Characterization of L1-[alpha]v[beta]3 interactions and signaling pathway in neurite outgrowth

The neural cell adhesion molecule L1 is a membrane glycoprotein which mediates intercellular adhesion and promotes neurite outgrowth from neurons. Mutations in the L1 gene are implicated in neurological disorders termed CRASH syndrome. L1 interacts via homophilic and heterophilic interactions, and its sixth immunoglobulin-like domain (L1-Ig6) contains an Arg-Gly-Asp (RGD) sequence which is recognized by integrin receptors. In vitro analysis of primary neurons from embryonic chick revealed that dorsal root ganglion neurons extended neurites in response to substrate-coated recombinant L1-Ig6. Peptide competition and mutagenesis experiments demonstrated that the RGD motif was a novel neuritogenic site. Using function-blocking antibodies, the integrin alphavbeta3 was identified as the neuritogenic receptor for L1-Ig6, which functioned independently of L1 homophilic binding.The rat pheochromocytoma PC12 cell line was identified as a model system for the biochemical analysis of neurite outgrowth involving L1-alphavbeta3 interaction. Although PC12 cells expressed functional L1 molecules on the cell surface which contributed to intercellular adhesion of PC12 cells, L1-homophilic interactions did not elicit neurite outgrowth. In contrast, L1 induced neurite outgrowth which was dependent on the interaction of L1-Ig6 with the alphavbeta3 integrin. Pharmacological studies suggested that downstream signaling molecules in this pathway included heterotrimeric G proteins, tyrosine kinases, and the mitogen-activated protein kinases.A role for the integrin-associated protein (IAP/CD47) in the L1-alphavbeta3 signaling pathway was examined. In NGF-primed PC12 cells, IAP colocalized with alphavbeta3 in the growth cones of extending neurites, and their association was demonstrated by co-immunoprecipitation experiments. Neurite outgrowth on L1-Ig6 was inhibited in the presence of recombinant IAP proteins and anti-IAP antibodies, suggesting that IAP is part of the alphavbeta3 receptor complex. The role of IAP in neurite outgrowth was studied in vivo using the developing mouse cerebellum. IAP was expressed in the molecular layer of the cerebellum along with the alphav, beta1, and beta3 subunits. Cerebellar granule cells extended neurites on substrates coated with L1-Ig6 in an alphavbeta3-dependent manner. Cerebellar neurons from IAP-/- mice, however, remained responsive to Ll-Ig6. In contrast, neurite outgrowth was abrogated for IAP-/- neurons cultured on vitronectin, suggesting that IAP differentially associates with integrins on cerebellar neurons to influence neurite outgrowth.