We have identified a close homologue of L1 (CHL1) in the mouse. CHL1 comprises an N-terminal signal sequence, six immunoglobulin (Ig)-like domains, 4.5 fibronectin type III (FN)-like repeats, a transmembrane domain and a C-terminal, most likely intracellular domain of approximately 100 amino acids. CHL1 is most similar in its extracellular domain to chicken Ng-CAM (approximately 40% amino acid identity), followed by mouse L1, chicken neurofascin, chicken Nr-CAM, Drosophila neuroglian and zebrafish L1.1 (37-28% amino acid identity), and mouse F3, rat TAG-1 and rat BIG-1 (approximately 27% amino acid identity). The similarity with other members of the Ig superfamily [e.g. neural cell adhesion molecule (N-CAM), DCC, HLAR, rse] is 16-11%. The intracellular domain is most similar to mouse and chicken Nr-CAM, mouse and rat neurofascin (approximately 60% amino acid identity) followed by chicken neurofascin and Ng-CAM, Drosophila neuroglian and zebrafish L1.1 and L1.2 (approximately 40% amino acid identity). Besides the high overall homology and conserved modular structure among previously recognized members of the L1 family (mouse/human L1/rat NILE; chicken Ng-CAM; chicken/mouse Nr-CAM; Drosophila neuroglian; zebrafish L1.1 and L1.2; chicken/mouse neurofascin/rat ankyrin-binding glycoprotein), criteria characteristic of L1 were identified with regard to the number of amino acids between positions of conserved amino acid residues defining distances within and between two adjacent Ig-like domains and FN-like repeats. These show a collinearity in the six Ig-like domains and four adjacent FN-like repeats that is remarkably conserved between L1 and molecules containing these modules (designated the L1 family cassette), including the GPI-linked forms of the F3 subgroup (mouse F3/chicken F11/human CNTN1; rat BIG-1/mouse PANG; rat TAG-1/mouse TAX-1/chicken axonin-1). The colorectal cancer molecule (DCC), previously introduced as an N-CAM-like molecule, conforms to the L1 family cassette. Other structural features of CHL 1 shared between members of the L1 family are a high degree of N-glycosidically linked carbohydrates (approximately 20% of its molecular mass), which include the HNK-1 carbohydrate structure, and a pattern of protein fragments comprising a major 185 kDa band and smaller fragments of 165 and 125 kDa. As for the other L1 family members, predominant expression of CHL1 is observed in the nervous system and at later developmental stages. In the central nervous system CHL1 is expressed by neurons, but, in contrast to L1, also by glial cells. Our findings suggest a common ancestral L1-like molecule which evolved via gene duplication to generate a diversity of structurally and functionally distinct yet similar molecules.

The neural cell adhesion molecule L1 is a multidomain protein that plays important roles in cell adhesion, migration, and neurite outgrowth. To analyze structure-function relationships of L1 in neurite outgrowth and cell body adhesion, we have expressed and purified a set of different fragments of the extracellular part of this glycoprotein in CHO cells and in Escherichia coli. When neurite outgrowth from small cerebellar neurons was measured on substrate-coated L1 or L1 fragments, neurite outgrowth was promoted by the immunoglobulin-like domains I-II, III-IV, and V-VI, and by the fibronectin type III homologous repeats 1-2, while the fibronectin type III homologous repeats 3-5 were ineffective. In contrast, cell bodies of small cerebellar neurons adhered mostly to the immunoglobulin-like domains I-II and V-VI, and to the fibronectin type III homologous repeats 3-5, but less to the immunoglobulin-like domains III-IV and fibronectin type III homologous repeats 1-2. In both assays, the neuronal cell surface receptor for all active protein fragments was identified as L1. No significant differences in functional activities were found between fragments with and without carbohydrate structures. These findings indicate that L1 uses several domains for homophilic interactions overlapping for the two functions analyzed here, but also showing some regional specialization. Furthermore, we show that a homophilic molecule uses several domains in one function, with neurite outgrowth requiring more domains than adhesion for maximal activity.

To determine the domains of the neural cell adhesion molecule L1 involved in neurite outgrowth, we have generated monoclonal antibodies against L1 and investigated their effects on neurite outgrowth of small cerebellar neurons in culture. When the 10 antibodies were coated as substrate, only antibody 557.B6, which recognizes an epitope represented by a synthetic peptide comprising amino acids 818 to 832 at the border between the fibronectin type III homologous repeats 2 and 3, was as efficacious as L1 in promoting neurite outgrowth, increasing intracellular levels of Ca2+, and stimulating the turnover of inositol phosphates. These findings suggest that neurite outgrowth and changes in these second messengers are correlated. Such a correlation was confirmed by the ability of Ca2+ channel antagonists and pertussis toxin to inhibit neurite outgrowth on L1 and antibody 557.B6. These observations indicate for the first time a distinct site on cell surface-bound L1 as a prominent signal-transducing domain through which the recognition events appear to be funneled to trigger neurite outgrowth, increase turnover of inositol phosphates, and elevate intracellular levels of Ca2+.

The neural cell adhesion molecule L1 is a multidomain protein that plays important roles in cell adhesion, migration, and neurite outgrowth. It can interact with itself by a self-binding, i.e., homophilic adhesion mechanism (Kadmon et al.: J Cell Biol 110: 193-208, 1990a). To determine the domains of L1 involved in homophilic binding, we have generated protein fragments of L1 in a prokaryotic and a eukaryotic expression system and used these covalently coupled to fluorescent microspheres to quantify aggregation between them by cytofluorometric analysis. Protein fragments containing the first and second Ig-like domains and the third fibronectin type III homologous repeat showed avid self-binding. Ig-like domains III and IV also showed some self-binding, whereas Ig-like domains V and VI and fibronectin type III homologous repeats 1 and 2 as well as 4 and 5 were less or not active. Binding between different domains was also observed: fibronectin type III homologous repeats 4 and 5 interacted with Ig-like domains I and II, and fibronectin type III homologous repeats 3-5 interacted with all Ig-like domains. These results were confirmed by experiments testing the binding of fragment-conjugated microspheres to substrate-coated L1 or to cell surface-expressed L1 on cultured neurons. Binding of L1 to itself was interfered with by all protein fragments tested, suggesting that also less avidly binding domains of L1 contribute to homophilic binding. These observations indicate prominent functional roles of both Ig-like domains and fibronectin type III homologous repeats in homophilic binding of L1.

Among the factors which influence neuronal morphology, the degree of substrate adhesivity has been suggested to play an important role in the growth and guidance of neurites. The present study was undertaken to investigate apparently contradictory results relating substrate adhesivity to the extent of neurite outgrowth. By using substrates coated with different concentrations of polyornithine to vary adhesivity, we could show that intermediate levels of neuron-to-substrate adhesive strength favored neurite outgrowth more than substrates of high or low adhesivity. However, when neurons were plated on substrates derived from the extracellular matrix, the strength of neuron-to-substrate adhesion was important for the growth of dendrite-like minor neurites, but not for the extension of axon-like major neurites, which grew independently of adhesive forces. On substrates of the cell adhesion molecule L1, growth of both major and minor neurites was adhesion-independent. Finally, in the presence of tenascin added to the culture medium, neurite growth was inhibited irrespective of the adhesivity of the substrate and the presence of substrate-bound extracellular matrix molecules or L1. These observations suggest that intermediate forces of adhesivity favor neurite growth in general, but that purely adhesive forces can be dominated by specific molecular instructions which differentially affect growth of major and minor neurites in positive and negative ways.