Neurite outgrowth is a major event in neural development being mediated by several members of the immunoglobulin superfamily of cell adhesion molecules, among them the neural cell adhesion molecule (NCAM).*
NCAM is expressed on the surface of almost all neural cells throughout the central and peripheral nervous system. It plays a pivotal role in early brain development, synaptic plasticity, and memory consolidation (Schachner, 1997
; Ronn et al., 1998
) and promotes neurite outgrowth upon homophilic or heterophilic engagement with other molecules on adjacent cell surfaces and in the extracellular matrix (for review see Crossin and Krushel, 2000
). This implicates NCAM both as a ligand and signal transducing receptor. Of the three major isoforms of NCAM (NCAM180, NCAM140, and NCAM120, named according to their apparent molecular masses), all have been found to serve as neuritogenic ligands (Doherty et al., 1989
) due to their identical extracellular domains.
First attempts to elucidate the molecular events underlying NCAM-mediated neuritogenesis have attributed a fundamental role to the fibroblast growth factor (FGF) receptor (Williams et al., 1995
). Overexpression of a truncated FGF receptor-1 with a deleted kinase domain inhibited neurite outgrowth of PC12 cells when cultured on NCAM-presenting fibroblasts (Saffell et al., 1997
). The ability of NCAM to promote neurite outgrowth was suggested therefore to depend solely on the interaction between the extracellular domains of NCAM and the FGF receptor. According to this concept, the interaction of NCAM with the FGF receptor activates the receptor tyrosine kinase with subsequent activation of PLCγ. As a final consequence, Ca2+
influx into the neurons results in neurite growth.
The view that the FGF receptor may not be the only mediator of NCAM-dependent signal transduction was indicated by data showing that NCAM-dependent neurite outgrowth is impaired in cultured neurons from mice deficient in the nonreceptor tyrosine kinase fyn (Beggs et al., 1994
). Moreover, immunoprecipitation studies revealed an association of NCAM140 but not NCAM180 with fyn (Beggs et al., 1997
). According to this model, NCAM clustering at the cell surface induces fyn phosphorylation with further recruitment of the focal adhesion kinase (FAK) to the NCAM–fyn complex. Activation of downstream kinases by this complex is then thought to be the initial step in NCAM-mediated neurite outgrowth.
Although the two signaling mechanisms would appear distinct at first sight, it is conceivable that both pathways could be operant in cells expressing NCAM isoforms as receptors for neurite outgrowth. The possibility also needs to be considered that activation of these signaling pathways could depend on the particular localization of the individual NCAM isoforms in different membrane subcompartments at the cell surface. Based on the observation that the intracellular domains of NCAM140 can be palmitoylated at four cysteine residues adjacent to the transmembrane domain (Little et al., 1998
), we asked whether localization to cholesterol-rich membrane microdomains, the so-called lipid rafts, or detergent-resistant microdomains could play a major role in signal transduction mediated by NCAM. These lipid rafts reveal high concentrations of cholesterol and sphingoglycolipids and thereby a higher resistance toward nonionic detergents such as Triton X-100 than other membrane compartments. Several studies have illuminated the involvement of lipid rafts in signaling of hematopoietic cells (Brown and London, 1998
), and there is growing evidence that transition of cellular receptors into rafts can enhance downstream signaling (Cinek and Horejsi, 1992
; Tansey et al., 2000
). Since these microdomains are enriched in potent effectors of signal transduction (e.g., src family kinases and growth-associated protein [GAP]-43) (Simons and Ikonen, 1997
), the accumulation of NCAM receptors in these microdomains might regulate downstream signaling in a particular manner. Therefore, we asked whether raft localization of particular NCAM isoforms was essential for downstream signaling leading to neurite outgrowth. In parallel, we intended to elucidate the relationship between NCAM-mediated signaling via lipid rafts and signal transduction via the FGF receptor that has been demonstrated to be present outside of rafts at the cell surface (Davy et al., 2000