prior to EGF stimulation blocks membrane sialidase activity as well as tyrosine phosphorylation. Of critical importance to this schematic is the finding that both Neu1 and MMP-9 co-immunoprecipitate with EGFR on the plasma membrane of both naïve and EGF-stimulated NIH3T3-hEGFR cells. Together, these findings reveal a novel EGFR activation mechanism in which cross-talk between Neu1 and MMP-9 plays a vital role in EGF-induced receptor activation.