A Peptide Motif from the Second Fibronectin Module of the Neural Cell Adhesion Molecule,NCAM, NLIKQDDGGSPIRHY,is a Binding Site for the FGF Receptor

The mechanism of fibroblast growth factor receptor (FGFR) activation by the neural cell adhesion molecule (NCAM) is not well understood. A motif in the second NCAM fibronectin type III (FN3) module, termed FGL, has by means of nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analyses been demonstrated to be involved in NCAM–FGFR interactions. An FGFR activation motif (FRM) in the first NCAM FN3 module also has been suggested to take part in NCAM interactions with FGFR. Here, we show for the first time that a peptide motif in the second NCAM FN3 module, different from the previously described FGL motif (NLIKQDDGGSPIRHY; termed BCL) binds and activates FGFR and induces FGFR-dependent neurite outgrowth in cultures of cerebellar granule neurons. Our results provide evidence that the BCL motif is one of the multiple FGFR binding sites in NCAM. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Alternative Splicing of the Cytoplasmic Domain of Neural Cell Adhesion Molecule Alters Its Ability to Act as a Substrate for Neurite Outgrowth

A full-length cDNA encoding 180-kDa neural cell adhesion molecule (NCAM 180) has been transfected into mouse NIH-3T3 fibroblasts, and stable clones expressing the transgene have been isolated and characterised. Transfection was associated with the expression of a major protein band of 180 kDa and a minor related band of 140 kDa. Antibodies reactive exclusively with human NCAM immunoprecipitated both proteins but failed to coprecipitate any other proteins. The ability of transfected NCAM to stimulate neurite outgrowth was determined by culturing rat cerebellar neurons on top of confluent monolayers of parental 3T3 cells or clones of transfected 3T3 cells expressing either NCAM 140 or NCAM 180. The results show that NCAM 180 is less able to act as a substrate for neurite outgrowth than NCAM 140.     

Equilibrium Binding Analysis of Neural Cell Adhesion Molecule Binding to Heparin

The kinetics of neural cell adhesion molecule (NCAM) binding to heparin were studied in a heparin-Sepharose-based solid-phase binding assay. The observed binding is time dependent and saturable. A binding constant of 5.2 +/- 1.4 X 10(-8) M is observed for binding of newborn rat NCAM to heparin. This is approximately 25 times lower than the binding constant determined for newborn rat NCAM homophilic binding. Both Scatchard and Hill plot analyses suggest the presence of only one binding site. Fab' fragments of antibodies to rat NCAM significantly inhibit binding, a result indicating that a specific site on NCAM is involved in binding to heparin. The binding is inhibited by heparin (IC50, approximately 5 micrograms/ml), whereas chondroitin sulfate is a less potent inhibitor (IC50, approximately 15 micrograms/ml).     

Casein Kinase II Phosphorylates the Neural Cell Adhesion Molecule L1

Abstract: L1 is an axonal cell adhesion molecule found primarily on projection axons of both the CNS and PNS. It is a phosphorylated membrane-spanning glycoprotein that can be immunoprecipitated from rat brain membranes in association with protein kinase activities. Western blot analysis demonstrates that casein kinase II (CKII), a ubiquitous serine/threonine kinase enriched in brain, is present in these immunoprecipitates. CKII preparations partially purified from PC12 cells are able to phosphorylate recombinant L1 cytoplasmic domain (L1CD), which consists of residues 1,144–1,257. Using these as well as more highly purified kinase preparations, phosphorylation assays of small peptides derived from the L1CD were performed. CKII was able to phosphorylate a peptide encompassing amino acids (aa) 1,173–1,185, as well as a related peptide representing an alternatively spliced nonneuronal L1 isoform that lacks aa 1,177–1,180. Both peptides were phosphorylated with similar kinetic profiles. Serine to alanine substitutions in these peptides indicate that the CKII phosphorylation site is at Ser^1,181. This is consistent with experiments in which L1CD was phosphorylated by these kinase preparations, digested, and the radiolabeled fragments sequenced. Furthermore, when L1 immunoprecipitates were used to phosphorylate L1CD, one of the residues phosphorylated is the same residue phosphorylated by CKII. Finally, in vivo radiolabeling indicates that Ser^1,181 is phosphorylated in newborn rat brain. These data show that CKII is associated with and able to phosphorylate L1. This phosphorylation may be important in regulating certain aspects of L1 function, such as adhesivity or signal transduction.     

Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Promotes Neuritogenesis and Cell Survival

The cell adhesion molecule L1 is a Lewis^x-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewis^x-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg⁶⁸⁷ yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system.     

Biochemical Characterization of Different Molecular Forms of the Neural Cell Adhesion Molecule L1

The neural cell adhesion molecule L1 is a phosphorylated, integral membrane glycoprotein that is recovered from adult mouse brain tissue by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, and 80 kilodaltons (L1–200, L1–180, L1–140, and L1–80, respectively). It has been shown that L1–140 and the phosphorylated L1–80 is generated from L1–200 by mild proteolytic treatment of intact cells. In the present study we have investigated the structural relationships between the different molecular forms of L1 and their location with regard to the surface membrane. We could show that L1–200 has two preferred cleavage sites, one that generates the amino terminal, extracellularly exposed L1–140 and the carboxy terminal L1–80 that spans the membrane. Cleavage at the other site leads to the generation of the amino terminally located L1–180 and the membrane-attached, phosphorylated carboxy terminal L1–30. This site is cleaved during treatment of live cultured cells with broad-spectrum, protease-free phospholipase C (but not phosphatidylinositol-specific phospholipase C) or exposure to sodium azide or cyanogen bromide. Other conditions that cause damage to cells do not lead to the generation of L1–180 and L1–30, suggesting a particular cell-intrinsic cleavage mechanism. L1–180 is truly soluble in aqueous solutions, since it can be recovered from culture supernatants and in the supernatant of a crude membrane fraction after incubation for 2 h at 37°C. Although trypsin treatment alone does not release L1–140 into the supernatant, combination of phospholipase C and mild tryptic treatment leads to the release of L1–140 and L1–50, the latter being most likely the extracellularly exposed domain of L1–80 that is complementary to the membrane-integrated phosphorylated L1–30. Phase separation experiments with Triton X-114 show that the released forms of L1–180 and L1–140 distribute into the aqueous phase, whereas they distribute into the detergent phase when in association with L1–200 or L1–80. However, when L1–80 is cleaved to yield the soluble L1–50 and membrane-anchored L1–30, L1–140 is released into the supernatant together with L1–50. A strong affinity of L1–200, L1–140, and L1–80 to each other is also indicated by the fact that they incorporate together into liposomes and separate only under strong detergent conditions. Also, a strong tendency to aggregate is observed for L1-containing liposomes, but not for those containing the adhesion molecules neural cell adhesion molecule and myelin-associated glycoprotein. Although the physiological roles of the soluble L1 forms, their mode of generation, and the strong affinity for each other remain to be investigated, the availability of soluble forms of L1 opens the possibility to use them as probes for the functional properties of L1 in assay systems involving live cells in vitro.     

Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix

A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37 degrees C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fibronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced. (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 X 10(-9) M, depending on the collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo.     

