Induction of Calcium Influx through TRPC5 Channels by Cross-Linking of GM1 Ganglioside Associated with α5β1 Integrin Initiates Neurite Outgrowth

摘要

Previous studies demonstrated that cross-linking of GM1 ganglioside with multivalent ligands, such as B subunit of cholera toxin (CtxB), induced Ca²⁺ influx through an unidentified, voltage-independent channel in several cell types. Application of CtxB to undifferentiated NG108-15 cells resulted in outgrowth of axon-like neurites in a Ca²⁺ influx-dependent manner. In this study, we demonstrate that CtxB-induced Ca²⁺ influx is mediated by TRPC5 channels, naturally expressed in these cells and primary neurons. Both Ca²⁺ influx and neurite induction were blocked by TRPC5 small interfering RNA (siRNA). Pretreatment of NG108-15 cells with neuraminidase increased cell-surface GM1 and greatly enhanced the signal. GM1 was not directly associated with TRPC5 but rather with α5β1 integrin, which opened the channel through a signaling sequence after cross-linking of the GM1/integrin complex. This cascade included autophosphorylation of focal adhesion kinase and subsequent activation of phospholipase Cγ (PLCγ) and phosphoinositide-3 kinase [PI(3)K]. Pharmacological blockers that inhibited tyrosine kinase, PLC, and PI(3)K suppressed both CtxB-induced Ca²⁺ influx and neurite outgrowth. These were also suppressed by SK&F96365, a nonspecific transient receptor potential channel blocker. Confocal immunocytochemistry revealed that GM1 cross-linking induced colocalization of GM1 with these signaling elements in sprouting regions of plasma membrane. In primary cerebellar granular neurons (CGNs), TRPC5 was detected at 2 d in vitro (2 DIV), a stage corresponding to CtxB-stimulated Ca²⁺ influx. Neurite outgrowth in CGNs, determined at 3 DIV, was accelerated by CtxB and suppressed by TRPC5 siRNA and the above blockers. The crucial role of GM1 was indicated with CGNs from ganglio-series null mice, in which growth of axons was significantly retarded.