Our results demonstrate a specific, functional, and direct physical interaction between L1-type CAMs and EGFR at cell contact sites, which results in the induction of the receptor tyrosine kinase activity in the absence of classical receptor agonists. Based on the observation that the artificial, GPI-anchored isoform of Nrg maintains its ability to generate a GOF phenotype in the absence of endogenous Nrg protein, this L1-mediated EGFR activation only requires the extracellular domain of the L1-type protein. Therefore, intracellular L1-binding proteins, such as the membrane skeleton component Ankyrin, do not appear to be essential for L1-dependent EGFR activation, although they may contribute to its modulation. An alternative model may involve a specific, but indirect interaction through a hypothetical “bridging” molecule expressed by S2 cells. However, for such a Drosophila linker protein to function in our experimental system, its binding specificities to EGFR and to L1- CAM would have to be conserved between flies and humans. Therefore, considering the evolutionary distance between humans and Drosophila, the present data are more consistent with a direct molecular interaction between L1- CAM and EGFR.
Our observation that the trans
-interaction between L1- CAM and EGFR does not induce the RTK activity () suggests that the stimulation of EGFR autophosphorylation by L1-mediated cell adhesion requires additional cis
-type interactions between the two proteins. We therefore postulate that a combination of trans
- and cis
-interactions between EGFR and L1-type CAMs functions in physically aligning RTK proteins and in inducing their tyrosine kinase activity in the absence of a traditional receptor ligand (). It remains to be elucidated which of the L1 and EGFR protein domains are involved in this interaction. Williams et al.
) postulated that specific cis
-interactions between neural CAMs and FGFR are responsible for the induction of FGFR signaling in neuronal cells. They provided some experimental evidence for the involvement of a specific, conserved amino acid motif (CAM homology domain) that is present in several neural CAMs and the vertebrate FGFR protein. However, structural studies of the FGFR ectodomain argue against the involvement of this particular amino acid motif in the activation by neural CAMs (Plotnikov et al., 1999
Figure 8. Model of the functional interactions between L1-type CAMs and EGF receptor proteins. L1-cell adhesion relies on trans-interactions between L1-type proteins, which are expressed on the surface of neighboring cells. The interaction between L1-type CAMs (more ...)
The current knowledge about neurite outgrowth, which was obtained mainly from in vitro studies, indicates that vertebrate FGFRs are also activated by a CAM-dependent mechanism that might be very similar to the one demonstrated here for EGFR. It remains to be determined whether EGFR and FGFR both act through the same phospholipase C-mediated pathway in neuronal cells or through different signaling cascades, which might result in differential effects on neuronal growth and differentiation. In Drosophila,
the Heartless FGFR and the DER EGFR-homologues seem to have at least partially overlapping specificities in mediating Nrg functionality (Garcia-Alonso et al., 2000
). Interestingly, Chen et al.
) demonstrated in vivo that the Caenorhabditis elegans
L1-type protein, LAD-1, is a substrate of an FGFR-dependent tyrosine kinase. This observation suggests the existence of a feedback loop, in which FGFR activity modulates the function of L1-type proteins through its intracellular domain and its association with the membrane skeleton.
In the Drosophila
nervous system, the importance of EGFR signaling is well established, especially during neurogenesis, eye disk, and midline development (Lage et al., 1997
; Okabe and Okano, 1997
; Dominguez et al., 1998
; Dumstrei et al., 1998
; Kumar et al., 1998
). Our finding that the functional interaction between L1-type molecules and EGFR is conserved in the human proteins suggests that such interactions also play important roles during vertebrate development. EGFR is widely expressed by many neuronal and glial cell types in vertebrates and appears to be involved in neuronal proliferation, cell fate choices, chemotactic cell migration, and neuronal survival (Yamada et al., 1997
). In addition, several studies report that EGF stimulates process outgrowth in primary cortical and cerebellar neurons (Morrison et al., 1988
; Kornblum et al., 1990
; Yamada et al., 1997
). However, Kornblum et al.
) found that when compared with EGF, basic FGF appears to be the more effective trophic agent for rat neocortical neurons in vitro.
In addition to the afore mentioned phospholipase C-mediated signaling pathway, which is triggered by the CAM- dependent activation of RTKs, the oligomerization of L1- type proteins induces neurite outgrowth that is initiated by the activation of the neuronal MAPK pathway (Panicker et al., 2003
). This mechanism appears to require the endocytosis of L1-CAM protein and the activation of nonreceptor tyrosine kinases, such as pp60 (c-src), and of small GTPases, such as Rac1 (Schaefer et al., 1999
; Schmid et al., 2000
). At least in the case of NCAM-mediated neuritogenesis, the activation of both receptor as well as nonreceptor tyrosine kinase pathways is required for a robust neurite outgrowth response (Niethammer et al., 2002
). This suggests that a similar cosignaling mechanism might also be involved in L1-induced axonal extension.
The results presented here demonstrate that L1-type CAMs directly induce the first step in the activation of one classical signaling pathway. With the exception of the experiment shown in , all S2 cell experiments were performed under serum-free conditions in the absence of EGF. Nevertheless, under in vivo conditions, classical vertebrate and Drosophila EGFR ligands (e.g., EGF, alpha-TGF, Spitz, Vein, and others) may still be required for achieving a full activation of neuronal EGFRs. In this case, neural CAMs might act in combination with these classical ligands to lower the activation threshold, rather than eliminating the requirement for them. In such a model, endogenous ligands and CAM-CAM interactions act synergistically during nervous system development and the activation of neuronal EGFR molecules would be the result of an integrative process, including different types of stimuli.