Up-regulation of the Homophilic Adhesion Molecule Sidekick-1 in Podocytes Contributes to Glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease worldwide. Although the mechanisms underlying this important disease are poorly understood, the glomerular podocyte clearly plays a central role in disease pathogenesis. In the current work, we demonstrate that the homophilic adhesion molecule sidekick-1 (sdk-1) is up-regulated in podocytes in FSGS both in rodent models and in human kidney biopsy samples. Transgenic mice that have podocyte-specific overexpression of sdk-1 develop gradually progressive heavy proteinuria and severe FSGS. We also show that sdk-1 associates with the slit diaphragm linker protein MAGI-1, which is already known to interact with several critical podocyte proteins including synaptopodin, α-actinin-4, nephrin, JAM4, and β-catenin. This interaction is mediated through a direct interaction between the carboxyl terminus of sdk-1 and specific PDZ domains of MAGI-1. In vitro expression of sdk-1 enables a dramatic recruitment of MAGI-1 to the cell membrane. Furthermore, a truncated version of sdk-1 that is unable to bind to MAGI-1 does not induce podocyte dysfunction when overexpressed. We conclude that the up-regulation of sdk-1 in podocytes is an important pathogenic factor in FSGS and that the mechanism involves disruption of the actin cytoskeleton possibly via alterations in MAGI-1 function.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

Lipid Raft-dependent Endocytosis of Close Homolog of Adhesion Molecule L1 (CHL1) Promotes Neuritogenesis

CHL1 plays a dual role by either promoting or inhibiting neuritogenesis. We report here that neuritogenesis-promoting ligand-dependent cell surface clustering of CHL1 induces palmitoylation and lipid raft-dependent endocytosis of CHL1. We identify βII spectrin as a binding partner of CHL1, and we show that partial disruption of the complex between CHL1 and βII spectrin accompanies CHL1 endocytosis. Inhibition of the association of CHL1 with lipid rafts by pharmacological disruption of lipid rafts or by mutation of cysteine 1102 within the intracellular domain of CHL1 reduces endocytosis of CHL1. Endocytosis of CHL1 is also reduced by nifedipine, an inhibitor of the L-type voltage-dependent Ca²⁺ channels. CHL1-dependent neurite outgrowth is reduced by inhibitors of lipid raft assembly, inhibitors of voltage-dependent Ca²⁺ channels, and overexpression of CHL1 with mutated cysteine Cys-1102. Our results suggest that ligand-induced and lipid raft-dependent regulation of CHL1 adhesion via Ca²⁺-dependent remodeling of the CHL1-βII spectrin complex and CHL1 endocytosis are required for CHL1-dependent neurite outgrowth.     

A Single Immunoglobulin-like Domain of the Human Neural Cell Adhesion Molecule L1 Supports Adhesion by Multiple Vascular and Platelet Integrins

The neural cell adhesion molecule L1 has been shown to function as a homophilic ligand in a variety of dynamic neurological processes. Here we demonstrate that the sixth immunoglobulin-like domain of human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin superfamily including α_vβ₃, α_vβ₁, α₅β₁, and α_IIbβ₃. The interaction between L1-Ig6 and α_IIbβ₃ was found to support the rapid attachment of activated human platelets, whereas a corresponding interaction with α_vβ₃ and α_vβ₁ supported the adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human L1-Ig6 effectively abrogated binding by the aforementioned integrins. A L1 peptide containing this RGD motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocked L1 integrin interactions and, as an immobilized ligand, supported adhesion via α_vβ₃, α_vβ₁, α₅β₁, and α_IIbβ₃. Whereas β₃ integrin binding to L1-Ig6 was evident in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺, a corresponding interaction with the β₁ integrins was only observed in the presence of Mn²⁺. Furthermore, such Mn²⁺-dependent binding by α₅β₁ and α_vβ₁ was significantly inhibited by exogenous Ca²⁺. Our findings suggest that physiological levels of calcium will impose a hierarchy of integrin binding to L1 such that α_vβ₃ or active α_IIbβ₃ > α_vβ₁ > α₅β₁. Given that L1 can interact with multiple vascular or platelet integrins it is significant that we also present evidence for de novo L1 expression on blood vessels associated with certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel role for L1 in vascular and thrombogenic processes.     

