Induction of calcium influx through TRPC5 channels by cross-linking of GM1 ganglioside associated with alpha5beta1 integrin initiates neurite outgrowth.
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 alpha5beta1 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 Cgamma (PLCgamma) 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.
Wu
Gusheng
G
Department of Neurology and Neurosciences, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA. gwu@umdnj.edu
Lu
Zi-Hua
ZH
Obukhov
Alexander G
AG
Nowycky
Martha C
MC
Ledeen
Robert W
RW
D000818:Animals; D001369:Axons / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000502:physiology; D002772:Cholera Toxin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004789:Enzyme Activation; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D039081:Integrin alpha5beta1 / Q000187:drug effects / Q000378:metabolism; D016023:Integrins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D016501:Neurites / Q000187:drug effects* / Q000502:physiology*; D009474:Neurons / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects; D050052:TRPC Cation Channels / Q000172:deficiency / Q000378:metabolism*; D014018:Tissue Distribution
D003432:Cross-Linking Reagents; D039081:Integrin alpha5beta1; D016023:Integrins; D050052:TRPC Cation Channels; C112021:Trpc5 protein, mouse; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; D019869:Phosphatidylinositol 3-Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051966:Phospholipase C gamma; D002118:Calcium