The neural cell adhesion molecule, N-CAM, appears on early embryonic cells and is important in the formation of cell collectives and their boundaries at sites of morphogenesis. Later in development it is found on various differentiated tissues and is a major CAM mediating adhesion among neurons and between neurons and muscle. To provide a molecular basis for understanding N-CAM function, the complete amino acid sequences of the three major polypeptides of N-CAM and most of the noncoding sequences of their messenger RNA's were determined from the analysis of complementary DNA clones and were verified by amino acid sequences of selected CNBr fragments and proteolytic fragments. The extracellular region of each N-CAM polypeptide includes five contiguous segments that are homologous in sequence to each other and to members of the immunoglobulin superfamily, suggesting that interactions among immunoglobulin-like domains form the basis for N-CAM homophilic binding. Although different in their membrane-associated and cytoplasmic domains, the amino acid sequences of the three polypeptides appear to be identical throughout this extracellular region (682 amino acids) where the binding site is located. Variations in N-CAM activity thus do not occur by changes in the amino acid sequence that alter the specificity of binding. Instead, regulation is achieved by cell surface modulation events that alter N-CAM affinity, prevalence, mobility, and distribution on the surface. A major mechanism for modulation is alternative RNA splicing resulting in N-CAM's with different cytoplasmic domains that differentially interact with the cell membrane. Such regulatory mechanisms may link N-CAM binding function with other primary cellular processes during the embryonic development of pattern.

Specialized paranodal junctions form between the axon and the closely apposed paranodal loops of myelinating glia. They are interposed between sodium channels at the nodes of Ranvier and potassium channels in the juxtaparanodal regions; their precise function and molecular composition have been elusive. We previously reported that Caspr (contactin-associated protein) is a major axonal constituent of these junctions (Einheber et al., 1997). We now report that contactin colocalizes and forms a cis complex with Caspr in the paranodes and juxtamesaxon. These proteins coextract and coprecipitate from neurons, myelinating cultures, and myelin preparations enriched in junctional markers; they fractionate on sucrose gradients as a high-molecular-weight complex, suggesting that other proteins may also be associated with this complex. Neurons express two contactin isoforms that differ in their extent of glycosylation: a lower-molecular-weight phosphatidylinositol phospholipase C (PI-PLC)-resistant form is associated specifically with Caspr in the paranodes, whereas a higher-molecular-weight form of contactin, not associated with Caspr, is present in central nodes of Ranvier. These results suggest that the targeting of contactin to different axonal domains may be determined, in part, via its association with Caspr. Treatment of myelinating cocultures of Schwann cells and neurons with RPTP␤-Fc, a soluble construct containing the carbonic anhydrase domain of the receptor protein tyrosine phosphatase ␤ (RPTP␤), a potential glial receptor for contactin, blocks the localization of the Caspr/contactin complex to the paranodes. These results strongly suggest that a preformed complex of Caspr and contactin is targeted to the paranodal junctions via extracellular interactions with myelinating glia.

Axonal growth cones respond to adhesion molecules and extracellular matrix components by rapid morphological changes and growth rate modification. Neurite outgrowth mediated by the neural cell adhesion molecule (NCAM) requires the src family tyrosine kinase p59 fyn in nerve growth cones, but the molecular basis for this interaction has not been defined. The NCAM140 isoform, which is found in migrating growth cones, selectively co-immunoprecipitated with p59fyn from nonionic detergent (Brij 96) extracts of early postnatal mouse cerebellum and transfected rat B35 neuroblastoma and COS-7 cells. p59 fyn did not associate significantly with the NCAM180 isoform, which is found at sites of stable neural cell contacts, or with the glycophosphatidylinositol-linked NCAM120 isoform. fak , and p59 fyn and activate the catalytic function of these tyrosine kinases, initiating a signaling cascade that may modulate growth cone migration.