Structural Analysis of the Sixth Immunoglobulin-Like Domain of Mouse Neural Cell Adhesion Molecule L1 and Its Interactions with αvβ3, αIIbβ3, and α5β1 Integrins

Abstract: Previous experiments suggested that the human cell adhesion molecule L1 interacts with different integrins via its sixth immunoglobulin-like domain in an RGD-dependent manner. Here we have described the expression of this domain from early postnatal mouse brain, analyzed the structure of the recombinant protein by circular dichroism and fluorescence spectroscopy, and performed solid-phase binding studies to αvβ3, αIIbβ3, and α5β1 integrins. The domain was found to have the expected β-sheet organization, which was lost in the presence of guanidine hydrochloride. The midpoint of the single-step transition occurred at 1.5 M guanidine hydrochloride. The sixth immunoglobulin-like domain of mouse brain L1 contains two RGD motifs and was found to bind in a concentration-dependent and saturable way to αvβ3, αIIbβ3, and α5β1 integrins, suggesting specific interactions with these ligands. However, only the interaction to αvβ3 could be inhibited in a concentration-dependent manner by an RGD-containing peptide, and the IC₅₀ was determined to be ∼20 n M . Mutants of the domain, which lack either one or both of the RGD sites, demonstrated that the RGD site comprising residues 562–564 is involved in the interaction to αvβ3. Our findings indicate an RGD-independent mechanism for the interactions to αIIbβ3 and α5β1, as no involvement of any RGD motif could be demonstrated.     

Developmental Expression of Neural Cell Adhesion Molecules of Oligodendrocytes In Vivo and in Culture

Abstract: Previously, we have shown that oligodendrocyte adhesion molecules are related to the 120,000–M_r neural cell adhesion molecule (NCAM-120). In this report, we present further evidence that the oligodendrocyte adhesion molecule is NCAM-120. Studies on the expression of NCAM-120 and other molecular forms of NCAM in vivo in rat brain, in vitro in primary mixed cultures, and in cultures enriched for oligodendrocytes are described. Western blot analysis of rat brain using anti-NCAM showed that NCAM-120 first appears at postnatal day 7 and increases in quantity thereafter, coincident with the development of oligodendrocytes in vivo and comparable to the expression of myelin basic protein. Purified oligodendrocytes from 4-week-old rat brains expressed only NCAM-120. Quantitation of various forms of NCAMs in rat brain showed marked age-related differences in the expression of three molecular forms of NCAM. Immunofluorescence analysis showed that oligodendrocytes, at all ages tested, expressed NCAM, but in older oligodendrocytes, the intensity of staining was less. Western blot analysis of oligodendrocyte-enriched cultures showed that from day 1 after isolation (12 days of age) through day 7 after isolation (18 days of age) only NCAM-120 is seen. A possible role for NCAM in myelination and remyelination is discussed.     

The Cell Adhesion Molecule L1 Is Developmentally Regulated in the Renal Epithelium and Is Involved in Kidney Branching Morphogenesis

We immunopurified a surface antigen specific for the collecting duct (CD) epithelium. Microsequencing of three polypeptides identified the antigen as the neuronal cell adhesion molecule L1, a member of the immunoglobulin superfamily. The kidney isoform showed a deletion of exon 3. L1 was expressed in the mesonephric duct and the metanephros throughout CD development. In the adult CD examined by electron microscopy, L1 was not expressed on intercalated cells but was restricted to CD principal cells and to the papilla tall cells. By contrast, L1 appeared late in the distal portion of the elongating nephron in the mesenchymally derived epithelium and decreased during postnatal development. Immunoblot analysis showed that expression, proteolytic cleavage, and the glycosylation pattern of L1 protein were regulated during renal development. L1 was not detected in epithelia of other organs developing by branching morphogenesis. Addition of anti-L1 antibody to kidney or lung organotypic cultures induced dysmorphogenesis of the ureteric bud epithelium but not of the lung. These results suggest a functional role for L1 in CD development in vitro. We further postulate that L1 may be involved in the guidance of developing distal tubule and in generation and maintenance of specialized cell phenotypes in CD.     

Glucocorticoids and Nerve Growth Factor Differentially Modulate Cell Adhesion Molecule L1 Expression in PC12 Cells

Abstract: The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one-half, whereas NGF increased L1 protein levels ∼2.3-fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10⁻⁷ M dexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down-regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adrenal gland.     

