Neurite outgrowth induced by NGF or L1CAM via activation of the TrkA receptor is sustained also by the exocytosis of enlargeosomes

NGF binding to its protein kinase receptor TrkA is known to induce neurite outgrowth and neural cell differentiation. The plasma membrane expansion, necessary for the process, was shown to be contributed by the VAMP7-dependent exocytosis of endocytic vesicles. Working with wild-type PC12 (wtPC12), a cell model widely used to investigate NGF-induced neurite outgrowth, we found that a fewhours of treatment with the neurotrophin (and to a lower extent with basic FGF and EGF) induces the appearance of enlargeosome vesicles competent for VAMP4-dependent exocytosis abundant in high REST-PC12 clones. Both the neurite length assay and the immunocytochemistry of enlargeosomes exocytosis revealed that activation of TrkA is induced not only by NGF, but also by the L1 adhesion protein, L1CAM, whose soluble construct binds the receptor with submicromolar affinity. In the intact wtPC12, the L1CAM construct induced autophosphorylation and internalization of TrkA followed by the activation of the PI3K, MEK, and PKCγ signaling cascades, analogous to the responses induced by NGF. Down-regulation of either VAMP7 or VAMP4 revealed the coparticipation of the two corresponding vesicles to the outgrowth responses induced by NGF and L1CAM. Finally, mixing experiments of wtPC12 cells rich in TrkA with high REST PC12 cells transfected with L1CAM documented the transactivation of the receptor by the adhesion protein surface-exposed in adjacent cells. In view of the known inhomogeneous surface distribution of both L1CAM and TrkA in various neural cells including neurons, their transcellular binding could be restricted to discrete sites, governing local signaling events distinct from those induced by soluble messengers.