Chemical analyses and binding studies have been correlated to clarify the relationship of structure to function in the neural cell adhesion molecule (N-CAM) from embryonic chicken brain. N-CAM isolated from the cell surface appears to include two closely related polypeptide chains. Treatment with neuraminidase of such preparations of N-CAM bound by antibodies on solid supports yielded components of Mr 140,000 and 170,000. These components each had the same amino-terminal sequence as N-CAM and gave nearly identical profiles on peptide maps. Immunoprecipitation of N-CAM from 9-day brain cells treated with tunicamycin yielded corresponding components of Mr 130,000 and 160,000, suggesting that the differences between these two components of N-CAM are in the polypeptide rather than the carbohydrate portions of the molecules. N-CAM appears to be oriented with the amino terminus extending away from the cell surface and with the bulk of the sialic acid near the middle of the peptide chain. As shown previously, incubation of N-CAM at 37 degrees C generates a fragment (Fr1) of Mr 65,000 that lacks most of the sialic acid. Treatment of membranes with Staphylococcus aureus V-8 protease released a fragment (Fr2) of N-CAM that contained most of the sialic acid; this fragment had an Mr of 108,000 after neuraminidase treatment. Both of these fragments contain the amino-terminal portion of the polypeptide chain. At least a portion of the N-CAM binding site was found to be located in the amino-terminal region of the peptide chain. Most or all of the sialic acid was not directly involved in binding, although it can influence binding, as indicated by the finding that neuraminidase-treated N-CAM (desialylated-N-CAM) bound to cells to a greater extent than untreated N-CAM. The Fr1 and the Fr2 fragments in solution did not bind to cells but were as effective as N-CAM and desialylated-N-CAM as competitors for N-CAM binding to cells. When fixed covalently to beads, N-CAM, desialylated-N-CAM, and the Fr1 and Fr2 fragments bound specifically to cells. In contrast, the N-CAM autolysis products released along with Fr1 neither bound to cells nor competed for N-CAM binding. In addition to suggesting a location for the N-CAM binding region, the accumulated results raise the possibility that valence may play a key role in N-CAM binding.

Abstract. We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human Ll-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L-and N-type calcium channels. Calcium influx into neurons induced by K + depolarization fully mimics the L1 response. Furthermore, we show that L1-and K+-dependent neurite outgrowth can be specifically inhibited by a reduction in extraceUular calcium to 0.25 ttM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM-and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.

Receptor protein tyrosine phosphatase β (RPTPβ) is expressed as soluble and receptor forms with common extracellular regions consisting of a carbonic anhydrase domain (C), a fibronectin type III repeat (F), and a unique region called S. We showed previously that a recombinant Fc fusion protein with the C domain (βC) binds to contactin and supports neuronal adhesion and neurite growth. As a substrate, βCFS was less effective in supporting cell adhesion, but it was a more effective promoter of neurite outgrowth than βCF. βS had no effect by itself, but it potentiated neurite growth when mixed with βCF. Neurite outgrowth induced by βCFS was inhibited by antibodies against Nr-CAM and contactin, and these cell adhesion molecules formed a complex that bound βCFS. NIH3T3 cells transfected to express βCFS on their surfaces induced neuronal differentiation in culture. These results suggest that binding of glial RPTPβ to the contactin/Nr-CAM complex is important for neurite growth and neuronal differentiation.

The neural cell adhesion molecule NCAM plays an important role in axonal growth, learning, and memory. A signaling pathway has been elucidated in which clustering of the NCAM140 isoform in the neural plasma membrane stimulated the activating phosphorylation of mitogen‐activated protein kinases (MAPKs) and the transcription factor cyclic AMP response‐element binding protein (CREB). NCAM clustering transiently induced dual phosphorylation (activation) of the MAPKs ERK1 and ERK2 (extracellular signal‐regulated kinases) by a pathway regulated by the focal adhesion kinase p125fak, p59fyn, Ras, and MAPK kinase. CREB phosphorylation at serine 133 induced by NCAM was dependent in part on an intact MAPK pathway. c‐Jun N‐terminal kinase, which is associated with apoptosis and cellular stress, was not activated by NCAM. Inhibition of the MAPK pathway in rat cerebellar neuron cultures selectively reduced NCAM‐stimulated neurite outgrowth. These results define an NCAM signal transduction mechanism with the potential for modulating the expression of genes needed for axonal growth, survival, and synaptic plasticity. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 542–558, 1999

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