Palmitoylation of Neurofascin at a Site in the Membrane-Spanning Domain Highly Conserved Among the L1 Family of Cell Adhesion Molecules

Abstract: This report presents the first evidence that a member of the L1 family of nervous system cell-adhesion molecules is covalently modified by thioesterification with palmitate, and identifies a highly conserved cysteine in the predicted membrane-spanning domain as the site of modification. Neurofascin is constitutively palmitoylated at cysteine-1213 at close to a 1:1 molar stoichiometry. Kinetics of palmitate incorporation into neurofascin expressed in resting neuroblastoma cells indicate that the palmitate modification has the same turnover rate as the polypeptide chain and does not affect the protein stability of neurofascin. Palmitoylation of neurofascin expressed in dorsal root ganglion neurons is not required for delivery of neurofascin to the plasma membrane or targeting to axons. Palmitoylation also has no effect on ankyrin-binding activity of neurofascin, on the oligomeric state of neurofascin in solution, or on cell-adhesion activity of neurofascin expressed in neuroblastoma cells. A significant difference between native and C1213L neurofascin is that these proteins were localized in distinct fractions within a low-density membrane population enriched in signaling molecules. These results indicate a palmitate-dependent targeting of neurofascin to a specialized membrane microdomain.     

The Role of Cytoplasmic Serine Residues of the Cell Adhesion Molecule L1 in Neurite Outgrowth,Endocytosis, and Cell Migration

Summary 1. The cell adhesion molecule L1 has been implicated in adhesion and migration of cells, in axon growth, guidance, and fasciculation, in myelination and synaptic plasticity. The cytoplasmic domain of neuronal L1 is highly conserved between species and has been shown to be phosphorylated at serine and tyrosine residues. 2. To investigate the significance of L1 serine phosphorylation, mutants of L1 were generated in which ser-1152, ser-1181, ser-1204, and ser-1248 were exchanged for leucine and rat B35 neuroblastoma cells were stably transfected with the L1-cDNA constructs. 3. Neurite outgrowth on poly-l-lysine (PLL) as substrate was determined either with or without differentiation into a neuronal phenotype with dbcAMP. In addition, antibody-induced endocytosis and cell migration were examined. 4. Our observations indicate that phosphorylation of single serine residues of the cytoplasmic domain of L1 contributes to neurite outgrowth through different mechanisms. Neurite growth is increased when ser-1152 or ser-1181 is replaced by a non-phosphorylatable leucine and decreased when ser-1204 or ser-1248 is mutated to leucine. Furthermore, mutation of ser-1181 to leucine results in strongly enhanced antibody-induced endocytosis of L1 and also in enhanced cell migration.     

Chondrocyte Apoptosis Induced by Aggrecan G1 Domain as a Result of Decreased Cell Adhesion

A major feature of cartilage deterioration during joint injury and disease is aggrecan degradation and the loss of proteoglycan. Most of the degraded fragments are released into the circulatory system except the G1 domain which accumulates locally in the synovial fluid and cartilage because of its hyaluronan-binding ability. In this study, our objective was to investigate the effects of G1 accumulation on chondrocyte function. We chose to mimic the accumulation of G1 domain by developing a method to express G1 in chondrocytes. We transiently and stably expressed aggrecan G1 domain in the cells and tested the effects of G1 in cell adhesion and apoptosis. Overexpression of the G1 construct induced apoptosis in adherent chondrocytes but not in chondrocytes maintained in suspension cultures. Higher levels of G1 expression caused greater reduction in cell-substratum interaction and induced more cell death. The effect was dose dependent. To corroborate our findings, the role of G1 in reducing adhesion and inducing apoptosis was further investigated in fibroblasts. We found that low adherent cultures also had high levels of apoptosis. Our results suggest that G1 induced apoptosis by destabilizing cell-substratum interaction.